SPG5, SPG8, SPG11 SPG12, SPG15, SPG16 SPG20, SPG21, SPG26 SPG22, SPG47, SPG48 SPG49, SPG50, SPG53 SPG54, SPG55, SPG56 SPG58, SPG63, SPG64 SPG65, SPG67, SPG74 SPG13 (HSPD1), RNASEH2B, ADAR1, IFIH, SPG2 Spg4

SPG10,SPG13, SPG17, SPG31 SPG36 Spg4 Spg8

Sca1

Liena Elbaghir

Omer Elsayed

Inaam N Mohammed

Ahlam Abd

Alrahman Ahmed Hamed

Maha Abdelmoneim Elseed

Adam Johnson

Mathilde Mairey

Hassab Elrasoul Siddig

Ali Mohamed

Mohamed Nagib Idris

Mustafa A M Salih

Sarah Misbah El-Sadig

Mahmoud E Koko

Ashraf Yahia

Osman Mohamed

Laure Raymond

Marie Coutelier

Frédéric Darios

Rayan Abubaker Siddig

Ahmed Khalid

Mohamed Albashir Ahmed

Arwa Mohammed

Abbker Babai

Hiba Mohamed

Osman Malik

Zulfa Mohammed Babikir

Mohammed Omer

Eman Osama

Eldirdiri Mohamed

Hanan Babikir Eltahir

Nasr Aldin

Ali Magboul

Elfatih Elfadl Bushara

Abdelrahman Elnour

Salah Mohamed

Abdel Rahim

Abdelmoneim Alattaya

Mustafa Idris Elbashir

Eltayeb Muntaser

Ibrahim

Alexandra Durr

Anjon Audhya

Professor Alexis Brice
email: alexis.brice@upmc.fr

Ammar Eltahir Ahmed

Giovanni Stevanin

linked Mito Mitochondrial SPGn Spastic paraplegia gene "n" ALS Amyotrophic lateral sclerosis CP Cerebral palsy SLS Sjogren-Larsson Syndrome MSS Marinesco Sjogren Syndrome ARSACS AR spastic ataxia of Charlevoix-Saguenay NGS Next generation sequencing LHON Leber hereditary optic neuropathy PLS Primary lateral sclerosis PD Parkinson disease CPSQ Cerebral palsy spastic quadriplegia IEM Inborn errors of metabolism E-Pyr Extrapyramidal syndrome PN Peripheral neuropathy PNS Peripheral nervous system CNS Central

Keywords: paraplegia, PLS: primary lateral sclerosis, ALS: amyotrophic lateral sclerosis, PN: peripheral neuropathy, CPSQ: Cerebral palsy spastic quadriplegia, NBIA: neurodegeneration with brain iron accumulation, PD: Parkinson disease. HSP Hereditary Ataxias Extrapyramidal syndromes (NBIA, PD, Dystonias, Choreas) Leukodystrophy Hereditary Spastic Paraplegia, SPG57/TFG, SPG11, SACS, ATL1, ALS2

Hereditary spastic paraplegias (HSP) are the second most frequent motor neuron diseases. We used next generation sequencing to screen 74 HSP genes on 51 patients from 23 consanguineous families from Sudan. Two other families were studied by candidate gene sequencing. We established a genetic diagnosis in seven families (28%) with autosomal recessive complex HSP. Truncating homozygous mutations were identified in SPG11 and SACS genes in three and one families, respectively. Three missense mutations were also found, each in one family, in TFG/SPG57, ATL1 and ALS2. All were novel except one in SPG11 previously reported with earlier onset compared to our patients. The TFG missense mutation (exon 2: c.64C>T p.Arg22Trp / PB1 domain) is the second identified worldwide, and we demonstrated its effect on TFG oligomerization in vitro. Patients did not present with visual impairment, as observed in the previously reported SPG57 family (p.Arg106Cys / coiled coil domain), suggesting unique contributions of the PB1 and coiled coil domains in TFG complex formation/function. Our seven families manifested inter-and intra-familial variations implying the possibility of modifier factors, complicated by high inbreeding that precipitated the phenomenon of multiple genes segregating in different subfamilies. Further genetic heterogeneity is expected in HSP Sudanese families.

III

Acknowledgement

I am grateful to the members of my thesis defense jury who have kindly accepted to read and assess my PhD manuscript. I would also like to express my sincere gratitude to my thesis directors Dr.Giovanni Stevanin and professor Ammar Eltahir Ahmed for kindly accepting to guide me throughout my journey in this challenging PhD project. They have been quite supportive at each step and have given me a great deal of independence which has allowed me to gain the required skills in project management and trouble shooting and be prepared for the next step. Dr.Giovanni Stevanin has struggled to get me the budget necessary for my experiments and has gently helped me at each step despite his rather busy schedule. I enjoyed much his humble friendly attitude despite his vast knowledge in our weekly discussions that we had in the last two months of my stay in France. Pr.Ammar Eltahir has kindly taken the burden of the neurophysiological investigations and opened his heart and clinic for all patients included in the cohort. He has been quite supportive and enthusiastic to help me by all means and efficiently contributed to the recruitment of new families.

I would like to thanks Pr.Alexis Brice for accepting to collaborate and acquaint me in his team and even taking all the effort to come to Sudan to finalize the collaboration. My gratitude is great for Prof. Victor Patterson who has practically started the collaboration by introducing me to Pr.Brice. He has been extremely supportive and encouraging.

All my appreciation is conduced to my mentors Pr.Muntaser Eltayeb Ibrahim and Pr.Mustafa Idris Elbashir who embraced and supported me from the beginning when the project was just an ambitious day dreaming.

Our collaborating Sudanese clinicians are the corner stone in this research. The three lively neuropaediatricians (Dr.Inaam Gashey, Dr.Maha Elseed and Dr.Ahlam Hamed ), the humane Dr.Sarah Misbah, Pr.Mustafa Salih, Pr.Mohamed Nagib and his team, Dr. Hassab Elrasoul Siddig, Pr. Farooq Yasin and all the referring physicians, the radiologists, the ophthalmologists and the neurophysiologists all have contributed significantly to establish this project. My sincere gratitude is conducted to the Sudanese patients and their families who were as kind as a Sudanese can be expected.

Prof. Alexandra Durr and Prof. Odile Boespflug-Tanguy (and her team especially Imen Dorboz) have not saved any effort to help me build my expertise in clinical neurogenetic and neuropaediatrics. Pr.Eric Leguern valuable tips with his sense of humour were priceless.

Prof. Anjon Audhya team (USA) has kindly performed all the biochemical functional studies for the TFG protein and with the Parkinson team (Dr.Suzanne Lesage and her team) has yielded our successful reports so thanks to their efforts.

The assistants of Alexis Brice team (Lydia Guennec, Marie-Luce Poupinel-Descambres and Antoine Guisier) have been professional and if not for the efforts of Lydia Guennec in my registration for the PhD I would have been unable to continue.

The French embassy in Sudan and Campus France and the University of Khartoum provided me with the scholarship that has enabled me to come and do the PhD study in France.

IV

My acknowledgement to the funding agents of the study: the France-Parkinson Association (to AB), the Agence Nationale de la Recherche (SPATAX-QUEST project, to GS), the Verum Foundation (to GS and AB), the Roger de Spoelberg Foundation (R12123DD, to AB), the French Academy of Sciences (to AB), the European Union (Neuromics projects, OMICS call, to AB) and the European Joint Programme -Neurodegenerative Disease Research (JPND-COURAGE-PD) project and Programme d'Investissement d'Avenir IHU-A-ICM (ANR-10-IAIHU-06).

I would like to transmit my gratitude to team of the DNA and Cell bank (with special regards to the friendly Sylvie Forlani and Christelle Dussert), the laboratory and bioinformatics staff of the sequencing platform (ICM) in which we performed all the next generation techniques and the bioinformatic analysis, the platform P3S where we had the homozygosity mapping experiments performed with the help of Dr.Wassila Carpentier (who has done big efforts not only to explain and train but also has given me valuable tips for my thesis writing).

Working with the team of Dr.Giovanni Stevanin has been a pleasure. They have become my second family. Professionally, Marie Coutelier and Christelle Tesson helped me in the first steps of my PhD and have been quite cooperative whenever I needed. The moments that I had with Marie were great and extremely pleasant. With Marie I discovered that Paris is truly described the city of light and because of that I stated to fall in love with the quiet lighted streets at night. The help that I have received from Mathilde Mairey with a beautiful smile and a comforting attitude of a friend cannot be described by words. Her efforts to help me in the last months of my PhD make me run out of expressions. Sawssan Ben Romdhan has been the support (with a friendly spirit) that I needed at a time when I was so desperate because of the accumulating work. Dr. Frédéric Darios and his professional group have helped and trained me on the basics of manipulations of cells and functional experiments. Special thanks are conducted to the nice Typhaine Esteves. Livia Parodi, Federica Barreca, Graziella Mongone and Paola have made me believe that Italians are all kind. The special memories, help, great laughter and the discussions I had with Khalid Hamid El-Hachimi, Livia Parodi, Celine Lustremant, Valérie Drouet, Claire Pujol, Claire Sopie Davoine, , Khadija Tahiri, Sara Morais, Oriane Trouillard, Annie Sittler, Remi Valter, Julien Branchu make me grateful to them and I feel sad and tearful when I imagine that we can't be together anymore as we used to be. Special thanks to Claire Pujol for helping me with the final editing of the manuscript. She helped me at the critical moments of extreme fatigue and exhaustion with unforgettable friendship spirit.

My team members in Sudan have been the scaffolding unit of all the work. Ashraf Kambalawy, Mahmoud Koko, Rayan Abubakar established the team with me and their professional attitude, hard working, highly committed and loyalty made me surmount all the obstacles. All the younger members of my team have worked extremely hard just to make our work successful without waiting for any reward and they have represented our team in conferences and workshops in a distinct way (Arwa Babai, Zulfa Mohamed, Hiba Malik, Israa Zuhair, Amal Salah, Ahmed Khalid, Elhami Abdalmuti, Lina Salah, Duaa Mustafa, Mayada Osama, Nabila Mohamed Hasan, Abdelhamid Mohamed and the two under-graduates Malka Osman and Eman Osama).

Finally but most importantly, I would like to say honestly and proudly say that if not for the support of my family I would have done literally nothing. They have set an example of a great family who would sacrifice for the sake of others. My husband I would describe as a great man. Not only has he sacrificed all his rights of a stable situation with a settled wife but also V he supported me psychologically and financially. My parents ElBaghir Omer Elsayed and Fatma Elsaraj have taken care of my kids and I would say my mother deserves a medal of honour for taking in charge the responsibilities of my kids' studies and have made them brilliant youngsters better than what I could have done. My kids (Tull, Abdelaziz and Yusr) have encouraged me to go on when I was in despair and they insisted that I continue to help the patients. My parents in law (Abdelaziz Tangasawi and Asia Abdallah) took in charge a significant deal of my responsibly towards my husband and kids in parenting manner. The precious professional advices of my father in law have been extremely useful.

My brother Omer Elbaghir has shown me what it means to be a brother. He supported me psychologically and supported my research financially with real love. His wife Dina Saklah used to call me to encourage me. I would like to thank all my dear sisters (Lubaba, Lubna and Shayamaa) and their families for the continuous supporting love. All my appreciation Shaymaa who helped my mother in her care for my kids in silent way.

Finally, my thanks are to other friends and family that I could not list all. The combined efforts of everyone have helped me overcome the sorrow that I used to experience upon separation from my family over long months I spent alone in France.

Liena Elbaghir Omer Elsayed

Introduction

Hereditary spastic paraplegias (HSP) are a heterogeneous group of neurodegenerative disorders with the core defining clinical features of insidiously progressive weakness and spasticity of the lower extremities. They impose diagnostic challenges due to the multiple overlaps with other neurological disorders in which spastic paraplegia/quadriplegia is present.

The rapidly expanding surge of new causative genes (named spastic paraplegia gene "n"or SPGn) that accompanied the emergence of multiple next generation sequencing (NGS) techniques further contributed to this diagnostic dilemma. In addition, the concept of neurodegeneration being a continuum of disorders is illustrated by 2 facts in HSP:

-Many clinico-genetic HSP entities are associated with other neurological signs that can be the major clinical features of overlapping diseases. For example, hereditary ataxias (HA) represent the other extreme of the spectrum of spinocerebellar neurodegenerative disorders.

HAs may sometimes be difficult to differentiate clinically from HSPs as cerebellar manifestations are present in more than 30 clinico-genetic SPG entities. Many HSP subtypes present with peripheral neuropathy [SPG3A, SPG5A, SPG11, SPG17, SPG30, SPG31, SPG57, SPG70, FAM134B]. A third example is leukodystrophy (non-neurodegenerative disorders). Although it is classified as a differential diagnosis of HSP that should be excluded in early steps of clinico-genetic investigations, white matter abnormalities are described as a complicating feature for at least 28 HSP subtypes, making differential diagnosis a real challenge.

-Mutations in multiple SPG genes can account for HSP but also for allelic phenotypes. This is the case for motor neuron disease [MND] [KIAA1840 (SPG11), ERLIN2 (SPG18)] and the reverse situation is also true (ALS2 is a major cause of amyotrophic lateral sclerosis but few HSP families have also been reported mutated in this gene). Moreover, mutations in seven SPG genes are associated with leukodystrophy as an allelic disorder [PLP1(SPG2), HSPD1 In this study, I explored the clinical varieties and genetic and molecular pathways behind spastic neurodegeneration in a familial Sudanese cohort. The Sudanese population is highly consanguineous with inter-and intra-generational consanguinity loops. Although a lot is known about it from clinical neurologic point of view, it remains almost completely unexplored from a neurogenetic point of view. Statistics estimates about incidence, prevalence and relative frequencies of HSP genes are not available to the present time as in most sub-Saharan countries.

With the help of clinicians from Sudan, we recruited 41 nuclear and extended Sudanese families representing 337 total number of individuals of whom 106 were affected patients.

First, full clinical examination was performed by myself and/or my colleagues in Sudan. Then I have established a DNA bank. To do so, saliva samples were collected for extraction of genomic DNA. When necessary for validation experiments, fibroblasts were also obtained from skin biopsies. The second aspect of my thesis consisted of deciphering the genetic bases explaining the disease in these families. Screening of several known SPG genes was done in five families using a phenotype-based candidate gene approach. I applied an NGS-based screening strategy for 74 SPG-related genes as the main screening method in all remaining 36 families and the two families with negative candidate gene approach result. Whole exome sequencing (WES) was then done in search for novel genes in families with negative screening results in which DNA of two or more patients were available. Homozygosity mapping using genotyping array was done to further consolidate gene search process. In certain conditions, functional studies were necessary, depending on the feasibility and novelty of expected outcome.

I identified the genetic cause in 17/41 families. In 12 families the genes where I found a segregating mutation, were known HSP genes. In three families, novel genes were identified mutated whereas five candidate genes were found segregating with disease in two other unrelated families requiring additional experiments to conclude. In these highly inbred families, multiple genes /genetic disorders were found running in parallel in different branches [five families] or in the same branch [three families] of the family. Analysis of the NGS screening panel and of WES data, thus imposed certain challenges specific to these families with elevated consanguinity rates. Another peculiar phenomenon was the appearance X of clinical differences between Sudanese patients and patients of other origins even when caused by mutations in the same genes and in rare conditions even when caused by the same variant which contributed to the phenotypic heterogeneity already reported in these disorders.

State of the art

Hereditary spastic paraplegia (HSP) is considered as the second most frequent motor neuron disease (MND) with a prevalence of 3-10/100000 in most populations (Noreau et al., 2014).

HSP is transmitted via all modes of inheritance which include: autosomal dominant (AD), autosomal recessive (AR), X-linked (X-L), mitochondrial/maternal (Mito) and mixed inheritance (AD/AR; AR/? AD). The Prevalence of (AD) HSP ranges between 0.5-5.5/100000 and (AR) HSP from 0.3-5.3/100000 (Ruano et al., 2014). HSP shows remarkable genetic and clinical heterogeneity [Figure , [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF], [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF]]. To date there are more than 88 loci/72 genes implicated in the pathogenesis of HSP. 75 clinico-genetic forms are assigned the designation spastic paraplegia gene "n"or SPGn but other forms remain unclassified yet (Tesson et al., 2015) [START_REF] Klebe | Clinical and genetic heterogeneity in hereditary spastic paraplegias: from SPG1 to SPG72 and still counting[END_REF]. The category of mixed inheritance has emerged recently with increasing number of genes showing this pattern (five genes with frank AD/AR inheritance: SPG3A, SPG9, SPG72, BICD2, SPG30 and two showing AR inheritance with milder/ partial phenotype in individuals with heterozygous genotype (possible dose dependent phenotype): SPG7,SPG58) [Table ].

Clinical spectrum of HSP 1.Associated signs and clinical phenotypes

There is huge phenotypic heterogeneity associated with HSP. The core clinical presentation of patients is with pyramidal syndrome which includes increased tone (spasticity with scissoring and clonus in its most severe form), hyperreflexia and extensor plantar response (Babinski sign). It can be classified as pure (uncomplicated) and complex (complicated) according to the absence or presence of additional neurological and extra-neurological manifestations [Figure [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF]]. Pure forms can be associated with involvement of the dorsal column and diminuted or even abolished vibration sense. Sphincteric involvement (mainly urinary urgency) can occur in pure HSP too due to increased bladder muscle tone (Fink, 2000) (Tesson et al., 2015) [START_REF] Klebe | Clinical and genetic heterogeneity in hereditary spastic paraplegias: from SPG1 to SPG72 and still counting[END_REF]. Complicating neurological clinical features include cognitive/mental function deterioration, cerebellar syndrome, peripheral neuropathy, features of lower motor neuron involvement (amyotrophy), signs of bulbar/ pseudobulbar palsy, optic neuropathy, psychiatric symptoms, extrapyramidal signs, auditory neuropathy as well as brain imaging abnormalities. MRI of the brain may show atrophy (focal: thin corpus callosum, atrophy of the cortex and cerebellum or generalized), features of dysmyelination (hypo-and de-myelination) and leukodystrophic changes (white matter hypersignal intensity lesions). Extraneurologic systems involved are the eye, heart, skeletal system, skin, hair and gastrointestinal system.

Cognitive and mental impairment are the most frequent clinical features complicating various HSP forms (≈38 forms) followed by cerebellar signs, peripheral neuropathy and/or amyotrophy and eye signs taken collectively (cataract, retinal/macular degeneration and optic atrophy) [Annexes: Tables 1 to 11] The rapidly expanding genetic heterogeneity has resulted in an even more increase of phenotypic heterogeneity. The spectrum of HSP has broadened largely to include additional atypical phenotypes that were not reported previously in the earlier cohorts of HSP [START_REF] Harding | Classification of the hereditary ataxias and paraplegias[END_REF] [Table ]. Non-exhaustive illustrative examples include tetraplegic cerebral palsy (SPG50) [START_REF] Verkerk | Mutation in the AP4M1 gene provides a model for neuroaxonal injury in cerebral palsy[END_REF], HSP with hyperbilirubinemia and persistent vomiting due to hiatus hernia (SPG29) [START_REF] Orlacchio | New locus for hereditary spastic paraplegia maps to chromosome 1p31.1-1p21.1[END_REF] and atypical HSP presenting with areflexia, ventriculomegaly, apnoea, hypoventilation and gastrooesophageal reflux disease associated with SPG49/ TECPR2 [START_REF] Oz-Levi | Mutation in TECPR2 Reveals a Role for Autophagy in Hereditary Spastic Paraparesis[END_REF].

The list of reported complicating neurological and non-neurological clinical signs has also extensively expanded to reach the present range. In addition to the signs associated with the above mentioned phenotypes; the mitochondrial genes (MT-ATP6, MT-TI) cause with HSP associated with cardiomyopathy [START_REF] Verny | Hereditary spastic paraplegia-like disorder due to a mitochondrial ATP6 gene point mutation[END_REF]. Another mitochondrial gene MT-CO3 has been linked to HSP but with a Leigh syndrome-like lesion in the brain [START_REF] Tiranti | A novel frameshift mutation of the mtDNA COIII gene leads to impaired assembly of cytochrome c oxidase in a patient affected by Leigh-like syndrome[END_REF].

The greatest part of clinical heterogeneity can be attributed to the AR HSP forms which mainly present as complicated HSP in contrast to the tendency of AD HSP for pure presentation [Figure [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF]] [Table ].

Age at onset

Age at onset of HSP varies widely. It ranges from birth in some HSP forms to more than 40 years (in some rare cases the disease started at the age of 80 years). The variability of age at onset not only occurs between various forms but also occurs within single HSP forms and even within families in patients carrying the same mutation [Figure [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF]].

It is often difficult to assess the age at onset precisely in HSP. This is sometimes attributed to the subtlety of the presenting motor symptoms particularly in complex forms. In pure forms, some patients do not complain of the disease but have clinical signs at examination. This results in an underestimated and overestimated incidence, prevalence of HSP.

The age at onset of AR HSP tends to cluster in childhood in 80% (45/57) of the genetic forms. More than 34 AR HSP forms as well as (3/5) X-linked forms occur in toddlers (0 -<5 years). AD forms do not show similar clustering but tend to have variable age at onset. Some forms show substantial variability. Of these forms with widely variable age at onset are four AD HSP subtypes [including three of the commonest AD HSP forms (SPG4, SPG3A, SPG10)], two AR HSP (SPG11 and SPG48) and an X-linked HSP (SPG2) [Figure 12345].

Disease progression

Most HSP subtypes are slowly progressive. However, significant variability exists regarding disease severity and progress. Some forms deteriorate rapidly and multiple subtypes are very slowly progressive that can be fairly considered as non-progressive. Surprisingly, late onset forms are more often associated with a more rapidly progressive evolution. In many cases the disease is progressive at the start of the disease then reach a static plateau. Rarely, the disease improves before reaching this plateau.

History of Spastic Neurogenetic Disorders

It is important to go back to the history of HSP and the evolution of its definition and classification over time in order to better understand the dimensions of the HSP. This will help identify the relations among all overlapping neurogenetic disorders which present with pyramidal syndrome as whole or part of their phenotypic spectrum.

HSPs were described first in 1883 by the German neurologist Adolph Strümpell and later described more extensively in 1888 by the French physician Maurice Lorrain.

In 1983, A. E. Harding suggested classification of hereditary ataxias (HAs) and HSPs.

In this classification, HSP was classified into pure and complicated HSP and each was further grouped into subtypes based on the mode of inheritance in the pure HSP and on clinical syndromic presentation in the complicated forms. It is noteworthy that many of the syndromes included in the complicated forms are reclassified at present as subtypes of other overlapping syndromes. Examples are Sjogren-Larsson Syndrome (SLS; now considered as leukodystrophy) and AR HA of Charlevoix-Saguenay syndrome (ARSACS; now considered as AR ataxia). Other subclasses were broadly described as could be predicted at that era when the genetic studies were just starting for more precise classification [START_REF] Harding | Classification of the hereditary ataxias and paraplegias[END_REF].

Only five loci and one gene were described until 1996 [START_REF] Fink | Hereditary spastic paraplegia: advances in genetic research. Hereditary Spastic Paraplegia Working group[END_REF]. By 1999, 12 loci, of which, 6 AD loci and two X-linked genes, were linked to HSP. [START_REF] Fink | Hereditary spastic paraplegia: genetic heterogeneity and genotype-phenotype correlation[END_REF] By 2003, 20 loci and 9 genes were identified [START_REF] Fink | Advances in the hereditary spastic paraplegias[END_REF]. The completion of the human genome sequence project (publication of the draft genome in 2001 and the completion of the sequence in 2004) heralded a new era of medical and genetic research and neurogenetic research was not an exception (Venter et al., 2001) (International Human Genome Sequencing [START_REF] Consortium | Finishing the euchromatic sequence of the human genome[END_REF] [START_REF] Stein | Human genome: end of the beginning[END_REF]. The recent advent of the next generation sequencing (NGS) technologies in the late 2000s has resulted in identification of a large number of genes, instead of loci in the previous linkage-dependent era, since 2010 [START_REF] Mardis | Next-Generation DNA Sequencing Methods[END_REF] [START_REF] Metzker | Sequencing technologiesthe next generation[END_REF].

A continuum of spastic neurogenetic disorders

Many hereditary spastic paraplegia genes (including mitochondrial genes) have been found to result in more than one allelic phenotype. These phenotypes have occasionally closely related clinical scenarios. Two obvious examples of genes that present with phenotypic alleles include two mitochondrial genes: MT-ATP6 and MT-CO3. MT-ATP6 as stated earlier has recently been related to HSP [START_REF] Verny | Hereditary spastic paraplegia-like disorder due to a mitochondrial ATP6 gene point mutation[END_REF]. Previously, MT-ATP6 has been linked to Leigh syndrome, Leber hereditary optic neuropathy (LHON) and NARP syndrome [Neuropathy, Ataxia, And Retinitis Pigmentosa [START_REF] Rahman | Leigh syndrome: clinical features and biochemical and DNA abnormalities[END_REF]) [START_REF] Lamminen | A mitochondrial mutation at nt 9101 in the ATP synthase 6 gene associated with deficient oxidative phosphorylation in a family with Leber hereditary optic neuroretinopathy[END_REF] [START_REF] Holt | A new mitochondrial disease associated with mitochondrial DNA heteroplasmy[END_REF]. Another example is the mitochondrial gene MT-CO3 has been linked to HSP but with a Leigh syndrome-like lesion in the brain [START_REF] Tiranti | A novel frameshift mutation of the mtDNA COIII gene leads to impaired assembly of cytochrome c oxidase in a patient affected by Leigh-like syndrome[END_REF]. MT-CO3 has also been associated with Leber optic atrophy [START_REF] Johns | Cytochrome c oxidase mutations in Leber hereditary optic neuropathy[END_REF]. Further examples are illustrated in [Figure [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF]].

There are also numerous genes that are located in the overlap zone with related clinical scenarios. These genes are classified to be related to HSP, HA, leukodystrophy, neurodegeneration with brain iron accumulation (NBIA), Parkinson disease/parkinsonism (PD), primary lateral sclerosis and amytrophic lateral sclerosis (PLS and ALS), cerebral palsy spastic quadriplegia (CPSQ), hereditary dystonia, mental retardation, peripheral neuropathy (PN) and even some inborn errors of metabolism (IEM) as illustrated earlier.

Clearly not all the above mentioned categories are neurodegenerative as exemplified by the overlapping metabolic disorders (eg. leukodystrophy and IEM). This phenomenon is sometimes inseparable from the above observation about one gene presenting with multiple phenotypic alleles. A number of HSP focused examples are illustrated in [Figure [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF]]

The recent genetic advances identified some genes that correspond to loci previously linked to HSP. SPG9 is a clear illustration for this fact. The identification of the gene and the study of the proposed protein function has demonstrated an amino acid metabolic pathway quite related to what has long been considered as a differential diagnosis of HSP (as the case of other genes/proteins eg ARG1 related to the urea cycle or other genes related to lipid metabolism) (Coutelier et al, 2015a) [START_REF] Coutelier | Reply: ALDH18A1 gene mutations cause dominant spastic paraplegia SPG9: loss of function effect and plausibility of a dominant negative mechanism[END_REF].

Consequent to all the above phenomena and if we consider all the above mentioned examples, we observe that multiple atypical forms of HSP have emerged either as a result of an atypical underlying cause or an atypical clinical phenotype [Table (1-1)].

There appears a lack of an objective case definition and too much complexity of the closely related spastic neurogenetic disorders. This has led to the rise of many reviews that highlight these overlaps [START_REF] Klebe | Clinical and genetic heterogeneity in hereditary spastic paraplegias: from SPG1 to SPG72 and still counting[END_REF] and suggest reclassification (Garcia-Cazorla et al., 2014) (Vallat et al., 2016) or even expert case definition and classification of some of these entities (leukodystrophies and leukoencephalopathies) [START_REF] Vanderver | Case definition and classification of leukodystrophies and leukoencephalopathies[END_REF]. HSP: hereditary spastic paraplegia, HA: hereditary ataxia, PN: peripheral neuropathy and E-Pyr.: extrapyramidal syndrome. Opaque curves stand for clinical presentation as pure category (HSP,HA,PN,E-Pyr.). Interrupted lines imply that the gene manifests the clinical category only in complex form



The core phenotypic features of HSP can be explained by alterations of the pyramidal motor system responsible for the voluntary movements in Human beings. The neurons of the pyramidal tracts extend from the cerebral motor cortex to innervate the skeletal muscles at the neuromuscular junctions. These neurons can be injured in a length dependent manner as travel very long way to their destination. The resulting axonal degeneration occurs mainly via a dying back mechanism of the longest neurons and therefore leads to clinical involvement of the lower limbs (LLs) and to a lesser extent of the upper limbs (ULs) [START_REF] Deluca | The extent of axonal loss in the long tracts in hereditary spastic paraplegia[END_REF]) [START_REF] Blackstone | Cellular Pathways of Hereditary Spastic Paraplegia *[END_REF].

 The pyramidal motor tracts are arranged in two stages:

1. The corticospinal spinal tract which originates from layer V of the motor cortex (neuronal cell bodies). The axons of these large pyramidal neurons pass down through the medullary pyramids where the majority of fibers decussate in the caudal part of medulla to descend as the lateral corticospinal tract within the spinal cord. Some of these upper motor neurons synapse directly with the lower motor neurons at the anterior horn of the spinal cord whereas the vast majority synapse with the spinal interneurons which then relay with the lower motor neurons.

2. The lower motor neurons terminate on the skeletal muscle at the neuromuscular junctions distributed all over the body with the exception of the areas innervated by cranial nerves which have other pathways [START_REF] Blackstone | Cellular Pathways of Hereditary Spastic Paraplegia *[END_REF].



In complex clinical phenotypes other regions of the central and peripheral nervous systems (CNS and PNS) as well as other extra-neurological organs and systems can also be affected.

HSPs are mainly due to neuronal degeneration

The contribution of oligodendrocytes and other glial cells to the pathology cannot be ignored. However, with the exception of certain genetic entities including those in which myelin proteins are affected, the main category of cells that degenerate in HSP are neurons. Neurons have two principal morphological, molecular and functional compartments: the axons and the somatodendritic compartment. Dendrites are multiple short and highly branched neurites that function in receiving and integrating electrical synaptic inputs from thousands of neurons. There is only a single axon responsible for transmitting the integrated information in the form of an action potential that travels along the axonal membrane. The axon has a unique cytoskeletal organization. It contains several specialized structures which include the axon initial segment (AIS) and presynaptic boutons [START_REF] Kevenaar | C mutations in two families with hereditary spastic paraparesis and cerebellar dysfunction --Dor et al[END_REF].

Specific proteins accumulate at the proximal part of the axon to assemble the AIS which separates the axon from the rest of the neuron. The AIS serves to maintain neuronal polarity. The assembly of presynaptic protein complexes leads to presynaptic differentiation [START_REF] Kevenaar | C mutations in two families with hereditary spastic paraparesis and cerebellar dysfunction --Dor et al[END_REF].

Pathways involved in HSP pathogenesis

Not only the genetic technologies but also the closely linked functional studies have (Noreau et al. in 2014) [Figure [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF]].

However; given the fact that a lot remains to be explained about the underlying pathogenesis of HSP, the possible odds for reclassification are unlimited.

Functional heterogeneity of HSP proteins

Many proteins produced from SPG genes are now found to be associated with multiple cellular functions and known to interact with various proteins involved in the same pathway (or inter-digitations of pathways) or sharing the same gene ontology function. This demonstrates how far we stand back from understanding the mechanisms of these disorders and why one gene/protein results in certain phenotype in an individual and with another phenotype when the background genetics changes.

Assessment and diagnostic approach of HSP patients

Positive family history is the main point that strongly suggests HSP. However, in sporadic cases, HSP remains a diagnosis of exclusion. The patient assessment requires proper history and clinical examination. Spastic Paraplegia Rating Scale (SPRS) is a scale used to assess the severity of the motor symptoms [START_REF] Schüle | The Spastic Paraplegia Rating Scale (SPRS) A reliable and valid measure of disease severity[END_REF]. [START_REF] Klebe | Clinical and genetic heterogeneity in hereditary spastic paraplegias: from SPG1 to SPG72 and still counting[END_REF].

As a result of the immense genetic/phenotypic heterogeneity it is becoming extremely difficult to suggest the gene beyond the HSP using candidate gene approach based on the clinical phenotype. With the advent of NGS techniques, multi-gene panels have become the favorite screening tool as they allow massive screening for genes responsible for spastic neurogenetic disorders. These panels are time/effort effective but pose the important question about cost effectiveness as with its use in other diseases [START_REF] Robson | Multigene Panel Testing: Planning the Next Generation of Research Studies in Clinical Cancer Genetics[END_REF].

Treatment options for HSP

Unfortunately there is no curative treatment for HSP and all the available options are symptomatic treatment. Simple walking aids and wheel chairs, hearing aids and air matrices can be of great use to improve the quality of life. Physiotherapy, speech therapy, occupational therapy are used based upon the clinical presentation. Two studies have been done recently on a new trend to support the use of robot gait training. Provisional evidence from one study suggests long term improvement of walking and balance in HSP patients [START_REF] Bertolucci | Robotic gait training improves motor skills and quality of life in hereditary spastic paraplegia[END_REF]. However, despite the improvement in gait in the second study there was no change in kinetics [START_REF] Seo | Robot-assisted gait training in a patient with hereditary spastic paraplegia[END_REF]. Oral medications of antispasticity drugs (baclofen, tizanidine, tolperisone or dantrolene) as well as intramuscular botulinum toxin injections and continuous intrathecal baclofen application (ITB) can decrease the functional disability especially when the spasticity is predominant over the weakness [START_REF] Klebe | Clinical and genetic heterogeneity in hereditary spastic paraplegias: from SPG1 to SPG72 and still counting[END_REF] [START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF].

ITB is a widely used in children as well as in adults for treatment of spastic disorders at variable doses. Recently provisional evidence has shown that spastic HSP may require low doses compared to other spastic disorders [START_REF] Dressler | Intrathecal Baclofen therapy in Germany: Proceedings of the IAB-Interdisciplinary Working Group for Movement Disorders Consensus Meeting[END_REF].

Encouraging results with significant improvement of SPRS in HSP patients have been observed in a recent study conducted on 12 patients to support the use of dalfampridine.

Dalfampridine is a potassium channel blocker that has recently been approved to improve walking in patient with multiple sclerosis [START_REF] Béreau | Dalfampridine in hereditary spastic paraplegia: a prospective, open study[END_REF].

Anti-cholinergic medications can help with the urinary urgency. Treatment for complicating clinical signs follows their own guidelines [START_REF] Klebe | Clinical and genetic heterogeneity in hereditary spastic paraplegias: from SPG1 to SPG72 and still counting[END_REF].

Surgerical interference can be implemented through orthopedic surgical procedures (for contractures) and ophthalmological surgeries (cataract extraction) to protect against augmentation of disability by blindness.

The emergence of multiple metabolic pathways associated with HSP may provide targets for more specific options in certain HSP subtypes.

Genetic counseling

Genetic counseling depends on the pattern of inheritance. High consanguinity in certain communities (Africa, Middle East..) can be an important pointer towards the tendency to have AR HSP over AD HSP although the latter can't be entirely excluded. Explanation of the expected risk of having an affected child associated with each pattern of inheritance depends mainly on good interpretation of family pedigree although it can still be ambiguous in certain cases until the causative gene/variant is found [START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF].

Materials and methods

Ethics

This study was approved by the Paris Necker Ethics Committee (France) and the Ethical Committee of the University of Khartoum, Medical Campus (Sudan). A written informed consent was obtained from all participants [Annex 13].

Subjects recruitment

Our cohort was composed of 106 patients and 337 healthy related individuals from 41 extended Sudanese families, almost all consanguineous with multiple consanguinity loops.

Patients were referred from several principal tertiary neurology and neuro-pediatric clinics in Khartoum (list of the departements…). They belong to various ethnic groups distributed in different regions of Sudan. For each kindred we recruited the proband, all other accessible diseased family members and, when possible, available unaffected first, second and third degree relatives.

Inclusion and exclusion Criteria

Inclusion Criteria:

1. Clinical presentation suggestive of spastic neurodegeneration.

2. Favorable criteria were familial consanguinity and positive family history of neurodegenerative disorders.

Exclusion criteria:

3. Alternative acquired causes of pyramidal syndrome should all be excluded.

4.

Other overlapping genetic disorders that clearly belong to a differential diagnosis as overt leukodystrophy/leukoencephalopathy are to be excluded when necessary data are available.

N.B

In born errors of metabolism (IEM) were excluded on clinical ground and metabolic screening when the test was feasible. However, it could not be included as an exclusion criteria as the tests were not always available.

Recruitment Rate:

The rate of recruitment was slower at the start. Later, as the neurogenetic team was created in Sudan, the recruitment became more efficient. We also managed to enhance the referral system by the help of the young doctors of the team who were distributed in various hospitals and tertiary neurology clinics in Khartoum to consolidate the principle of referral to our neurogenetic clinic/team and provided the physicians with printed referral forms that we prepared.

Clinical phenotyping

Sampling

Two milliliters of saliva were collected using Oragene.Discover® DNA self-collection kits (OGR-500) from each patients and healthy related and unrelated controls. For infants and markedly disabled patients Oragene.Discover® DNA assisted-collection kits (OGR-575) was used to collect around one milliliter of saliva (Genotek Inc., Canada).

Skin biopsies were collected in certain cases where an interesting genetic result was obtained and needed further functional studies. They were taken from the inner part of the upper arm after 20-30 minutes of local anesthesia batch using sterilized disposable kits.

DNA Extraction, and quality/quantity check 1. DNA Extraction:

DNA purification out of 0.5 ml of saliva was done according to the prepIT®.L2P manual protocol provided by the producer.

DNA quality/quantity check:

DNA quality control were performed using three laboratory methods: the standard agarose gel electrophoresis, Caliper® bioanalyzer to check for degradation of high molecular weight DNA and the NanoDrop® 8000Uv-Vis spectrophotometer (Thermo Scientific, Wilmington, USA) to assess the purity of the DNA.

DNA quantity was measured with the use of Qubit® fluorometer (Promega®, Wisconsin, USA) as well as the NanoDrop® 8000Uv-Vis spectrophotometer (Thermo Scientific, Wilmington, USA).

Standard Agarose gel electrophoresis

Agarose gel electrophoresis allows the separation and identification of 0.5-to 25-kb DNA fragments. It can thus be used for quality checking of genomic DNA to assess its integrity and to exclude its degradation. It was also used to separate PCR products (see below) according to their precise molecular weight.

1.

The protocol summary can be divided into three stages:

preparation of an agarose gel concentration appropriate for the size of DNA fragments to be separated the DNA samples are loaded together with a graded band size ladder into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation -Visualization of gel under UV illuminator and capture of necessary images 2.

In practice:

1% gel was prepared using standard method: heating one gram of agarose powder dissolved in 100 mL of (0.5-1x) TBE (Tris/Boris/EDTA) buffer solution in a microwave oven for 3-5 minutes. Addition of 5 µL (concentration=10 mg/mL in H2O) of an intercalating stain as Ethidium bromide or cyber safe stain under the safety hood after cooling the solution to less than 60° C using all good laboratory practice guidelines for safety use of biohazardous substances. The buffer improves the conductivity of the DNA by adding negative charges to it and thus improves the DNA migration and protects the DNA through its EDTA component from degradation.

We used 0.8% gel for genomic DNA control and 1% agarose gel was used for PCR products analysis. A higher agarose percentage enhances v. Sample concentration is given as QF value (an option to provide calculation of final concentration is available if the value of the starting volume of the sample is entered.

Genetic linkage analysis using traditional microsatellite markers

We performed linkage analysis to screen two families (10 patients and 31 healthy related controls) for candidate loci. For each gene we used four microsatellite markers flanking:

CYP7B1 (SPG5), KIAA1840 (SPG11), ZFYVE26 (SPG15), GBA2 (SPG46), CYP2U1
(SPG49 or SPG56 according to HUGO and NCBI nomenclatures respectively) and SACS genes. Classical procedures were used for the PCR amplification. Allelic resolution was run in an ABI3730 sequencer (Applied Biosystems, US). Peak Scanner® software (Applied Biosystems®, USA) was then utilized for analysis of product length which varied according to the number of nucleotide repeats. Haplotypes were then manually reconstructed for each locus and pedigree in order to obtain the minimal number of recombination events.

Primer design, PCR amplification and control 1. Primer Design

Specific primers were designed using primer3plus software (ref). The sequence used as an input was obtained from the reference sequence downloaded from any of the genome browsers (ensembl genome browser was preferentially used).

Primer specificity was tested using the BLAT and BLAST tools of the genome browsers (UCSC and NCBI). In difficult cases where specificity was doubted, the in silico PCR tool (UCSC genome browser) was used.

PCR

amplification of microsatellite markers or of specific exons/regions of specific genes was performed using a classical procedure that included preparation of a master mix composed of a buffer, dNTPs, Taq polymerase, specific primers (forward/F and reverse/R), H 2 O and the DNA template (DNA sample).

 Touchdown protocol was used to amplify the whole exon where the variant of interest was located or, if that was too big to get the optimal amplification we amplify around 100-150 bp up-and down-stream of the variant. The touchdown protocol is, as any standard PCR protocol, composed of three steps:

1. Denaturation (94-95°C)

2. Hybridization (annealing) (Range including the melting temperature (Tm) of the primer), typically 65°C to 54°C

Extension (synthesis) (72°C)

The advantage of the touchdown is that the first 10-30 cycles are performed at high annealing temperature to maintain the specificity of the polymerization and then reduced it gradually to cover a wider range of annealing temperatures making it easier to obtain a PCR product in adequate quantity.

PCR products were then checked for specific amplification at the expected size using a caliper ® Lapchip ® GX bioanalyzer (Hopkinton, Massachusetts, USA).

2.8. Genome-wide genotyping, homozygosity mapping and whole genome linkage analysis The compatible iScan® technology was used to obtain the necessary SNP images.

The SNP image data sets obtained allowed us to perform a variety of downstream applications, including the search for loss of heterozygosity (mapping of homozygous regions) and CNV detection studies. In addition the data analysis would allow identification of common variants (including in mtDNA), ancestry determination, loss-of-variant and indel identification, as well as sex confirmation.

Laboratory protocol :

2.8.2.1. Practical summary of the principal steps of the process:

1. DNA Amplification (Pre-Amp):

After the DNA samples are run on gel electrophoresis to be sure that they are not degreaded and a test PCR is done to exclude the presence of PCR inhibitors. The DNA samples are chemically denatured and neutralized to prepare them for amplification. The denatured DNA is isothermally amplified in an overnight step. As the process is isothermal (non-PCR amplification) the whole-genome amplification uniformly increases the amount of the DNA sample by several thousand-fold without significant amplification bias.

2. DNA Fragmentation (Post-Amp):

The fragmentation is a controlled enzymatic process which fragments the amplified product. The process uses an endpoint fragmentation to avoid overfragmenting the sample.

DNA Precipitation (Post-Amp):

This is achieved via isopropanol precipitation followed by centrifugation at 4°C to collect the fragmented DNA.

DNA Resuspension (Post-Amp):

The precipitated DNA is resuspended in hybridization buffer. 6. BeadChip Wash (Post-Amp):

The aim of this step is to prepare the BeadChip for staining and extension.

All unhybridized and non-specifically hybridized DNA are washed away to leave only the specifically hybridized DNA fragments.

7. BeadChip Extend and Stain(XStain) (Post-Amp):

This includes single-base extension of the oligos on the BeadChip, using the captured DNA as a template. It incorporates the detectable labels on the BeadChip and determines the genotype call for the sample. X-Stain occurrs in a capillary flow-through chamber.

8. BeadChip Imaging (Post-Amp):

The Illumina iScan System is used to scan the BeadChip. It uses a laser to excite the fluorophore of the single-base extension product on the beads. The scanner records high resolution images of the light emitted from the fluorophores.

Bioinformatic analysis (downstream):

GenomeStudio V2011.1 (1) software was used for data analysis. After genotyping quality check, the genotypes for all markers were determined. Two additional plugin and algorithm (CNV region and cnvpartition, respectively) were used for the analysis of CNVs and loss of heterozygosity regions.

Screening HSP or

End Repair:

In this step both and 3 end of the fragmented dsDNA are turned into blunt ends with the use of Kapa® end repair master mix. The mix which contains an end repair enzyme and buffer is incubated in a thermocycler with the DNA fragments. The fragments are then cleaned using Agencourt AMPure XP reagent®. This is a kit that employs the highly efficient PCR cleaning system that utilizes the Solid Phase Reversible Immobilization (SPRI) magnetic-based technology with the help of paramagnetic beads.

The cleaning system allows easy removal of contaminants as primers, primer dimers, salts and dNTPs to leave only the proper amplified dsDNA fragments.

A-tailing:

It is an intermediate step necessary for the adapter ligation (the step that follows). Addition of a stretch of dAMP to the 3 end of the amplified and cleaned dsDNA fragments is performed using a Kapa® A-tailing buffer and enzyme in a thermocycler based step. An SPRI cleaning step followed.

Adapter ligation:

SeqCap® adapter Kits A and B (with 12 adapters each) allows indexing of the 24 samples with 24 different adapters. The index adapters are linkers specific to sequencing platforms that allow library fragments to attach to the flow cell surface called P5 and P7. Each adapter has an index that contains a unique sequence that identifies samples at sequencing and allows their multiplexing. A mix of KAPA® T4 DNA Ligase and buffer is then incubated with dsDNA sample and the specific index adapter in a thermocycler at 20˚. This is followed by an SPRI cleaning step.

Dual-SPRI ® size selection:

Fragments with size between 250-450 bp are selected by dual binding to the magnetic beads. This is important because this size range corresponds to the target sequences that can be processed in the MiSeq sequencing system. ). An overnight incubation in a thermocycler at 47°C (with heated lid turned on at 57°C) allows the hybridization to take place overnight.

v. First capture: capture of the sample hybridized to biotinylated probes is performed using Streptavidin Dynabeads ® incubated with the sample at 47°C for 45 min followed by a wash process.

vi. Middle-captured LM-PCR followed by SPRI cleaning step.

vii. Second hybridization reaction with biotinylated probes at 47°C for overnight.

viii. Second capture with streptavidin capture bead followed by a wash process.

ix. Post-captured LM-PCR followed by SPRI cleaning step.

7. The product is measured using NanoDrop® spectrophotometer to determine the concentration. The proposed sample library yield is expected to be around 0. μg ( 00 ng), with an A260/ A280 between 1.7 and 2.0.

The quantity is then confirmed by Qubit® fluorometer and run in Caliper®

Bioanalyzer DNA 1000 chip to assure that the fragments size ranges between 150-500 bp (average 300bp).

Library preparation for Miseq sequencing:

This is based on the concentrations obtained with Qubit® fluorometer. The different libraries are combined in one tube with the more concentrated multiplexed amplified captured sample being diluted to the concentration of the less concentrated one (C1*V1=C2*V2). Then with the use of the elution buffer tween (EBT), the library is diluted to 4nM to obtain the appropriate concentration of the Miseq sampling using a formula provided by the manufacturer. The sample is chemically denatured using NaOH followed by addition of a special hybridization buffer (HT1) supplied with the Miseq sequencing kit to dilute the library to a final concentration of 9 pM. The library is then combined with Phix library control (internal quality control for cluster generation, sequencing and alignment) and loaded into the designated reservoir in the prefilled cartridge reagent provided by Illumina.

Miseq sequencing

The Illumina reagent cartridge (Illumina reagent kit v2), the thoroughly

Bioinformatic analysis

Read mapping and variant detection

The Miseq output data is in the form of two FastQ files per sample corresponding to Read 1 and Read2 data (≈forward and reverse reads). The files are exported to be analyzed by CLC Genomics Workbench 7.0 software (CLC Bio ® , Denmark). This software has a re-sequencing tool that allows alignment of the genetic variation data to a reference sequence. The software first maps the reads to the reference sequence (based on GRCh37 assembly) and thus the coverage of each region is determined as well as the on-target specificity. Consequently, two coverage files are produced per sample:

-Coverage report which provides a general summary about each sample coverage including the mean coverage of all regions/sample.

-Coverage analysis (OA) which provides detailed coverage data about each region.

End Repair:

In this step both and 3 end of the fragmented dsDNA are turned into blunt ends with the use of Kapa® end repair master mix.

A-tailing:

Addition of a stretch of dAMP to the 3 end of the amplified and cleaned dsDNA fragments is performed using a Kapa® A-tailing buffer and enzyme in a thermocycler based step. An SPRI cleaning step follows.

Adapter ligation:

SeqCap® adapter Kit A and B (with 12 adapters each) allows indexing of the 10 samples with 10 different adapters.

Dual-SPRI size selection:

Fragments with size between 250-450 bp are selected.

Capture steps:

Illustration is provided in [Figure ].

i. Pre-Captured LM-PCR is followed by SPRI cleaning step.

ii. Measurment of the product using NanoDrop ® spectrophotometer to determine the concentration. The proposed sample library yield is > 1 μg, with a A260/ A280 between 1.7 and 2.0. The quantity is rechecked by Qubit ® fluorometer for more accurate measurement.

It is run in Caliper ® Bioanalyzer DNA 1000 chip to assure that the fragments size ranged between 150-500 bp (average 300 bp).

iii Incubation in a thermocycler at 47°C (with heated lid turned on at 57°C) allows the hybridization to take place over 64-72 hours.

v. Capture: capture of the sample hybridized with biotinylated probes by Streptavidin Dynabeads ® incubated with the sample at 47°C for 45 min followed by a wash process.

vi. Post-captured LM-PCR followed by SPRI cleaning step.

7. The product is measured using NanoDrop ® spectrophotometer and run in Caliper ® Bioanalyzer DNA 1000 chip to assure that the fragments size ranged between 150-500 bp (average 300bp).

8. Library preparation for NextSeq-500 sequencing:

9.

Based on the concentrations obtained with Qubit® fluorometer the more concentrated multiplexed amplified captured sample is diluted to the concentration of the less concentrated one (C1*V1=C2*V2). Then with the use of the elution buffer tween (EBT), the library is diluted to 4nM to obtain the appropriate concentration of the NextSeq sampling using a formula provided by the manufacturer. The sample is chemically denatured using NaOH followed by addition of a special hybridization buffer (HT1) supplied with the NextSeq sequencing kit to dilute the library to a final concentration of 1.8 pM. The library is then loaded into the designated reservoir in the prefilled cartridge reagent provided by Illumina.

10. NextSeq-500 sequencing:

The Illumina reagent cartridge (NextSeq 500 HighOutput kit 150 cycles), the thoroughly washed and dried flow cell and the buffer cartridge are loaded into the NextSeq500 machine and the sequencing process is launched after assuring that the waste cartridge is empty. This allowed the EI to remove single stranded primers, DNA and PCR product. It also allowed the AP to remove the phosphates of the remaining dNTP to prevent their incorporation at the sequencing reaction leading to background noise.

Sanger Sequencing

Sanger sequencing is still the gold standard in sequencing. This is the reason why ssubsequent to all approaches, we performed Sanger sequencing for validation of selected variants and to confirm cosegregation of the appropriate zygosity of the variant with the disease distribution in the family. Sanger sequencing is also used to sequence regions of low coverage in certain genes suggested by the clinical phenotype. Sanger sequencing is done at the GATC® platform. One sense primer solution (forward or reverse) at 5 pM concentration combined with a diluted PCR product/ sample are prepared in 98-well plate and sent to the external facility. The BIGDYE chemistry on an ABI3730 sequencer (Applied Biosystems) is used for sequencing. Bioinformatic sequence analysis is performed using Seqscape® (Applied Biosystems®, USA) and

Chromas lite® software (Technelysium, Australia).

Basic Functional Studies (provisional post-genetic workup)

1. Biochemistry:

Through our collaborator in USA biochemical tests were achieved to illustrate the effect of a mutation on TFG protein (Elsayed et al., under revision):

Recombinant proteins were expressed as fusions to GST using BL21 (DE3) Escherichia coli, and purifications were conducted using glutathione agarose beads. Proteins were cleaved from resin with GST-HRV3C protease overnight at 4°C and purified further using ion exchange chromatography. Light-scattering data were collected. Purified proteins were applied onto a high-resolution size-exclusion chromatography column (Wyatt WTC-030S5), which was coupled to a Wyatt miniDAWN TREOS three-angle light-scattering detector. Data were collected at a flow rate of 0.5 mL/min and analyzed (ASTRA software) to determine the molecular mass of proteins (Elsayed et al., under revision).

Culture of fibroblasts

Skin biopsies:

They are collected in culture medium for growth of fibroblasts.

Components of the medium are indicated in [Table ]: Fibroblast culture is the base for most of the downstream procedures in functional studies such as immune-fluorescence, protein extraction and protein based processes as western blotting, immunoprecipitation.

The processing is all conducted in a sterile atmosphere under sterile hood:

1. The skin biopsy is cut into small pieces in a Petri dish with the use of two scalpels.

2. 3 mL of the culture medium is added and the fragments are homogenized by pipetting up and down.

3. 2 mL of the suspension is transferred into a culture flask T25.

4. The flasks are incubated in 37 degrees incubator. 4. The flask is observed under microscope for check of cells viability.

5. The flask is incubated again in 37 degrees and observed daily.

6. The process is repeated whenever cells are confluent.

Results

General features of the cohort

Our cohort consisted of 41 nuclear and extended Sudanese families. The cohort included 23 families with predominant pyramidal signs (more like a classical HSP) (23/41; ≈ 7%).

There were families where other neurological features (mainly ataxia including spastic ataxia cases) were as severe as the upper motor neuron lesion (UMNL) signs (12/41; ≈ 29%). There were as well families with the pyramidal tract likely affected as part of other diseases (juvenile onset Parkinson disease -JOPD, inborn errors of metabolism -IEM or NBIA) (6/41; ≈14%).

There was a rather high consanguinity rate (37/41: 90%) [Figure ]. The families originated from almost all regions of Sudan but correlated with the regions/tribes where the traditional culture of consanguineous marriages was the rule for many generations. The highly inbred communities and tribes from Al Jazira (central Sudan) predominated our cohort as could well be predicted (eight families), followed by the White Nile region (An Nil Alabyad) and Northern region (Ash Shamalyah) [Figure ]. In the four nonconsanguineous families (2AD, 1 sporadic and one AR), the parents were not related but consanguinity existed in both maternal/paternal branches, separately.

;

Figure (3-2):

The political map of the Sudan showing the origin of 41 families included in the cohort.

In ten families, there were sporadic cases (24%) whereas the pattern of inheritance was autosomal recessive in 27 families (66%) and autosomal dominant in four families (10%).

Branch 2 of family FM7 showed possible AD inheritance but was not included in the four AD families [Figure ]. However, pseudo-autosomal dominant could not be excluded in the majority of AD families. Pure HSP constituted only 10% (4/41) of the cohort, while complicated HSP with minimal complexity (one additional complicating feature only) was only found in two families (5%). The remaining 35 families (85%) were complicated with at least two additional clinical signs (on top of the pyramidal features) which made the high 51 level of complexity the rule in the cohort. 17 out of the 35 families with high complexity clinical picture (17/41; 41.5%) showed classical HSP whereas 18 families were atypical (43.5%) [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF]] [Table (3-1)]. Age at onset was in childhood (<18 years) in 80% of families, adulthood (>18 years) in 16% and variable among related patients (childhood to adulthood) in 4 %. The two families within the latter category of variable age at onset were both dominant with anticipation which could explain the large variability [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF]].

[Table (3-2)].

Genetic results

Identification of mutations in known genes

According to inheritance and clinical presentation, 41 families were screened for mutations in known genes as a preliminary step. In 3 families, because of their complex phenotype, WES was performed. In one additional case, the phenotype was compatible with a spinocerebellar ataxia due to nucleotide expansion and a genotyping approach was then used.

Identification of families mutated in known HSP genes

a) Summary: We tested 65 patients from 25 consanguineous HSP families originating from Sudan for HSP candidate genes using next generation sequencing on patients from 23 families and candidate gene sequencing in two families. We could establish a genetic diagnosis in seven families (28%) with autosomal recessive complex HSP. Truncating homozygous mutations were identified in SPG11 and in SACS genes in three and one families, respectively.

Three missense mutations were also found in one family each in TFG/SPG57, Identification of novel SPG57 mutation affecting TFG oligomerization and description of HSP mutations in Sudan. EJHG 2016 (under revision). Introduction: Pure Hereditary Spastic Paraplegias (HSP) and Hereditary ataxias (HA) represent the extremes of the spectrum of spinocerebellar neurodegenerative disorders. They are often correlated and impose diagnostic difficulty specially with other neurodegenerative disorders with close clinical presentation as motor neurone disease (MND).1 They can be transmitted by 70 all modes of inheritance.1-5 HSP is a group of disorders with the core defining clinical features of insidiously progressive weakness and spasticity of the lower extremities. It is the second most important motor neuron disease with a prevalence of 3-10 per 100000 in most populations.6 Prevalence of autosomal dominant (AD) HSP ranges between 0.5-5.5 per 100000 individuals and that of autosomal recessive (AR) HSP between 0.3-5.3 per 100000 individuals.7 HSP can be pure (uncomplicated) or complex according to the absence or presence of additional neurological and non neurological manifestations. To date there are more than 67 known HSP genes.1 AD HSP forms are the most frequent in western populations. SPG4, SPG3A, SPG10 and SPG31 account for up to 50% of AD HSP. SPG3A (ATL1) alone accounts for 10% of AD HSP, with a frequency depending on the age at onset.8 On the contrary, AR HSP predominates in highly consanguineous communities.7,9,10 Thin corpus callosum (TCC)associated HSP represents a distinct subgroup of HSP accounting for approximately a third of AR HSP.11 At least nine genes have been identified to be responsible for this category. They include SPG1, SPG11, SPG15, SPG18, SPG21, SPG32, SPG46 and SPG49/56 (SPG49 according to HUGO and SPG56 according to OMIM nomenclature) and in rare cases, SPG7. Mutations in SPG11 (also known as KIAA1840 and FLJ21439) (OMIM gene *: 610844) constitute the most frequent cause of TCC-associated HSP (41-77%).12 SPG11 is responsible for 10-20% of all AR HSP [Spastic Paraplegia 11; OMIM phenotype #: 604360], particularly in the Mediterranean basin. [START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF] Next generation sequencing aided the identification of rare genes causing HSP.1,17 TFG/SPG57 stands as an example of this genetic surge. Mutations in TFG/SPG57 were implicated in SPOAN-like HSP (spastic paraplegia, optic atrophy, neuropathy)18 and hereditary sensory motor neuropathy with proximal predominant involvement (HSMN4P). [START_REF] Ishiura | The TRK-Fused Gene Is Mutated in Hereditary Motor and Sensory Neuropathy with Proximal Dominant Involvement[END_REF] The TFG protein is a highly conserved regulator of protein secretion, which functions at the interface between the endoplasmic reticulum (ER) and ER-Golgi intermediate compartments. It acts as an oligomer in vivo.18 Spastic ataxias now stand as a distinct category with few genes recognized to date. AR spastic ataxia of Charlevoix Saguenay (ARSACS) (OMIM phenotype #: 270550) is a challenging phenotypic overlap between HSP and HA, with a remarkable clinical diversity.20-22 ALS2 (Alsin) (OMIM gene*: 606352), first described by Yang et al., is another prominent example of a gene implicated in pyramidal syndrome, with overlapping roles in other neurodegenerative conditions, including amyotrophic lateral sclerosis.23 Relatively little is known about the genetics of spinocerebellar degenerations (SCD) in the Sudanese population. [START_REF] Salih | Genetic Disorders in Sudan[END_REF] In this article, we describe 25 unrelated families with progressive spastic neurodegenerative disorders and their genetic profiling to determine the genes involved. We report the first seven Sudanese families carrying novel and rare mutations in KIAA1840 (SPG11), TFG/SPG57, ATL1 (SPG3A), ALS2 and SACS genes. Furthermore, we demonstrate the pathological effect of the novel TFG mutation reported here using in vitro experiments. Further genetic heterogeneity of HSP is also highlighted by our study.

ATL1

Materials and Methods:

Ethics approval: This study was prospectively reviewed and approved by the Paris Necker Ethics Committee (France) and the Ethical Committee of the University of Khartoum, Medical Campus (Sudan). It was conducted in accordance with the recommendation of the Helsinki declaration. A written informed consent was obtained from all participants. Subjects Recruitment: Our cohort was composed of 65 patients from 25 extended Sudanese families, all consanguineous with multiple consanguinity loops belonging to various ethnic groups distributed in different regions of Sudan. For each kindred we recruited the proband, all other accessible diseased family members and, when possible, available unaffected first, second and third degree relatives. Inclusion criteria for probands were clinical presentation suggestive of spastic neurodegeneration, preferably with familial consanguinity and positive family history of neurodegenerative disorders. Clinical Phenotyping: Patients were clinically examined and diagnosed by the referring consultant neurologist/neuro-pediatrician, followed by a second scrutinized phenotyping by the research team. We examined all available healthy related controls to exclude subtle disorders. Patients were assessed for motor disability using a Seven-Stage Disability Score (supplementary I and II). Magnetic resonance imaging (MRI) of the brain was performed for at least one patient per family in most families.

Sampling and DNA Purification: Two milliliters of saliva were collected using Oragene.Discover DNA collection kits (Genotek Inc., Canada). DNA purification was done according to the prepIT.L2P manual protocol provided by the manufacturer. DNA quality and quantity control were performed using standard Agarose gel electrophoresis as well as the NanoDrop spectrophotometer (Thermo Scientific, Wilmington, USA) and the Qubit fluorometer (Promega, Wisconsin, USA).

Genetic linkage analysis:

We performed linkage analysis to screen two families ( 10patients and 31 healthy related controls) for candidate loci based on patients' clinical and brain imaging presentation or based on their known relative frequencies. We used microsatellite markers (list available upon request) flanking the CYP7B1 (SPG5), KIAA1840 (SPG11), ZFYVE26 (SPG15), GBA2 (SPG46), CYP2U1 (SPG49 or SPG56 according to HUGO and NCBI nomenclatures respectively) and SACS genes. Classical procedures were used for their PCR amplification and allelic resolution in an ABI3730 sequencer (Applied Biosystems, CA, USA). Peak Scanner software (Applied Biosystems, CA, USA) was utilized for analysis of product length. Haplotypes were then manually reconstructed for each locus and pedigree in order to obtain the minimal number of recombination events.

Targeted Next Generation Sequencing Screening Panel (NGS Panel):

We used a multi-gene targeted next generation sequencing panel to screen 23 index patients for 67 genes known to be related to a variety of spastic disorders1 and 7 unpublished candidate genes (Supplementary III). NimbleGen/SeqCap Ez enrichment system was used (Roche, CA, USA). Sequencing was performed using MiSeq platform (Illumina, San Diego, USA) on 250 bp paired-end reads. Base calling and quality control were performed using Genomics Workbench (CLC Bio, Denmark). Variants were detected using probability-based and quality-based algorithms. For gene/variant prioritization, stop codons, frameshift, splice site and missense variants with a minimum depth of 30x were selected. Minor allele frequency (MAF) cut-off of 1.5% was used first then decreased to 0.1% for more stringency. Five pathogenicity prediction algorithms were used for missense variants (SIFT, Polyphen-2, Mutation Taster, Align GVGD, KD4v). PhastCons and PhyloP conservation scores were used. For splicing sites prioritization, five tools (Human Splicing Finder, SpliceSiteFinder-Like, Gene Splicer, NNSPLICE, MaxEntScan) were used. All tools were collectively provided by AlamutVisual software (Interactive Biosoftware, France). Sanger Sequencing: We performed Sanger sequencing using the BIGDYE chemistry on an ABI3730 sequencer (Applied Biosystems, CA, USA) to confirm the segregation of culprit variants with the disease. Sequences were analyzed using Seqscape (Applied Biosystems, CA, USA) and Chromas lite software (Technelysium, Australia). Biochemistry: Recombinant proteins were expressed as fusions to GST using BL21 (DE3) Escherichia coli, and purifications were conducted using glutathione agarose beads. Proteins were cleaved from resin with GST-HRV3C protease overnight at 4°C and purified further using ion exchange chromatography. Light-scattering data were collected as described previously.18 Briefly, purified proteins were applied onto a high resolution size-exclusion chromatography column (Wyatt WTC-030S5), which was coupled to a Wyatt miniDAWN TREOS three-angle light-scattering detector. Data were collected at a flow rate of 0.5 mL/min and analyzed (ASTRA software) to determine the molecular mass of proteins.

Results: Genetic Results:

In 25 studies families, autosomal recessive inheritance was observed in 60% (15/25 families). Seven cases (28%) were sporadic and three were from autosomal dominant pedigrees (12%). In two families, we first performed linkage analysis using microsatellites for seven AR HSP loci and found homozygous haplotypes segregating with the disease surrounding the SPG11 locus. Sanger sequencing confirmed the segregation of a homozygous mutation in SPG11 with the disease distribution in these two families (F1 subfamily I and F6). The other 23 families were screened for mutations in 74 HSP genes using targeted capture followed by massive parallel sequencing. In total, genetic diagnosis was established in seven of the 25 families (28%). Causative mutations were identified in five genes: SPG11 (three families), TFG/SPG57, ATL1, ALS2 and SACS (one family each). The SPG11 mutations were two homozygous nonsense mutations and a homozygous frameshift single base deletion (Figure ( 1)). In three genes (TFG/SPG57, ATL1, ALS2), missense mutations were identified as the cause of the disease (Figure [START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF] and Figure ( 3)). A nonsense mutation was detected in exon 10 of the SACS gene (Figure ( 3)). Details of these mutations are found in table [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF]. All the variants showed segregation with the disease and were absent from large sets of exomes (n=68,251) in local data base, 1000 Genomes, EVS and Broad institute ExAC cohorts. The missense variants were predicted to be deleterious by at least three pathogenicity prediction algorithms and affected conserved amino-acids in highly conserved protein regions (Table 1). All mutations were novel except a frameshift deletion in SPG11 (c.6709del, p.Ala2237Glnfs*7) that was previously reported in Somalia.25 Biochemical effect of TFG novel missense mutation (p.Arg22Trp): This is the second TFG/SPG57 mutation in HSP families. This p.Arg22Trp mutation lies within the Phox and Bem1p (PB1) domain of the TFG protein. The PB1 domain has been shown previously to self-associate in yeast 2-hybrid studies.26 To determine whether the mutation impacts the homo-oligomeric properties of the TFG PB1 domain, we conducted a series of size exclusion chromatography studies, coupled to multi-angle light scattering, which enables an accurate determination of absolute protein molecular mass. Consistent with a previous structural characterization of TFG,27 we found that the PB1 domain, at concentrations ranging from 18-45 μM, formed octamers in solution (~92 kD). In contrast, the p.Arg22Trp mutant of the PB1 domain exhibited a significantly reduced molecular mass of ~24 kD throughout the same range of protein concentrations, indicating it was only capable of forming dimers. Additionally, the elution time of the p.Arg22Trp mutant PB1 domain was extended relative to the wild type protein, consistent with the idea that the mutant failed to self-assemble properly (Figure ( 2)). Together, these studies demonstrate that the p.Arg22Trp mutation disrupts the ability of the PB1 domain to oligomerize highlightING a mechanistic basis for HSP.

Phenotype-genotype correlations: A complex phenotype was predominant in this cohort (21/25 families, 84%). Clinical data from 23 patients (7 families) with established genetic diagnosis is presented in table [START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF] and table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF]. A summary of the MRI and electro-physiological investigations is provided in table [START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF].

In three nuclear families (F34, FM5 and F19), with mutations in ATL1, ALS2 and TFG respectively, we observed homogeneous clinical patterns in all patients. Family F34 represented a rare example of both pure HSP phenotype and AD inheritance in our cohort, despite the high consanguinity in this family. The presentation (Table 3)matched the classical clinical pattern and the early age at onset associated with SPG3A. [START_REF] Hedera | Spastic Paraplegia 3A[END_REF] In family FM5, four patients from two branches presented with infantile onset progressive pyramidal syndrome with pseudobulbar palsy. This is in conformity with what was reported in Infantile onset ascending spastic paralysis (IAHSP) associated with Alsin mutations (OMIM #607225) (Table 3).29-31 Interestingly, phenotypes in family F19 extended the signs associated with mutations in TFG/SPG57. The mean age at onset in the three affected sibs was around one year and mild cognitive impairment was also observed. They presented with signs of progressive pyramidal tract involvement. In contrast to the SPOAN-like HSP reported by Beetz et al.18 in the first SPG57 family (c.316C>T, p.R106C), patients in this family had intact optic nerve and normal fundi.

The electrophysiological studies confirmed variable degrees of motor neuronal demyelination in addition to motor axonal degeneration matching the previous report.18

Although brain MRI revealed no significant abnormalities in the first family, we report thinning of the corpus callosum, variable degrees of cerebellar atrophy and mild white matter hyperintensities, as part of the SPG57 clinical spectrum.

In other families, the phenotypic presentation varied between patients from different branches. Patients in family F14 carried a SACS truncating mutation. Age at onset was late childhood, in agreement with Baets et al.32 and higher than what was reported in Tunisian patients.33 Although this was not genetically analyzed, it is strongly suggested that there were at least two different segregating disease genes within the same nuclear sibship as our index patient had a family history of three older brothers who died all from renal failure and were clinically diagnosed as Laurence Moon Biedl (LMB) syndrome years ago. Furthermore, the two diseased direct cousins who had the same damaging SACS mutations showed interesting clinical differences. They both had spastic ataxia. However, the proband had predominant ataxia and peripheral neuropathy whereas the cousin had clinical features of a predominant spastic paraparesis. In line with the studies done on other inbred communities,9,10 we found that autosomal recessive HSP inheritance is predominant in Sudan. However, in our series, the complex phenotype is predominant (84%), in disagreement with Coutinho et al. who elicited a predominant pure phenotype,9 but in agreement with a study in Tunisia10 that reported 69% of complex cases. Such complex forms are often misdiagnosed due to overlapping symptoms with other diseases. The use of a targeted NGS panel provided an efficient and reliable screening method to test HSP and HSP-related genes. We screened 53 patients from 21 families and identified culprit variants in five families. Mutations in known genes were identified in 28% of index cases (7/25) in line with other reports using candidate gene screening or exome sequencing in heterogeneous diseases. [START_REF] Abu-Shahba | Expanding the Clinical Spectrum of SPG11 Gene Mutations in Recessive Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF] All mutations, except one, are novel with convincing evidence of pathogenecity. The cohort of Sudanese families with autosomal recessive HSP showed high frequency of SPG11 mutations (3/22; 13%), in conformity with other populations.15 Of note, SPG7 was absent in the present cohort where SPG57, ARSACS, and ALS2 accounted for 1 family each (4%). Regarding dominant forms, SPG3 was seen in one family while SPG4 was absent from our series. While the phenotype was homogeneous between patients in several families (F34, F19, FM5 showing mutations in ATL1, TFG/SPG57 and ALS2 respectively), it was more often heterogeneous in other extended families (F1, F6, F14). These differences ranged between subtle and overt, including motor and cognitive domains, onset and progression, and were evident within nuclear and extended families.

Of major interest, we report the second SPG57 family with three siblings carrying the novel c.64C>T, p.Arg22Trp mutation. We observed significant clinical differences that may be attributed to the different domains affected in the two families and the functional sequel.

Whole exome sequencing approach

In three complex HSP families, WES was used as a first approach.

 Family F15

Family F15 was an extended family composed of three branches. The age at onset ranged between one to three years. The patients had history of regression of milestones following a normal early development till the age at onset which ranged from one to three years. The clinical picture was dominated by spastic quadriplegia complicated by cognitive impairment in the majority of patients and epilepsy in two patients. Despite the homogeneity of the clinical presentation in all branches, stereotypic clapping distinguished the patient from branch 1 [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF]] [Table ] [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF]].

Using WES in the five patients and one healthy parent, only one homozygous The first case in whom the genetic basis of argininemia was characterized was a Japanese patient with compound heterozygous mutations [START_REF] Haraguchi | Molecular basis of argininemia. Identification of two discrete frame-shift deletions in the liver-type arginase gene[END_REF] The combination of progressive spastic tertraplegia, regression of milestones, epilepsy in our patients is in agreement with what was reported about the presentation of a loss of function mutation on the Arginase 1 [START_REF] Wu | Seven patients of argininemia with spastic tetraplegia as the first and major symptom and prenatal diagnosis of two fetuses with high risk[END_REF] (Schlune et al., 2015) [START_REF] Sin | Arginase-1 deficiency[END_REF] . In contrast to the Japanese patient described by Haraguchi in 1990, the five Sudanese patients in family F15 did not have microcephaly which was reported in many patients of hyperargininemia [START_REF] Haraguchi | Molecular basis of argininemia. Identification of two discrete frame-shift deletions in the liver-type arginase gene[END_REF]) (Wong et al., 2015). In contrast to what was reported in five Chinese patients with poor physical growth associated with argininemia the Sudanese patients had normal physical growth despite the mental impairment with variable severity witnessed in all of them [START_REF] Wu | Five Novel Mutations in ARG1 Gene in Chinese Patients of Argininemia[END_REF] Biochemistry assessment of amino acid levels in the serum of the patients showed an increased level of Arginine in all patients in a first assessment but it was normal in another occasion. A laboratory error was considered in the second occasion as arginine was not reported as normal in patients with ARG1 mutation to suggest oscillating level of arginine with occasionally normal level and peaks. In a set of Brazilian patients the level of arginine was approximately near borderline in some patients [START_REF] Carvalho | Analysis of novel ARG1 mutations causing hyperargininemia and correlation with arginase I activity in erythrocytes[END_REF]. Retesting of erythrocyte Arginase activity and plasma Arginine level is planned in the near future to further confirm the diagnosis due to the availability of treatment and protection options. In the absence of other evidence of the involvement of any other variants, I considered the ARG1 mutation as causative in this family. WES in patient 210 combined to homozygosity mapping analysis identified three variants novel or rare with convincing evidence of pathogenicity. Among these, the novel variant (c.1427+2T>C) in the PLA2G6 gene acting as a splice site donor was the most convincing and the clinical scenario of the patients matched with what was reported in the literature about the presentation of PLA2G6. The mutation very likely led to the skipping of exon10 [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF]]. The predicted change at the donor site, which is 2 bps upstream, is -100.0% (MaxEnt: -100.0%, NNSPLICE: -100.0% and HSF: -100.0%). The mutation was found to cosegregate with the disease distribution. It was found in a homozygous state in two patients and homozygous reference or heterozygous in five healthy related individuals [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF]].

The clinical presentation of the two patients was compatible with infantile neuroaxonal dystrophy (INAD) due to neurodegeneration associated with mutations in PLA2G6 gene (PLAN) [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF]].

In INAD the age at onset is between six months and three years. Its less frequent variant has later onset: atypical neuroaxonal dystrophy (atypical NAD). The hallmark in both conditions is the presence of cerebellar atrophy and optic atrophy

 Family F30

The four patients (sisters) showed homogeneous clinical scenarios with age at onset of motor symptoms at around 2 years and the age at development of cataract at 4-6 years. They had delayed motor and normal intellectual development. The main clinical features were ataxia complicated by short stature, cataract, pyramidal signs, mild cognitive impairment and hypogonadism. The pyramidal syndrome was more prominent in LL than in UL. There was mild cognitive impairment and clinical evidence of hypogonadism in three sibs (lack of menarche in one and early menopause in the 2nd decade of life in two of them) whereas the fourth was too young to assess [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF]] [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF]].

An extremely rare missense variant in the SIL1 gene was found among seven others that were identified upon analysis of WES of two patients (258,259). It was the only one that was found to co-segregate with the disease distribution (homozygous in four patients whereas five healthy related individuals were homozygous reference or heterozygous) [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF]].

The variant was extremely rare and was found only once in a heterozygous state and never in the homozygous state in the 60,706 WES in the broad institute ExAC browser. It was not found in EVS, dbSNP or 1000 genome databases. The mutation [Exon 8: g.138356887 G>A, c.740C>T / p.(Ala247Val)] was a transition mutation of a highly conserved nucleotide. The variation was predicted pathogenic 345678 in MSS in male and females in many studies [START_REF] Senderek | Mutations in SIL1 cause Marinesco-Sjögren syndrome, a cerebellar ataxia with cataract and myopathy[END_REF] [START_REF] Karim | A novel mutation in BAP/SIL1 gene causes Marinesco-Sjögren syndrome in an extended pedigree[END_REF]. The spasticity is a rare manifestation of MSS as it was only reported in three Japanese sibs [START_REF] Takahata | Novel mutations in the SIL1 gene in a Japanese pedigree with the Marinesco-Sjögren syndrome[END_REF]. We could not detect any evidence of myopathy and no supporting investigations could be performed (serum creatine kinese, EMG/NCS and muscle biopsy). Serum immunoglobulin levels could not be measured although low serum IgG and IgA had been reported in MSS [START_REF] Hasegawa | Whole-exome sequence analysis of ataxia telangiectasia-like phenotype[END_REF].

Genotyping approach

 Family F26

Because of the specific association of pyramidal signs with AD cerebellar ataxia and macular degeneration [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF]], the DNA of the proband of this family was sent to the diagnostic unit in the hospital (Dr. Cécile Cazeneuve, UF de neurogénétique, APHP) for expansions in known AD cerebellar ataxias (SCA1, SCA2, SCA3, SCA6, SCA7, SCA17 and DRPLA) using a PCR-based approach followed by genotyping to determine the number of CAG repeats carried by each genes. A deleterious allele with 60 +/-3 CAG trinucleotide repeats in a heterozygous state in the ATXN7 gene was found [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF]].

Clear anticipation was observed with the age at onset 18.5 years younger in the second generation (son and daughter) than the first generation (mother) and the disease showing more severe and rapidly progressive course in the kids [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF]]. The anticipation is well known in SCA7 although it was more linked to paternal transmission [START_REF] Benomar | Autosomal-dominant cerebellar ataxia with retinal degeneration (ADCA type II) is genetically different from ADCA type I[END_REF]. The clinical presentation, summarized as a combination of AD ataxia, pyramidal signs, extrapyramidal features and macular degeneration [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF]], matched what was reported in the literature about SCA7. SCA7 was referred as progressive cerebellar ataxia with pigmentary macular degeneration (type II autosomal dominant cerebellar ataxia (ADCA) in the classification of Harding in 1983. It was found to be due to unstable CAG repeat in the gene ATXN7 [START_REF] David | Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion[END_REF].

Comment

In 12 families the genetic cause was found to be a gene previously reported to cause spastic syndrome. Three of them were found through the classical candidate gene approach. These included two families (F1 and F6) with SPG11 (Elsayed et al, in revision) and one family (F26/SCA7) through the analysis of pathological expansions in spinocerebellar ataxia genes (part 3.2.1.4). The remaining nine were

Identification of new causative HSP Genes

Ten families previously excluded through gene panel explorations were subjected to WES in search for new potential genes. These families were selected on the basis of their informativity (available full clinical data, multiple sampled patients to allow segregation analysis). In families where the pedigree suggested AR inheritance WES was performed in one patient only per family or family branch in case of extended families. In families where AD inheritance was suggested WES was done in two patients at least. There were rare conditions in which we chose to perform WES to more than one individual per branch.

ABHD16A mutation in family F37

Case report

The affected individuals were two boys (314, 315) who were born to first degree consanguineous parents after an uneventful pregnancy and delivery. The two sibs showed homogeneous disease progression. A little after birth, the family observed some delay in their psychomotor development followed by significant regression of the poorly acquired milestones at around 3-4 years. They were not floppy at birth or at their early months. They had delayed acquisition of minimal speech skill. They started to walk late unaided but limited. When they were at 3-4 years of age they started to show regression of the limited walking and poor speaking skills they acquired where they reverted to crawling (314) and aided walking (315). At around 7-10 years of age they became both wheel chair bound. They acquired sphincter control at 2 and 3 years of age (younger and older sib, respectively).

They did not develop sphincter disturbance with the disease progression.

Last clinical examination:

LL examination could not be performed for the older sib (314) because of postcorrective surgery for ankle deformity. The younger sib (315) had talipes equinovarus more pronounced on the left leg. Both brothers had spastic paraplegia presenting with severe bilateral spasticity, hyperreflexia, extensor plantar response and motor deficit in the LL (distal more than proximal). Extrapyramidal signs in form of significant choreoathetosis were detected. They were both mentally retarded. They had psychiatric signs in form of irritability, distractability, bouts of crying and the ease of getting upset and frightened. Sterotypic behavior were marked and included clapping and head slapping. LL wasting both peripherally and distally was a sign of lower motor neuron pathology [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF]].

Clinical Summary:

1-Complicated HSP (TCC HSP)

2-Age at onset of two sibs: within the 1st year of life. 8-Psychiatric symptoms (distractability, irritability, bouts of crying, ease of upset and frightened attitude) 9-Brain MRI: TCC, subcortical and periventricular WMH signals.

Gene identification

Genetic exploration using WES in patient (314), focusing on rare/novel gene with direct effect on the protein/mRNA, identified 13 rare variants at the homozygous state . Only one segregated with the disease in the family, a nonsense mutation in the ABHD16A gene: [Exon 5: g.31668722G>A, c.340C>T, p.Arg114*].

112

The mutation was found to completely co-segregate with the disease distribution where it was found in a homozygous state in the two affected siblings. The parents were confirmed to be obligate heterozygous carriers ].

This mutation was predicted to result in nonsense mediated decay (NMD) of the mRNA and absence of the protein with complete loss of function in the homozygous state [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF]].

 Functional interest of the gene ABHD16A (also known as Human lymphocyte antigen B-associated transcript 5 (BAT5))is located on the short arm of chromosome 6 (6p21.33) [Ensembl: Chromosome 6: 31,686,703,444 (GRCh38); Chromosome 6: 31,654,671,221 (GRCh37)].

ABHD16A (Abhydrolase domain containing 16A) has recently been shown to be the main Phosphatidyl serine lipase in the brain and constitutes the second protein in the ABHD12/ABHD16 axis [START_REF] Kamat | Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay[END_REF].

ABHD16A is correlated in STRING network with three of the proteins that are included in the ataxia interactome (SAFB, PTGS2, and RNF5) which can be considered as an added pointer high probability of its involvement in a neurodegenerative disorder [START_REF] Lim | A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration[END_REF]. He was not dry by day and sustained anal control till recently and he has still nocturia.

Anal sphincter was well controlled. His visual acuity diminished since the age of 7 months and at 2 years he developed bilateral cataract and was operated. He had been diagnosed with absence epilepsy (age not specified). Just after two years the parents noted that he started to develop imbalance and then progressively more motor symptoms started to occur. They noted that his neck fell back as if the patient lost neck control. At 10 years they noted that he developed some tremors when reached for an object. The eldest and youngest sisters had basically the same natural history of the disease but cataract developed earlier at 7 months and 1 year 8 months, respectively. They had both excessive bouts of crying at <2 months and 4 months, respectively. While the youngest sib did not have any type of seizures the oldest had history of febrile convulsions once at the age of 4 years.

Last clinical examination:

The index case had dysmorphic features including elongated face and malocclusion of mouth. Some pigmented spots were noted in the trunk ("café au lait") as well as some pustules which were described as repetitive by the parents.

Skeletal deformities were found including scoliosis and enhanced lumber lordosis. Lower motor neuron features were found (trunkal hypotonia and marked UL and LL wasting both proximally and distally). He had pyramidal signs (LL predominantly with spasticity and hyperreflexia but flexor plantar response) and features of cerebellar dysfunction in UL, wide based gait and eye (saccadic pursuit). Extrapyramidal signs in form of significant choreoathetosis and dystonia (retrocolis) as well as titubation of the whole upper half of the body and pill rolling (like) tremor were detected. There was cognitive impairment and psychiatric signs as irritability and the ease of getting upset.

Only the youngest sister was available for examination. She was irritable and had bouts of continuous crying. She had the same trunkal "café au lait" but no pustules. She had left eye divergent squint. Lumber lordosis was also found. She had some dysmorphic features including minor prominent upper and lower jaws and some milder malocclusion of the mouth. She had trunkal hypotonia but the tone of the limbs was normal. There was UL and LL hyperreflexia with flexor plantar responses. Mild cerebellar ataxia of the UL was observed on reaching for objects [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF]].

Clinical Summary:

1-Complicated spastic ataxia WES analysis identified 3 rare variants at the homozygous state that segregated with the disease in genes MINK1, ZNF433 and CAMSAP3.

CAMSAP3

The most convincing variant was a known missense variant rs180741228 [exon [START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF]683,190(GRCh37)] and stands for calmodulin regulated spectrin-associated protein family, member 3.

The variant rs180741228 passed the GATK filter with high confidence and was covered at a depth of 41x. The variant had an ExAC frequency of 0.09773% and was present only at the heterozygous state. The variant fully co-segregated with the disease distribution in family F41 and was homozygous in the two sampled affected siblings whereas other five healthy related individuals were heterozygous carriers or homozygous reference as in controls [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF]].

The mutation resulted in a change from the basic amino acid arginine to the hydrophobic amino acid tryptophan (moderate physicochemical difference [Grantham distance of 101 (0-215)]). Arginine is moderately conserved considering 18 species, but conserved in mammals.

The change was predicted as pathogenic by three different algorithms (SIFT, Polyphen2 and Mutation taster) [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF]].

MINK1

A 17: 4,833,898,061 (GRCh38);17: 4,736,801,356 (GRCh37)].

The variant completely co-segregated with the disease distribution where it was homozygous in the affected sibs and heterozygous carriers or homozygous reference in three healthy related individuals [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF]].

The variant passed the GATK filter with high confidence and coverage at depth of 24X. The mutation of this moderately conserved nucleotide [PhastCons : 1.0, (phyloP: 3.35 (-14.1;6.4)] was predicted pathogenic by MutationTaster which designated it as disease causing (p-value: 1) [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF]]. There is a moderate physicochemical difference [Grantham distance of 29 (0-215)] between both hydrophilic amino acid glutamate and glutamine. The amino acid is a highly conserved amino acid in all mammals and vertebrates up to the Tetraodon nigroviridis.

ZNF433

A mutation in exon 4 of the gene ZNF433 [g.12126975, c.707G>A, p.Ser236Asn] (based on the longest transcript NM_001080411.2) cosegregated with the disease distribution in family F41.

ZNF433 (zinc finger protein 433) is also known as FLJ40981. It is located on the short arm of chromosome 19 (19p13.2) [Ensembl: 19: 12,014,035,741 (GRCh38);19: 12,125,146,556 (GRCh37)].

The variant passed the GATK filter with high confidence and was covered at a depth of 52x. The variant has an ExAC frequency of 0.00083% only at the heterozygous state. The variant completely co-segregated with the disease distribution where it was homozygous in the two sampled affected sibs and heterozygous carriers or homozygous reference in two healthy related individuals [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF]].

The mutation of this weakly conserved nucleotide [PhastCons : 1.0, (phyloP:

1.01 (-14.1;6.4)] was predicted possibly damaging by PolyPhen2 (p-value: 1)

[Table 3456789

MRI of the brain Cerebellar atrophy Cerebellar atrophy

Electrophysiological Studies (NCS) ND ND imbalance) when she was 3 years whereas the younger had predominant cognitive impairment and psychological symptoms at the same age.

3.3.3.2.Last clinical examination:

On assessment of the patients they both had convergent squint. They were both wheel-chair bound. The older had talipes equina varus. They were tetraspastic with hyperreflexia. The plantar response was extensor in the younger sib. The motor deficit in both patients was mild and did not match the spasticity in severity.

Unfortunately the cerebellar function could not be assessed properly in the younger sib but signs of cerebellar ataxia were found in the older girl who had ataxia in UL, LL, at gait and in the eyes (saccadic pursuit). The cerebellar ataxia was less prominent than the pyramidal features. Horizontal ophthalmoplegia was found in the two sibs but the younger (403) had oculomotor apraxia in addition.

The younger sib had marked mental retardation, drooling of saliva and significant psychiatric signs which include irritability, emotional lability and persistent crying.

On the contrary, the older showed only minor mental subnormality and blunt mood. None of them had signs of extrapyramidal or lower motor neuron lesions

[Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF]].

3.3.3.3.Clinical Summary:

1-Complicated HSP

3.3.3.4.Genetic exploration:

WES filtering in patient (402) identified ten variants at the homozygous state and predicted deleterious. Only two variants from two genes (BIRC5 and C21ORF91) co-segregated with the disease distribution.

BIRC5

The most convincing one was a missense transversion mutation in BIRC5 BIRC5 is located on the long arm of chromosome 17 (17q25.3) [Ensembl: 17: 78,214,225,636 (GRCh38) 76,210,221,717(GRCh37)].

The variant had passed the GATK filter with high confidence and good coverage with 79x depth). Co-segregation with the disease distribution in family F50 was confirmed where it was homozygous in the two sampled affected siblings whereas both parents were heterozygous carriers [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF]].

The mutation resulted in a change from phenylalanine to leucine. The two are branched hydrophobic amino acids. There is a small physicochemical difference [Grantham distance is 22 (0-215)] but leucine lacks the aromatic ring. The amino acid is located in the core of the zinc finger of the conserved baculoviral inhibition of apoptosis protein (IAP) repeat domain. This change was predicted pathogenic by SIFT, Polyphen2 and Mutation taster [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF]].

The concerned nucleotide has a PhastCons conservation score of 1.00 (0-1) and a PhyloP score of 2.06 (-14.1-6.

C21ORF91

A known but rare variant rs36095555 in exon 3 of the gene C21ORF91 [g. 21 [START_REF] Ishiura | The TRK-Fused Gene Is Mutated in Hereditary Motor and Sensory Neuropathy with Proximal Dominant Involvement[END_REF]161,191,703 (GRCh37)].

The rs36095555 had an ExAC frequency of 0.0629 but existed only at the heterozygous state. The variant completely co-segregated with the disease distribution where it was homozygous in the two sampled affected siblings whereas both parents were heterozygous carriers [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF]].

The variant passed the GATK filter with high confidence and coverage at depth of 61X. The mutation of this moderately conserved nucleotide [PhastCons : 1.0, (phyloP: 2.63 (-14.1;6.4)] was predicted pathogenic by both SIFT (deleterious; Score :0) and MutationTaster which designated it as disease causing (p-value: 1) [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF]]. There is a small physicochemical difference [Grantham distance of 27 (0-215)] between both hydrophilic amino acid alanine and proline. The amino acid is a highly conserved amino acid considering 12 species up to the frog.

127

C21ORF91 is relatively tolerant to missense (ExAC z score = 0.19) and LOF mutations (ExAC pLI = 0.002) (0-1; 1 = the maximum intolerant).

In 60,706 WES in ExAC browser, the total number of observed missense mutations is more than the expected (82 observed variants compared to 78.6 expected). Speech skill was not acquired and there was also obvious poverty of movement.

Sphincter control was acquired normally. There was evidence of mental retardation and psychological symptoms in form of blunt mood. There was no evidence of dysmorphism.

3.3.4.2.Last clinical examination:

On assessment of the patients they were aphasic. They had predominant pyramidal signs (UL and LL spasticity, hyperreflexia and bilateral extensor plantar response). All three had skeletal deformity in form of pes cavus and mild scoliosis.

They had generalized sever dystonia and bradykinesia. There was no sign of cerebellar syndrome. Provisional ophthalmoscopy showed bilateral optic atrophy in all three patients but this was not reassessed by the ophthalmologist. They had marked mental retardation, drooling of saliva and they had blunt mood and lack of interaction with others [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF]].

3.3.4.3.Clinical Summary:

1-Complicated HSP with Dystonia. ST7L is located on the short arm of chromosome 1 (1p13.2) [Ensembl: 1: 112,523,620,825 (GRCh38);1: 113,066,163,447 (GRCh37)].

The variant completely co-segregated with the disease distribution where it was homozygous in the affected sibs and heterozygous carriers or homozygous reference in four healthy related individuals ].

The variant passed the GATK filter with high confidence and coverage (depth of 26X). The mutation of this moderately conserved nucleotide [PhastCons : 1.0, (phyloP: 2.95 (-14.1;6.4)] was predicted pathogenic by MutationTaster which designated it as disease causing (p-value: 1) [Table 3456789 ND Normal ND

PANK4 mutation in branch 1 of family FM7

In this family (FM7), there were two branches. Patients presented with a complex neurological disorder that we found different despite the multiple shared points. In branch 1 there were five affected sibs whereas in branch 2 there were two patients (a father and his daughter). The two branches were far related few generations upstream [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF][START_REF] Novarino | Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders[END_REF][START_REF] Beetz | Inhibition of TFG Function Causes Hereditary Axon Degeneration by Impairing Endoplasmic Reticulum Structure[END_REF]].

Family FM7/ Branch 1

3.3.5.1.Case report

The affected individuals were five sibs (2047,2048,2049,2050,2051) presented to the clinic at the ages ranging between 13 to 29 years. They were born to consanguineous parents. The parents noted the disease onset since birth : they didn't cry and were then suspected to be mute. The two had difficult prolonged delivery (2050, 2051) and there was antepartum hemorrhage in late pregnancy of the individual (2051). All other sibs were born after an uneventful pregnancy.

With the exception of patient ( 2048 The two boys (2050, 2051) were diagnosed with epilepsy at the age of 12 years.

3.3.5.2.Last clinical examination:

On assessment of the patients they were all deaf or almost deaf, mute and with apparent mental retardation. In three of them there was microcephaly. Psychiatric signs included remarkable euphoria in four patients (2047,2049,2050,2051) and blunt mood in their sister (2048). All patients had cerebellar ataxia and the patients with microcephaly had additional pyramidal signs (UL and LL spasticity, hyperreflexia) [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF]].

3.3.5.3.Clinical Summary:

1-Complicated HA/ HSP

Family FM7 branch 2

In this branch the affected case were a father and his daughter who presented at the ages of 29 and 9 years, respectively. They did not complain of any neurological symptoms and were found upon interrogation of the members of first branch about family history of deafness and mutism. They were mute and had deep hearing impairment and mild cerebellar ataxia. They had normal development and were intellectually sound. No additional symptoms or signs were detected.

Genetic exploration:

A single known missense transition mutation (rs151240697) in exon15:

g.2442812C>T, c.1822G>A, p.(Val608Met) of PANK4 (based on the longest transcript: NM_018216.2) was found in patient 2049 and 2051.

Co-segregation with the disease distribution in branch 1 of family FM7 was confirmed where it was homozygous in all the five affected siblings and heterozygous or homozygous reference in seven healthy individuals [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF][START_REF] Novarino | Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders[END_REF][START_REF] Beetz | Inhibition of TFG Function Causes Hereditary Axon Degeneration by Impairing Endoplasmic Reticulum Structure[END_REF]].

The variant was not the cause in branch 2 of family FM7 with a different clinical phenotype despite some shared signs. In this branch, the two patients where a homozygous reference and heterozygous carrier [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF][START_REF] Novarino | Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders[END_REF][START_REF] Beetz | Inhibition of TFG Function Causes Hereditary Axon Degeneration by Impairing Endoplasmic Reticulum Structure[END_REF]].

Further analysis using homozygosity mapping data was done. HZM did not show any shared region of homozygosity shared between patients of both branches although a low threshold for detection of homozygous region of 100000 bp was utilized. No genomic rearrangement was found too.

Causes unidentified

A summary of the families in which the genetic cause was not identified is in [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF]] and statistics represented in [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF][START_REF] Novarino | Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders[END_REF][START_REF] Beetz | Inhibition of TFG Function Causes Hereditary Axon Degeneration by Impairing Endoplasmic Reticulum Structure[END_REF][START_REF] Ishiura | The TRK-Fused Gene Is Mutated in Hereditary Motor and Sensory Neuropathy with Proximal Dominant Involvement[END_REF]].

The cause was not identified in 24 families in the cohort. Negative results upon analysis of WES data were found in five families. In the remaining 19 families the cause was the absence of WES data due to the families prioritization policy that we followed where only high yielding families, in which two or more affected individuals were sampled, were selected for WES. Five families were exception as they were not included in the cohort for WES because of logistic reasons such as the cost and the time factor.  Family 27, syndromic presentation Among the 24 families without genetic results, one family emerged, with a peculiar phenotype, suggesting a common genetic origin:

Clinical summary

In this family, three sibs presented at the ages of 15, 6.5 and 5 years of age with homogenous clinical picture of globally retarded growth with apparent developmental entrapment/arrest since around 1-3 years of age. They had facial dysmorphism more prominent in the older sib. Hair colour was lighter than their parents and healthy sibs sibs and hair texture smoother. They had scaly seasonal hypopigmented skin rash. Mental retardation and psychiatric signs of irritability and emotional lability were observed. Remarkably, the three sibs were extremely friendly, interactive and welcoming. The neurological syndrome was of complex spastic ataxia with bradykinesia (extrapyramidal signs). Ophthalmological assessment revealed optic atrophy with retinitis punctata albescence (in all three sibs) (mottled retina and a variant from retinitis pigmentosa) and hypermetropia in the older sib with most advanced disease. The older girl was diabetic. In one occasion the boy showed altered liver function test but there was no further investigations to exclude other causes nor a follow up tests ] [Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF][START_REF] Novarino | Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders[END_REF]].

This phenotype presentation has never been reported previously, to our knowledge, and might represent a new syndrome.

Comments and summary

The overall genetic diagnostic power was 41% since a genetic diagnosis was established in 17 families (known, potential new genes) out of the 41 Sudanese families included in the cohort (in two families the causative gene/variant was identified in a branch of the family: F1 and FM7) [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF][START_REF] Novarino | Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders[END_REF][START_REF] Beetz | Inhibition of TFG Function Causes Hereditary Axon Degeneration by Impairing Endoplasmic Reticulum Structure[END_REF][START_REF] Ishiura | The TRK-Fused Gene Is Mutated in Hereditary Motor and Sensory Neuropathy with Proximal Dominant Involvement[END_REF][START_REF] Takiyama | Sacsinopathies: Sacsin-Related Ataxia[END_REF][START_REF] Gregianin | A Novel SACS Mutation Results in Non-Ataxic Spastic Paraplegia and Peripheral Neuropathy[END_REF]] [A summary of the thesis method and genetic results is in [Figure ].

WES was collectively performed in 14 families. In three of them, the genetic diagnoses were mutations in genes known to cause spastic neurogenetic disorders. In five families we had potential new genes for further confirmation of pathogenic effect through functional studies [Figure [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF][START_REF] Novarino | Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders[END_REF][START_REF] Beetz | Inhibition of TFG Function Causes Hereditary Axon Degeneration by Impairing Endoplasmic Reticulum Structure[END_REF][START_REF] Ishiura | The TRK-Fused Gene Is Mutated in Hereditary Motor and Sensory Neuropathy with Proximal Dominant Involvement[END_REF][START_REF] Takiyama | Sacsinopathies: Sacsin-Related Ataxia[END_REF][START_REF] Gregianin | A Novel SACS Mutation Results in Non-Ataxic Spastic Paraplegia and Peripheral Neuropathy[END_REF](22)].

. Overview of the cohort

The cohort is heterogeneous but within limits that could be expected from the broad inclusion criteria as well as the huge phenotypic and genetic heterogeneity of the HSP and its overlap with other closely related differential diagnoses. The core clinical characteristic of the Sudanese familial cohort is the increased level of complexity which is noted in the majority of families.

The most frequent complicating clinical features are signs of cerebellar syndrome, cognitive impairment and various eye involvement (neuronal and non neuronal). This matches what we found in our analysis of the clinical presentation of various forms of HSP. Peripheral neuropathy is underestimated given the fact that EMG/NCS has not been performed in many patients and the clinical examination alone is not satisfactory especially on assessment of pediatric and mentally impaired patients [Figure ] [Table (4-1)].

Extrapyramidal signs and more specifically chorea/athetosis are more prevalent in our cohort (4/41 families: F28, F37, F41 and F47) than what we found on analyzing the known HSP subtypes. To date, SPG58 is the only type of HSP in which chorea was reported. MRI of the brain was not performed in all families but in the cases where it was done it showed complex abnormalities [Figure ] [Figure ] [Table ]. In the methodology chapter we explained in details, the limitations that we encountered with the brain imaging.

Examples have been illustrated where we have broadened the phenotypic spectrum associated with certain known genes [F19/TFG (SPG57), FM4/DNAJC6 (JOPD), F30/SIL1 (MSS), F15/ARG1 (hyperargininemia)]. In rare conditions even the same mutation is associated with minor differences in their clinical presentation (family F6/SPG11).

The scene is further complicated by the fact that in at least seven families multiple phenotypes coexisted. In at least three of these families (F1, F6, FM7) we could provide provisional proof that these phenotypes are distinct genetically. This is not always easy especially when the candidate genes in these families are new (FM7). Yet when the family (or branch) under question is sporadic the decision will be even harder (  Family F27, a peculiar syndrome An intriguing condition was found in family F27. In this family a peculiar syndrome was observed as indicated in the results. Preliminary clinical assessment implied a new clinical syndrome described and to be reported after full work up. The gene behind the family was not identified. WES has been performed; analysis excluded homozygous variants in all protein coding regions of genes covered in the WES. As the family showed recessive inheritance with three sibs affected and the parents are nonconsanguineous, compound heterozygous variants were a possibility. Yet although none was identified, this possibility cannot be excluded given the fact that the second incriminated variant may be located in non-coding region or in region not well covered. Proper complete phenotyping is required including reexamination of the eyes, orthopedic consultation, standard IQ testing, endocrinological assessment, cardiac assessment, full metabolic screening, test and repeat liver functions for all patients, electrophysiological studies (visual and auditory evoked potentials, EMG/NCS, EEG)

and follow up by brain imaging. Reanalysis of the WES and functional assays for the fibroblasts just to assess the organellar morphology even in absence of genetic diagnosis will be considered.

Diagnostic NGS-based multigene panels for HSP

Phenotype directed choice of culprit genes can be used in this era of NGS to facilitate the selection of probable genes when more than one variant is obtained on bioinformatic analysis of output data of the diagnostic panels.

Below is a proposed flow chart based on phenotype/ genotype correlation data to aid in the selection of variants/genes [Figure [START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF]].

Evidently, this approach can mask unexpected phenotypes or inheritance models associated to mutations in a given gene. Therefore, the use of flow charts has to be done with caution in typical cases and is of interest mainly when the geneticist has to deal with various putative mutations in various known genes.

Comparison of the yield of genetic results

As described earlier, the overall success rate was 41% whereas in 24/41 families (59%) the cause could not be identified. In 5/41 families (12%), the causing genes were not associated with the disease before (potential new genes).The genes identified were known in 12/41 families (29%).

This power went significantly higher with the use of WES as it reached 57% (3.5x the diagnostic kit approach and 2.7x the combined screening methods approach). This could be explained by the percentage of families with potentially new genes causing the disease which was 36% of the diagnosed families (5/14 families). The failure rate of the WES approach was 43% and this could be attributed to the fact that in these families the variant causing the disease was located in a noncoding/intronic region or a compound heterozygous with one of the heterozygous mutations present in a badly covered region or a noncoding region. In all cases genomic rearrangements could not be completely excluded though preliminary results on coverage evaluation in WES data showed that they were unlikely. The rates are comparable to what has been reported about success rates in other studies ] (Novarino et al., 2014).

Figure (4-7):

Comparison of the success rates of approaches that were utilized in genetic investigation. WES: whole exome sequencing, HSP:hereditary spastic paraplegia.

Towards the identification of new genes

We identified variants in eight new genes corresponding to five novel genetic isoforms of HSP. Although additional genetic and functional data are required to definitely validate their causality on the spastic paraplegia segregating in patients, the function of some of them can be linked to physiopathology of HSP.

4.4.1.

ABHD16A and complex TCC HSP in family F37 (The flip coin of

ABHD12/PHARC)

The single mutation segregating in family F37 is a loss of function mutation in ABHD16A.

 Gene/protein isoforms and splice variants of ABHD16A

This gene has 16 transcripts (splice variants) but only two are protein coding (two isoforms)

NM_021160 is the transcript that codes for the canonical isoform1 (identifier: O95870-1) which is composed of 20 exons and is 558 amino acid long and 63,243 in Mass (Da).

 Function

Abhydrolase domain containing 16A (ABHD16A) is a phosphatidylserine (PS) lipase that generates lyso-PS in mammalian systems. It has been suggested

that it is the main PS lipase in the CNS (brain and spinal cord).

Lysophosphatidylserines (lyso-PSs) are a category of lipids that are involved in regulation of immunological processes [START_REF] Kamat | Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay[END_REF].

ABHD16A is localized on the cytoplasmic face of cellular membranes. This is consistent with its role as a PS lipase, PS are mainly confined to the inner part of the lipid bilayer in healthy cells [START_REF] Kamat | Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay[END_REF]. ABHD16A represent the one of the two pillars of the ABHD16A/ABHD12 axis [Figure [START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF]]. This axis dynamically regulates lyso-PS metabolism in vivo ( Kim, 2015) [START_REF] Kamat | Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay[END_REF]. Lyso-PS is generated by ABHD16A and becomes accessible to hydrolytic degradation by ABHD12 (OMIM *613599). ABHD12 is an integral membrane protein whose active site is predicted to face the lumen/extracellular space [START_REF] Blankman | A Comprehensive Profile of Brain Enzymes that Hydrolyze the Endocannabinoid 2-Arachidonoylglycerol[END_REF]. Disruption of ABHD12 and ABHD16A in mouse macrophages causes an increase and a decrease in both lipopolysaccharide-induced cytokine production and lyso-PSs, respectively. C18:0 lyso-PS is a lyso-PS that is considered to be the most abundant species. It is secreted by macrophages and potentiates the above mentioned release of cytokines [START_REF] Kamat | Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay[END_REF]. As expected, ABHD16A-/-Knockout mice have decreased brain lyso-PSs whereas ABHD12-/-Knockout mice have increased brain lyso-PSs. ABHD16A-/mice were born at a much lower frequency than expected for Mendelian distribution.

≈30% of the knock out mice are smaller than the ABHD16A+/+ and +/-mice.

Nevertheless, they appeared normal in their cage behavior with no observed evidence of increased postnatal lethality in these animals.

ABHD12 has been associated with PHARC (OMIM #612674) (PHARC stands for Polyneuropathy, Hearing loss, Ataxia, Retinitis pigmentosa, and cataract) a rare syndrome of AR complex HA [START_REF] Fiskerstrand | Mutations in ABHD12 Cause the Neurodegenerative Disease PHARC: An Inborn Error of Endocannabinoid Metabolism[END_REF]. PHARC is a neurodegenerative syndrome presumably attributed to a neuroinflammatory process accompanying the elevated level of lyso-PS and the production of the lipopolysaccharide-induced cytokines. The phenotypes associated with ABHD12

were later expanded to nonsyndromic presentation [START_REF] Nishiguchi | Exome sequencing extends the phenotypic spectrum for ABHD12 mutations: from syndromic to nonsyndromic retinal degeneration[END_REF]).

On the contrary to ABHD12 related clinical phenotypes [START_REF] Kamat | Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay[END_REF], loss of function mutation in the Sudanese family [ABHD16A (-/-)] lead to complex HSP with TCC with no evidence of cerebellar involvement.

If the PHARC is due to an exaggerated neuroinflammatory response due to accumulation of lyso-PS, the question asked is why the decrease in lyso-PS results in TCC HSP. Lyso-PS is necessary for [Figure [START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF]]:

-mast cell degranulation-stimulating activity. This is not induced by other -Nerve Growth Factor (NGF)-induced neurite outgrowth in PC12 cells. Neurite outgrowth is one of the important pathways involved in HSP. It is the major function involved in the X-linked SPG1 and involved in axonal guidance. The presentation of SPG1 resembles to some extent the presentation of our family as both are associated with TCC (hypoplasia of CC) and mental retardation. Neurite outgrowth is also indirectly associated with the functions of many of the spastic paraplegia proteins.

Lyso-PS is required for nerve growth factor (NGF)-induced secretion of histamine from rat mast cells. Lyso-PS alone is ineffective in that process but strongly promoted the NGF-induced differentiation. Lyso-PS colocalizes with NGF in sites of tissue damage and therefore may be a naturally-occurring modifier of neuronal structure and/or function [START_REF] Lourenssen | Lysophosphatidylserine potentiates nerve growth factor-induced differentiation of PC12 cells[END_REF]. -Lyso-PS also regulates cytochrome P450 activity. Lyso-PS interaction with P450 1A2 (CYP1A2) and 2E1 (CYP2E1) increases their phospholipase D (PLD) activity in a concentration-dependent manner and decreases the monooxygenase (MMO) activity of both CYPs [START_REF] Cho | Lysophosphatidylserineinduced functional switch of human cytochrome P450 1A2 and 2E1 from monooxygenase to phospholipase D[END_REF]. Of note, cytochrome P450 activity was recently linked to SPG49/56 (CYP2U1) which is also associated with TCC and mental impairment [START_REF] Tesson | Alteration of fatty-acid-metabolizing enzymes affects mitochondrial form and function in hereditary spastic paraplegia[END_REF].

-Lyso-PS (C18:1/OH lyso-PLs bearing the phosphoserine) also enhances apoptotic cell-engulfment by macrophages by activation of human neutrophil through binding and mobilization of G2A latent in the plasma membrane/secretory vesicle fraction [START_REF] Frasch | Lysophospholipids of Different Classes Mobilize Neutrophil Secretory Vesicles and Induce Redundant Signaling through G2A[END_REF] It is quite evident that lyso-PS induces various cellular responses in a Lyso-PSspecific manner, these actions may be mediated by different Lyso-PS receptors [START_REF] Makide | Novel lysophosphoplipid receptors: their structure and function[END_REF].

-stimulates migration of fibroblasts (Park et al., 2006).

 Potential Treatment ?

As was speculated that PHARC can be treated by inhibition of ABHD16A to decrease the Lyso-PS level. ABHD16A associated TCC HSP could be potentially treated by blocking the degradation of lyso-PS by ABHD12 or possibly by blocking the ABHD16A-counteracting acyltransferase enzyme. Unfortunately to present no selective inhibitor of ABHD12 has been characterized [START_REF] Hoover | Selectivity of Inhibitors of Endocannabinoid Biosynthesis Evaluated by Activity-Based Protein Profiling[END_REF][START_REF] Navia-Paldanius | Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12)[END_REF].

Tetrahydrolipstatin (THL/orlistat) is a drug of clinical utility as an anti-obesity agent as it functions as a pancreatic lipases blocker in the intestine. It has an inhibitory effect on ABHD12 but it is non-selective. It exerts its inhibition on other enzymes including ABHD16A [START_REF] Hoover | Selectivity of Inhibitors of Endocannabinoid Biosynthesis Evaluated by Activity-Based Protein Profiling[END_REF].

Another inhibitor, KC02, has significant inhibitory effect on ABHD12 but has two partial off-targets which are ABHD11 (94%) and LYPLA1 (63%) [START_REF] Kamat | Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay[END_REF].

In conclusion, the genetic and function data strongly support the implication of the loss of function mutations in ABHD16A in family F37. Further exploration of fibroblasts and macrophages of patients would be necessary to provide cellular functional argument in support of what we have hypothesized as underlying mechanisms.

CAMSAP3, MINK1, ZNF334 candidate genes in family F41

Three missense variants with convincing genetic arguments are segregating with the disease in family F41. Additional biological data, from fibroblasts of patients, will be required to determine which mutation is responsible for the phenotype. The function of these 3 genes is also informative and will define which mechanisms to carefully look for in the fibroblasts.

CAMPSAP3

This gene has 4 transcripts (splice variants) but only two are protein coding ). As these were provisional data, verification for more phenotypic details may be needed.

In our patients there were skin pigmentations that resembled the "café au lait" spots, cataract, some dysmorphic features including protrusion of upper and lower jaws and malocclusion of mouth. These features are then reminiscent of what is described in the KO mouse model.

 Expression

CAMSAP3 is a cytoplasmic protein mainly localized along the zonula adherens [START_REF] Meng | Anchorage of Microtubule Minus Ends to Adherens Junctions Regulates Epithelial Cell-Cell Contacts[END_REF]. Very high expression was detected in adult and fetal brain and liver in addition to adult ovary, kidney, testis and pancreas.

Very high expression was also detected in all regions examined of the adult brain. There is low expression in adult heart, lung, skeletal muscle, spleen, and spinal cord [START_REF] Nagase | Prediction of the coding sequences of unidentified human genes. XVII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro[END_REF].

 Identification and structure of CAMSAP3

No crystal structure is available at present. Interestingly, CAMSAP3 protein was identified de novo and renamed several times:

First it was identified as KIAA1543, which was named Nezha after a deity in Chinese mythology, as a PLEKHA7-binding protein. The human Nezha protein contained an N-terminal calponin homology domain (CH domain), two central coiled-coil regions, and a DUF1781 domain at the C-terminal end of the protein. In human Caco-2 cell line, major and minor Nezha proteins were detected using western blot analysis. They had molecular masses around 150 and 160 kD, respectively [START_REF] Meng | Anchorage of Microtubule Minus Ends to Adherens Junctions Regulates Epithelial Cell-Cell Contacts[END_REF].

Later, KIAA1543 was re-identified and called CAMSAP3. The deduced protein contains a calponin homology domain, 2 coiled-coil regions, followed by a proline-rich region, a coiled-coil region, and a C-terminal DUF1781 domain. The DUF1781 domain was renamed as the CKK domain, since it is found in KIAA1078 (CAMSAP1L1), CAMSAP1 and KIAA1543 (CAMSAP3) [START_REF] Baines | The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins[END_REF]. The protein was also independently discovered and named patronin by another group [START_REF] Goodwin | Patronin regulates the microtubule network by protecting microtubule minus ends[END_REF].

 Function

CAMSAP3 acts as a microtubule minus-end binding protein. It acts as a regulator of non-centrosomal microtubule dynamics and organization. It is required for the biogenesis and the maintenance of zonula adherens. It anchors the minus-end of microtubules to zonula adherens. It does so by recruiting the kinesin KIFC3 to the junctional sites. CAMSAP3 may regulate the nucleation and the polymerization of microtubules [START_REF] Meng | Anchorage of Microtubule Minus Ends to Adherens Junctions Regulates Epithelial Cell-Cell Contacts[END_REF]. CAMSAP3 was later found to bind to the minus-end of MTs through its CKK domain [START_REF] Baines | The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins[END_REF]. Indirectly, through the microtubule cytoskeleton, the protein may regulate the organization of cellular organelles including the Golgi and the early endosomes (genecards). Based upon differences in patterns of expression of marshalin seen in the organ of Corti (sense organ for hearing), eight isoforms were identified. Their size ranged from 863 to 1280 amino acids.

Marshalin-L and marshalin-S isoforms induced different MT-bundle structures and/or composition. The two isoforms differ by 416 aa, which include proteinprotein interaction domains CC and PR [START_REF] Zheng | Marshalin, a microtubule minus-end binding protein, regulates cytoskeletal structure in the organ of Corti[END_REF]. The Golgi network, normally located near the nucleus, was found to be disrupted and separated into fragments in cells expressing marshalin-Ld. These Golgi-membrane fragments colocalized with marshalin protein. This was justified by the fact that the distribution and the localization of the Golgi network is connected closely to the cytoskeleton [START_REF] Sandoval | Role of microtubules in the organization and localization of the Golgi apparatus[END_REF], Golgi disruption could be due to the changes in MTs which were caused by marshalin-Ld expression. Marshalin had multiple isoforms. It acted as a scaffolding protein and was capable of modifying the cytoskeletal networks, and consequently organelle positioning like Golgi apparatus, through interactions with various protein partners present in different cells [START_REF] Zheng | Marshalin, a microtubule minus-end binding protein, regulates cytoskeletal structure in the organ of Corti[END_REF]. This is not the first gene in which microtubules and Golgi apparatus are associated with complex spastic paraplegia. Microtubules are involved in the pathogenesis of some HSP subtypes, particularly SPG4. Subcellular organelles and more specifically the Golgi apparatus (as part of the ER-Golgi axis) are behind the occurrence of SPG21, SPG57, SPG58 and SPG associated with FAM134B.

Further functional studies will illustrate the real mechanisms of CAMSAP3 that we can consider to represent a good candidate in family 41.

MINK1

MINK1 (Misshapen-like kinase1) is a germinal center kinase. It has been found that MINK1 is composed of a kinase domain at the N-terminus, a proline-rich region, and a GCK homology region at the C-terminus. It is highly expressed in the developing mouse brain (Dan et al., 2000). MINK1 is involved in several crucial biological roles in the cells. It has been found to be necessary for completion of cytokinesis [START_REF] Hyodo | Misshapen-like kinase 1 (MINK1) is a novel component of striatin-interacting phosphatase and kinase (STRIPAK) and is required for the completion of cytokinesis[END_REF]. It plays a role in the activation of p38 pathways and cJun N-terminal kinase (JNK) (Dan et al., 2000). MINK1 is involved in cell motility and reorganization of cytoskeleton.

It colocalized with golgi apparatus [START_REF] Hu | Identification and functional characterization of a novel human misshapen/Nck interacting kinase-related kinase, hMINK beta[END_REF]. Cytokinesis has been associated with HSP (Hazan et al., 1999) as well as with the main role of some HSP genes in organization of cytoskeleton.

ZNF433

Little is known about ZNF433. It is shown to be expressed in the cerebral cortex (http://www.proteinatlas.org/ENSG00000197647-ZNF433/tissue). In one study it has been shown that ZNF433 together with another new gene VAV2

have suggestive evidence of association with multiple sclerosis but did not show genome-wide significance level [START_REF] Nischwitz | Evidence for VAV2 and ZNF433 as susceptibility genes for multiple sclerosis[END_REF].

Conclusion

The CAMSAP3 mouse phenotype similarity to some features we have had in our patients supports its role in pathogenesis, rather than MINK1. Knockdown and rescue experiments in zebrafish could help to determine which variant is causal.

4.4.3.

BIRC5 and C21ORF91 variants in family F50

BIRC5

Two missense variants with convincing genetic arguments are segregating with the disease in family F50. Additional biological data, based on the known function of the genes, is required to determine which mutation is responsible for the phenotype. Fibroblasts of patients will be used for such exploration.

 Gene/protein isoforms and splice variants of BIRC5 BIRC5 has 11 transcripts of which only 6 are protein coding. The canonical isoform 1 of the protein is 142 amino acids in length and is the product of the transcript NM_001168.2 (4 exons). The longest transcript (NM_001012271.1 ) is composed of 5 exons and encodes for variant 3/ isoform 3 of the protein with 165 aa also known as Survivin 28 but is not well characterized.

 Biochemical structure of survivin

Survivin is considered the smallest member of the proteins family of inhibitor of apoptosis (IAP). Unlike other members of IAP family it contains only one N-terminal Baculovirus IAP repeat (BIR) domain and lacks the C-terminal ring finger domain. This domain is necessary for ubiquitination dependant proteosomal degradation of caspases (Srinivasula, S. M. & Ashwell, 2008). In survivin, a c-terminal coiled-coil α-helix domain replaces the ring finger domain, which in turn is responsible for its regulation of cell division (Wheatley, S. P, 2015).

The overall shape of a survivin has been studied using x-ray crystallography [START_REF] Chantalat | Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions[END_REF]) [START_REF] Muchmore | Crystal structure and mutagenic analysis of the inhibitor-of-apoptosis protein survivin[END_REF]. The BIR and Helix domains has been found packed tightly together. The BIR domain is characterized by a zinc-coordinating Cys/His motif shown to be stabilized by hydrophobic interactions involving the amino acid residues: F22, W25, F27, M38, F43, F58, L64, W67, and F86. The BIR domain in survivin is used for homodimerization and interaction with other chromosome passenger proteins [START_REF] Verdecia | Structure of the human anti-apoptotic protein survivin reveals a dimeric arrangement[END_REF]. The BIR motif as well plays an important antiapoptotic role [START_REF] Sun | NMR structure and mutagenesis of the inhibitorof-apoptosis protein XIAP[END_REF].

The mutation we report here changes the above mentioned phenylalanine (F86) into leucine (L86). Although both amino acids are branched hydrophobic with small physiochemical difference, leucine lacks the aromatic ring of phenylalanine and this could well modify the crystal structure of the survivin protein. Further modeling workup of the effect of the p.Phe86Leu is required to demonstrate if that change will disrupt the above mentioned hydrophobic interactions and affects the stability of the core of the zinc finger domain with alteration of its functions.

Function

BIRC5/Survivin is a multitasking protein with a myriad of protein-protein interactions and then with potentially multiple functions. It has dual roles in preventing apoptosis and promoting cell mitotic proliferation. It has been demonstrated that survivin cannot inhibit apoptosis by directly binding to caspases under physiological conditions [START_REF] Eckelman | Human inhibitor of apoptosis proteins: why XIAP is the black sheep of the family[END_REF]. However, it inhibits apoptosis indirectly through interaction with both pro-and anti-apoptotic proteins that promotes cell survival (Dohi, et al, 2004[START_REF] Ceballos-Cancino | Regulation of mitochondrial Smac/DIABLO-selective release by survivin[END_REF].

Survivin is possibly implicated in the process of DNA repair [START_REF] Chakravarti | Survivin enhances radiation resistance in primary human glioblastoma cells via caspase-independent mechanisms[END_REF]. The multiplicy of survivin functions have been attributed in part to differences in cells types and the variable subcellular localization of the protein (Altieri, D., 2008). It has been postulated that mitochondrial Survivin is associated to antiapoptotic function and nuclear survivin has been connected to the role in mitosis and protection against ionizing radiation [START_REF] Colnaghi | Separating the antiapoptotic and mitotic roles of survivin[END_REF].

Survivin has a short half-life of about 30 minutes [START_REF] Zhao | The ubiquitinproteasome pathway regulates survivin degradation in a cell cycle-dependent manner[END_REF]. Its expression is dynamic and can be modified in response to many intracellular and extracellular stimuli. Examples for factors that can alter survivin expression include cell cycle stage [START_REF] Altieri | The case for survivin as a regulator of microtubule dynamics and celldeath decisions[END_REF] cytokines [START_REF] Mahboubi | Interleukin-11 up-regulates survivin expression in endothelial cells through a signal transducer and activator of transcription-3 pathway[END_REF] hormones [START_REF] Nabilsi | Sex hormone regulation of survivin gene expression[END_REF], certain bacteria [START_REF] Valenzuela | Helicobacter pylori-induced loss of survivin and gastric cell viability is attributable to secreted bacterial gamma-glutamyl transpeptidase activity[END_REF] noise [START_REF] Knauer | An otoprotective role for the apoptosis inhibitor protein survivin[END_REF],hypoxia [START_REF] Conway | Survivin-dependent angiogenesis in ischemic brain: molecular mechanisms of hypoxia-induced up-regulation[END_REF], medications [START_REF] Cheng | Effect of topiramate on apoptosis-related protein expression of hippocampus in model rats with Alzheimers disease[END_REF], radiation [START_REF] Dallaglio | Survivin: a dual player in healthy and diseased skin[END_REF], and DNA damage [START_REF] Capalbo | Radiation-induced survivin nuclear accumulation is linked to DNA damage repair[END_REF] among others.

Targeted deletion of survivin gene in neural progenitor cells in mouse developing brain has shown that survivin is critically important for early CNS development [START_REF] Jiang | Essential role for survivin in early brain development[END_REF]. This supports the suggested effect of the p.Phe86Leu incriminated in the pathogenesis of F50 with early onset at 1-2 years of age. Further accumulating evidence supports the possible involvement of survivin in causing neurological disorder with complex and severe manifestations. Deletion of survivin gene is associated with apoptosis which is severe and multifocal involving the retina and many regions of the central nervous system including the spinal cord, cerebrum, cerebellum and brain steam [START_REF] Jiang | Essential role for survivin in early brain development[END_REF]. In a transgenic mouse model, postnatal expression of survivin was detected in two key sites of adult neurogenesis (the subventricular zone (SVZ) and the subgranular zone (SGZ)) [START_REF] Coremans | Impaired neurogenesis, learning and memory and low seizure threshold associated with loss of neural precursor cell survivin[END_REF]. Lack of expression of survivin in the neural precursor cells during embryonic development, was associated with profound postnatal defects in neurogenesis and loss of interneurons that manifest by major deficits in learning and memory, and augmented sensitivity to seizures [START_REF] Coremans | Impaired neurogenesis, learning and memory and low seizure threshold associated with loss of neural precursor cell survivin[END_REF]. In human adults survivin expression has been detected in the cochlea and its expression was found to be dysregulated in a transcriptomic study in HSP patients with SPAST mutations [START_REF] Abrahamsen | A patient-derived stem cell model of hereditary spastic paraplegia with SPAST mutations[END_REF]. The study found that in the patient derived cells, there was a major dysregulation of gene expression with 57% of all mRNA transcripts affected including BIRC5 among other genes with functions related to microtubules dynamics [START_REF] Abrahamsen | A patient-derived stem cell model of hereditary spastic paraplegia with SPAST mutations[END_REF]. All this goes in concert with the presentation of our two sibs with complicated HSP. From another point of view, defective DNA repair may be one of the mechanisms by which survivin is implicated in causing HSP complicated with cerebellar syndrome. Compared to other tissues, the developing brain is particularly sensitive to DNA damage [START_REF] O'driscoll | The role of the DNA damage response pathways in brain development and microcephaly: insight from human disorders[END_REF]. Neurons are metabolically active and are susceptible to DNA oxidative damage [START_REF] Jackson | The DNA-damage response in human biology and disease[END_REF]. Accumulation of DNA damage in the terminally differentiated, irreplaceable neurons increases the vulnerability of the nervous system to DNA damaging insults [START_REF] Jackson | The DNA-damage response in human biology and disease[END_REF] .

Furthermore, being in G0, neurons do not repair DNA double strand breaks by homologous recombination and use the more error prone non-homologous end joining (NHEJ) mechanism [START_REF] Jackson | The DNA-damage response in human biology and disease[END_REF].

Defects in DNA repair mechanisms can usually present with problems in the nervous, reproductive and immune systems in addition to premature aging and increased susceptibility to cancer [START_REF] Ciccia | The DNA damage response: making it safe to play with knives[END_REF]. Neurons of the cerebellum account for roughly 50% of brain neurons [START_REF] Katyal | DNA strand breaks, neurodegeneration and aging in the brain[END_REF]. Many defective DNA repair syndromes present with degeneration of cerebellar neurons [START_REF] Ciccia | The DNA damage response: making it safe to play with knives[END_REF].

Survivin is thought to have a role in DNA double strand repair mechanism, particularly non-homologous end joining (NHEJ) repair machinery through accumulation of survivin into the nucleus and its physical interaction with the mediator of DNA damage checkpoint protein 1 (MDC1), the histone protein γ-H2AX in addition to the DNA dependant protein kinase catalytic subunit (DNAPKcs) [START_REF] Capalbo | Radiation-induced survivin nuclear accumulation is linked to DNA damage repair[END_REF] .

Survivin, now known to be a component of the chromosome passage protein complex (CPC) which is crucial for chromosome alignment and segregation during cytokinesis and mitosis [START_REF] Lens | The case for Survivin as mitotic regulator[END_REF]. Although its role as promitotic was particularly proved in cancer cells, we can never ignore the fact that this may apply to the highly proliferating fetal cell and this may have contributed to the importance of survivin for the survival of developing central nervous system [START_REF] Jiang | Essential role for survivin in early brain development[END_REF]. Further cellular and functional studies will allow us to shed light on the role of survivin that lead to HSP in the two sibs opening a new passage between cancer and neurodegeneration.

C21orf91

 Gene/protein isoforms and splice variants of C21orf91

It codes for the protein EURL homolog. The gene has 6 transcripts (splice variants) but only four are protein coding.

NM_001100420 is the transcript that codes for the canonical isoform1

(identifier: Q9NYK6-1). The transcript is composed of 5 coding exons and is 297 amino acid long and 33,948 in Mass (Da).

 Function

Not much is known about C21ORF91 and the protein EURL homolog function. The protein is a cytosolic protein with unknown function. The gene was found to be strongly correlated with the infection with Herpes simplex labialis (cold sore) [START_REF] Kriesel | C21orf91 genotypes correlate with herpes simplex labialis (cold sore) frequency: description of a cold sore susceptibility gene[END_REF]. In a study about hepatocellular carcinoma, C21ORF91 was found as a target for miR-194 among a few other genes [START_REF] Bao | NF-κB signaling relieves negative regulation by miR-194 in hepatocellular carcinoma by suppressing the transcription factor HNF-1α[END_REF]. In a genome-wide association study about conducted in African Americans to identify candidate genes associated with systolic and diastolic blood pressure and (SBP/DBP). rs2258119 in the gene C21orf91transmitted the strongest signal for SBP [START_REF] Fox | Association of genetic variation with systolic and diastolic blood pressure among African Americans: the Candidate Gene Association Resource study[END_REF]

Conclusion

Given what is known about the functions of the 2 candidates genes, the genetic and functional elements are in support of the involvement of BIRC5 variant in HSP pathology in family F50.

ST7L unique mutation responsible for the pathology in family FM3

In family FM3, a single rare and predicted pathogenic variant was segregating, in ST7L.

This gene has 28 transcripts (splice variants). Of them 8 are protein coding (for 8 isoforms produced by alternative splicing) (Uniprot: Q8TDW4).

NM_017744.4 longest transcript: which is composed of 15 exons and codes for the canonical isoform 1 (Uniprot identifier: Q8TDW4-1). The isoform is 575 amino acids and is 64,779 Da in Mass.

Unfortunately little is known about the structure and the functions of ST7L. It is considered a multi-pass membrane protein as it is predicted to be composed of two helical transmembrane domains. (Uniprot: Q8TDW4).

With the use of northern blotting ST7R mRNA was found in various normal tissues, and especially large amounts in testis.

In the Fantom 5 project (fetal) ST7L was found relatively highly expressed in the spinal cord, various regions of the brain (occipital, parietal and temporal lobes), skeletal muscles and the eye. The Fantom 5 project (adult) showed that the gene expression expanded to involve the cerebellum, basal ganglia, diencephalon and thalamus. It was not detected in the eye, skeletal muscle and temporal lobe (http://www.ensembl.org/Homo_sapiens/Gene/ExpressionAtlas?db=core;g=ENS G00000076826;r=19:7595902-7618304).

Broadly speaking, ST7L seems involved in negative regulation of cell growth (GO:0030308).

In a single study, luciferase reporter assay provided evidence that the expression of ST7L is suppressed by overexpression of miR-24 and that led to enhanced cell proliferation and invasion. The study demonstrated through sequence analysis that miR-24 could form complementary base pairing with the 3 prime UTR region of ST7L. Apoptosis assay performed showed that the apoptotic cell fractions were significantly increased when ST7L was restored compared to control cells, as observed after miR-24 suppression. The hypothesis suggested that ST7L decreases cell proliferation and increased apoptosis. The tumor suppressor effects of ST7L were in part mediated by negative regulation of bcatenin/Tcf4 signaling [START_REF] Chen | MiR-24 regulates the proliferation and invasion of glioma by ST7L via β-catenin/Tcf-4 signaling[END_REF].

Murine erythroleukemia (MEL) cells were used to study erythroid differentiation due to their ability of in vitro terminal differentiation in response to chemical induction. ST7L was identified among several genes that could be used as markers for erythropoiesis with the use of semi-quantitative qPCR. They suggested that these genetic markers could act as potential regulators in cellular functional studies of erythroid differentiation, or studied as straightforward cell type marker. [START_REF] Heo | Microarray profiling of genes differentially expressed during erythroid differentiation of murine erythroleukemia cells[END_REF] The onset of the disease in family FM3 was just after birth and in one individual the onset was suggested to be prenatal implying a defect in development occurring in the proliferating neuronal cell lines. Many neurological disorders especially leukodystrophies have been associated with haemopoeitic cancer. In some (adrenoleukodystrophy) stem cell therapy may help to improve even the neurological symptoms and signs.

In the absence of other variants or rearrangement, we can reasonably consider the ST7L variant as causative in the family but further biological elements are required to firmly validate its involvement in the pathology and decipher the underlying mechanism.

PANK4 unique variant in branch1 of family FM7

In branch 1 of family FM7, the single rare and pathogenic variant remaining after bioinformatics filtering was found in PANK4. The variant did not segregate in branch 2 of family FM7 and thus we hypothesize that it was not involved in causation of the disease in this branch. Although both branches had ataxia and hearing defect and mutism, we could distinguish them as two different phenotypes. As mentioned earlier this is not the only example of extended family in which patients from different branches present with closely related clinic-genetic entities. In family F1 spasticity and ataxia were shared clinical features in all four branches (A, B, C and D). SPG11 was found to cause the disease in patients of branch A/B with TCC HSP but not in the relatives branch C and D for whom the natural history was different (Elsayed et al., under revision).

 Gene/protein isoforms and splice variants

This gene has 12 transcripts (splice variants) but only two are protein coding.

NM_018216 is the transcript that generate the canonical isoform1 (identifier: Q9NVE7-1) which is composed of 19 exons and is 773 amino acid long and 85,991 in Mass (Da).

PanK4 belongs to the type II pantothenate kinase family. Unfortunately no crystal structure is available.

 Pantothenate kinases expression and function

There are four isoforms of pantothenate kinase: PanK1, PanK2, PanK3 and PanK4 [START_REF] Li | High glucose upregulates pantothenate kinase 4 (PanK4) and thus affects M2-type pyruvate kinase (Pkm2)[END_REF]. include the tricarboxylic acid cycle (TCA), biosynthesis of sterol, metabolism of amino acid, and both anabolic and catabolic pathways of fatty acids and complex lipids [START_REF] Leonardi | Coenzyme A: back in action[END_REF].

Although putative PanK4 was claimed to be catalytically inactive [START_REF] Zhang | Chemical knockout of pantothenate kinase reveals the metabolic and genetic program responsible for hepatic coenzyme A homeostasis[END_REF], evidence is progressively accumulating for its in vivo activity. [START_REF] Zhou | A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome[END_REF] PanK4 has been shown to be upregulated in glucose-challenged rat muscles suggesting that PanK4 expression is stimulated to allow flux at a higher rate through the TCA [START_REF] Li | High glucose upregulates pantothenate kinase 4 (PanK4) and thus affects M2-type pyruvate kinase (Pkm2)[END_REF].

The role that carbohydate metabolism plays in normal brain development and proper function can't be ignored. This has been extensively studied over decades.

Glucose is an essential energy source for the adult human brain. In the adult brain, neurons have the highest energy requirements [START_REF] Dienel | Fueling and imaging brain activation[END_REF]. Neurons as well as cancer cells are among the cell types that depend on glucose metabolism almost completely for energy generation.

Tight regulation of glucose metabolism is essential for brain physiology.

Disruption of normal glucose metabolism and its correlation with cell death pathways forms the basis for many brain disorders. Not only glucose is required for provision of the ATP as energy source for their actions and for production of the precursors for neurotransmitter synthesis but also to satisfy the brain's energy demands. Synaptic activity accounts for most of the energy consumption of the brain [START_REF] Mergenthaler | Sugar for the brain: the role of glucose in physiological and pathological brain function[END_REF]. The human cortex alone requires approximately 3×1023 ATP /s/m3 [START_REF] Dienel | Fueling and imaging brain activation[END_REF].

Aerobic glycolysis (AG) was shown to support developmental processes, and especially important for synapse growth and formation. Meta-analysis of brain glucose and oxygen metabolism studies demonstrated that AG increases during childhood, corresponding to the highest synaptic growth. High AG, was found to correlate with increased expression of genes related to formation and growth of synapses. In contrast, regions with elevated oxidative glucose metabolism showed high expression of genes more related to mitochondria and synaptic transmission [START_REF] Goyal | Aerobic glycolysis in the human brain is associated with development and neotenous gene expression[END_REF] Interestingly, paralog PANK2 is incriminated in Hallervorden-Spatz syndrome (HSS)/ pantothenate kinase associated neurodegeneration (PKAN). HSS is an AR neurodegenerative disorder associated with brain iron accumulation. Histologic studies for HSS reveals iron accumulation in basal ganglia via secondary metabolite accumulation. The differential expression of PanK genes discussed earlier may explain why PANK2 causes HSS.

In family FM7 branch 1, the affected sibs presented with developmental defect and age at onset since birth. We therefore suggest a different mechanism behind the pathogenesis of the disorder. PanK4 has been associated with glucose metabolism in muscles in addition to its role partially compensating PanK2 deficiency. In all cases

Pank4 is highly unexplored and a lot of functional work may be required to prove this hypothetical role in development and to explain the variable clinical severity witnessed in our five affected siblings. Zebrafish modeling may represent a good opportunity to explore the normal and pathological functions at the same time.

Cellular pathogenic mechanisms proposed for the novel genes: a resumé

In the following paragraph we will highlight the pathways proposed earlier for the above mentioned novel genes.

Microtubules/cytoskeletal related cellular mechanisms

Microtubules is a major component of the cytoskeleton that support the subcellular organization of organelles especially Golgi apparatus. Disruption of the organization and dynamics of the microtubules is the proposed pathogenetic mechanism for CAMSAP3.

BIRC5 is proposed to cause the disease through antiapoptotic, promitotic and DNA repair associated with microtubules formation and dynamics.

MINK1 has been associated with organization of cell cytoskeleton and motility.

Neurite outgrowth

This mechanism is the major process for neuronal cell formation and development of CNS. It is involved in a family while a mutation in ABHD16A together with possible effects on neuroinflammatory response.

Glucose metabolism

Glucose metabolism and defect in synthesis of CoA is suggested as the mechanism behind PanK4 despite the lack of sufficient studies. Its paralog, Pank2 is responsible for another neurodegenerative condition.

Apoptosis and regulation of cell growth

Minimal data and studies are available about ST7L. The only evidence designates ST7L as proapoptotic and negative regulator of cell growth (decreases cell proliferation).

Cytokinesis

MINK1 has been shown to plays a crucial role in many cellular processes including cytokinesis, a process overlapping microtubules/cytoskeletal dynamics.

A comprehensive overview of pathogenetic Mechanisms of HSP

The rapidly expanding surge of discovery of new genes revealed that we can just see the top of the iceberg and that we are lagging far beyond the appreciation of the full picture that links the disorders along the spectrum of the neurogenetic disorders and their mechanisms. As mentioned earlier multiple trials have been done to regroup and create categories of the functions attributed to HSP genes and their protein products. This is always not an easy task as HSP genes/proteins have been implicated in a wide variety of functions and what is unknown seems to exceed what we managed to disclose. In this section an attempt is done to create a modified version of functional categorization [Figure [START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF]] [Table ].

Around 11 major functional themes can be distinguished based on the known ultimate role HSP genes play/modify in the cell. Most of the HSP genes/proteins participate in 1 to 3 functional themes ]. An overall primary and a secondary (when relevant) functional category has been assigned to each HSP protein to summarize the principal functional theme of the SPG gene/protein [Table ]. As the phenotypic features has been almost exhaustively listed (Annexe Tables 1234567891011), a step further has been taken to observe the possible links between the pathways and the clinical presentation (pure versus complex), signs, age at onset and patterns of inheritance. This has been done using descriptive observations with basic statistical regression analysis to create provisional correlations. In this regression, the functional themes have been considered as the independent variables (X) and the phenotypes and signs have been treated as the dependent variables (Y). The statistical significance is set as P-value < 0.05. It is quite clear that this analysis will just provide basic crude correlations but it can be regarded as the first step to deduce preliminary clues to guide a second more specialized analysis with more functional data (cell types where the gene is expressed, putative function based on partners of these proteins…) which if accompanied with better fine tuning of the data analysed and may lead to more conclusive results. A problem encountered is the small number of forms included in certain functional categories. This point of weakness may suggest that some biased conclusions may occur from a statistical point of view.

General Observations:

 Age at onset:

With the exception of the tendency for early onset associated with lipid and nucleotide metabolism, age at onset is not obviously linked to any functional category. This might well be due to the role of all HSP genes during the development of the nervous system.

 Mode of inheritance:

-AR forms are observed to predominate the metabolic pathways (lipids and nucleotide metabolism) as well as degradation pathways and endomembrane trafficking.

-AD HSP are mainly clustering in the organelle shaping, development, microtubule dynamics and active cellular transport (often with mixed transmission) functional categories. Significant correlation exists ] between microtubules dynamics and three other functions: the development, organellar shaping and transport. This can be well explained by the role that the microtubules play in the neurons with regard to these functions:

1. MTs keep the organellar organization and thus maintain the shape of the subcellular organelles [START_REF] Zheng | Marshalin, a microtubule minus-end binding protein, regulates cytoskeletal structure in the organ of Corti[END_REF] 2. MTs play an essential role in neuronal development through various models that have been suggested including the role of MTs in polarized axonal transport [START_REF] Faivre | The microtubular apparatus of cerebellar purkinje cell dendrites during postnatal development of the rat: The density and cold-stability of microtu[END_REF] [START_REF] Dent | Reorganization and movement of microtubules in axonal growth cones and developing interstitial branches[END_REF] [START_REF] Brown | Slow axonal transport: stop and go traffic in the axon[END_REF] ( [START_REF] Nakata | Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head[END_REF]) [START_REF] Witte | Microtubule stabilization specifies initial neuronal polarization[END_REF] [START_REF] Hoogenraad | Control of neuronal polarity and plasticity--a renaissance for microtubules?[END_REF]. In addition, MTs contribute to the neuronal and synaptic plasticity in the process of memory and learning [START_REF] Penazzi | Chapter Three -Microtubule Dynamics in Neuronal Development, Plasticity, and Neurodegeneration[END_REF] On a clinical point of view, microtubule dynamics shows significant correlation with PN and deafness. The role of microtubules in AD HSP (SPG4, SPG10 and SPG31), which are mainly complicated with PN, can explain this association. The correlation of MT dynamics and hearing loss has been described in many studies about deafness and its genetic causes [START_REF] Hasson | Unconventional myosins, the basis for deafness in mouse and man[END_REF] Deafness, on the other hand, is significantly correlated with cataract, although linked to amino acid metabolism and organelle shaping but not to microtubule dynamics. As cataract is a common clinical feature in IEM, this makes the link between amino acid and metabolism logical.

Transport and microtubule dynamics:

Cellular transport is linked to the pure phenotype. This may well be because of the predominance of the AD forms where the pure presentation is the commonest.

Endomembrane trafficking:

Significant links can be observed between CI and other phenotypes including TCC, PN, cerebellar ataxia as well as with the complex presentation per se. This observation is expected given the fact that these are the commonest complicating signs in HSP as mentioned earlier in the introduction (Annex 1-11). Interestingly, CI has shown significant correlation with the endomembrane trafficking.

Endomembrane trafficking is associated with 14 HSP forms. all with a complex presentation with five forms rarely presenting as pure HSP (SPG11, SPG15, SPG48, SPG6 and SPG8). Moreover, although both AD and AR inheritance can be observed in this category, the AR HSPs are predominant (11/14 forms).

There is obvious overlap between the degradation pathway and the endomembrane trafficking. This overlap occurs in five HSP forms [SPG11, SPG15, SPG48/AP5Z1, SPG59/USP8 and VCP spastic paraplegia].

Degradation pathways:

Of the 10 forms associated with degradation pathway, one form presents as pure HSP and nine are complex (three of them has an alternative pure phenotype). The majority had AR inheritance (9/10) and present cerebellar ataxia (6/10).

The association between cerebellar syndrome and the degradation pathways is well known in the pathogenesis of AD cerebellar ataxias. Degradation of cellular inclusion is the basis of many types of HAs especially the polyglutamine AD cerebellar ataxias (PolyQ: SCA). In these disorders, the proteasomal and lysosomal degradation pathways are the main cellular defense mechanisms against the aggregates (the hallmark of the polyQ disorders) [START_REF] Seidel | Polyglutamine aggregation in Huntington's disease and spinocerebellar ataxia type 3: similar mechanisms in aggregate formation[END_REF]) [START_REF] Mori | G protein-coupled receptor 26 immunoreactivity in intranuclear inclusions associated with polyglutamine and intranuclear inclusion body diseases[END_REF] 4.6.2.5. Mitochondrial functions:

Out of 12 forms associated with mitochondrial functions, 11 have complex, with four having additional pure phenotypes.

There is no apparent tendency for association with certain age at onset or inheritance pattern. But there is statistically significant association with optic atrophy (p value : 0.007) and PN with amytrophy. This apparently matches what is known about the association in many mitochondrial inherited disorders between mitochondrial dysfunction and optic atrophy.

Lipid metabolism influence on myelination:

An obvious but interesting statistically significant observation with well known theoretical background is the effect of lipid metabolism on myelin. Such observations that emerged upon analysis would give a positive control to give the remaining links some sort of confidence.

"Take home" message:

Not all functions of the HSP proteins are known. Based on the state of the art in 2016, it is clear that:

 Age at onset is not discriminant of altered functions except for lipid and nucleotide metabolism, probably because of need during development.

 There is not a clear clustering of functions according to inherence with the exception of enzymatic functions in AR HSP. by the big number of genes discovered through international collaborations between the highly privileged teams with vast neurogenetic facilities and teams/specialists from developing countries. An easy working frame for neurogenetic disorders will increase the chance of even faster discovery rates for genes, better understanding of the physiopathological background and emergence of new phenotypes and will ensure at the same time that proper science will be provided the thing that will have great repercussions on our understanding of these disorders.

Cohort building

The first step that was required to start the study was the cohort building. This comprises the building of a network of specialists, a referral system (as illustrated earlier), a trained neurogenetic team and optimized clinical examination/sampling method. All these steps required hard work and keen determination to be achieved and to build the first neurogenetic team in Sudan and to continue despite all the challenges in a field that was previously unexplored.

Continuous Optimization of the Phenotyping Process:

1. Questionnaire:

As mentioned earlier we had to adapt the comprehensive standard questionnaire of the SPATAX network (https://spatax.wordpress.com/) to our local needs indicated by the nature of our cohort.

Video Documentation:

With the help of the small team that I created in Sudan, we managed to optimize the video documentation system to create video archiving system for the major and minor clinical signs that we encountered since the start of our recruitment. This was especially important for diagnosis establishment by the second viewer in order to eliminate alternate disorders. This had many advantages including increasing our expertise and training of the new members that joined our team to warrantee that they would be more efficient and accurate when they perform the clinical assessment.

i. Neuroradiology:

With the help of three neuroradiology teams and departments, we managed to set a new magnetic resonance imaging allowing storing them in databases. We also obtained a set of free MRIs to be dedicated to poor patients as a part of collaboration between our team representing the university of Khartoum/ faculty of medicine and the radiology departments of two governmental hospitals: Alamal Hospital, Police hospital (check the list of collaborations above). We also managed to provide means of transportation for several patients far from medical centers. These two steps allowed us to partially solve the two main problems that we encountered in MRI: unavailability due to financial inability and the badly conserved or lost films.

o Unsolved part of the problem of MRI:

1. We had to accept films in patients who already did the MRI and were reluctant to repeat them because of many reasons including:

-Logistic difficulties as lack of time for families living in the peripheral regions in Sudan and came to the capital for short time only -Fear of anesthesia in kids and mentally subnormal patients who could not stay still during the imaging process.

2. Anesthesia staff unavailable in certain imaging centers.

Patients far from equipped hospitals and unable to travel

ii. Other Fields:

The collaboration with the ophthalmology and neurophysiology departments allowed us to establish a steady referral system of our patients to three of the most advanced centers in our settings for free specialized assessment.

Diagnostic Capacities

Most importantly was building our clinical judgment capacity and the improvement of our diagnostic approach. As I was in charge of the final analysis of the clinical assessment this was a burden especially that most of our cohort was pediatric and needed special training considering my background training of adult neurology. Even in adult cases the neurogenetic disorders needed vast knowledge and expertise in that branch of neurology. The pediatric team in Sudan as well as the clinical neurogeneticist/ leukodystrophy (neuropediatrics) experts in France provided me with invaluable expertise.

Huge work / One pair of hands/ Time

The time factor was rather stressing as a lot of tasks had to be done in short time.

Almost half of the study time was dedicated to the cohort recruitment and organization of the work flow to make it smoother. In the remaining half, all laboratory work from the DNA extraction to the bioinformatic analysis and confirmation of results as well as some functional workup had to be achieved. As we needed to do functional studies, skin biopsy system had to be started and this needed training of our team, optimization and coordination with the DNA bank in

France in order to be launched successfully. The formation of the team in Sudan and the teaming with cooperative colleagues in France especially towards the end of the study had the best effect on the yield. This reinforces the notion that the best results are achieved via team work.

Budget management

As the case with every research project finance was an obstacle.

Sudan

It was a challenge to provide budget for the research cost in Sudan. This included the expenses for sampling sessions (gloves, sterilizers, needles .. etc) as well as providing travel/transportation cost to the poor families to the sampling center [and to the team members in case the reverse (home visits) occurs] in the peripheral regions of the country. It also included the sample shipment and custom cost. It included as well the cost for investigations for the poor noninsured patients (Imaging, electrophysiological studies and specialist consultation (ophthalmologists, audiologists). While the latter could be partially covered by building collaborations, the former had to be provided from the researcher personal wages and familial support.

France

There was no specific finance for the French/Sudanese collaborative project so it was an extra-burden for both the researcher and staff. To overcome the restricted budget for WES, a serious decision had to be taken for AR families. That was to do one WES per family or per family branch in case of extended families. This strategy worked quite well since we looked for homozygous variants manly. In AD families we had to do WES for two affected individuals. Families had to be prioritized for WES based on predicted yield. WES was thus not performed in sporadic families.

WES analysis

In addition to the difficulties that are usually encountered in analyzing WES data for prioritization of variants and genes, WES data analysis had to be adapted to the particular nature of the Sudanese families. One of the most important criterion that had to be considered is the probability of occurrence of two clinical entities in the same families. This probability increases when analyzing patients belonging to two branches of the same family or even when analyzing data of affected siblings. In fact, rather rarely that we find a causative mutation segregating in more than one branch of our extended families with more than one branch despite the closely related phenotypic presentation.

Administrative processes

This thesis was the first joint project between University of Khartoum (UofK)

and the University of Paris 6 (UPMC). Moreover, it was the first joint PhD approved by the senate of the UofK. This conferred certain difficulty to the PhD administrative process. However, this thesis demonstrates the feasibility of such European-African collaborative works and opens the way for increase collaborations and exchange of researchers in the near future.

Conclusion and prospective work

HSP in Sudan is a rare condition. We are far from the full characterization of all cases but considering that we collected 106 patients in an area of 30 million inhabitants, the minimal prevalence can be established as ≈ 0.4/100000 (≈ 0.3/100000 if families with pyramidal syndrome complicating PD, SCA7 are excluded). These roughly calculated figures although won't reflect the true epidemiological prevalence but they are in accordance with what has been reported about the prevalence of HSP in other countries (even more compatible with the reports of AR HSP being almost all AR HSP) (Ruano et al., 2014) 41% of Sudanese families were solved through gene screening, a frequency also similar to other studies of the host laboratory on European cases. In the case of F15, the identification of the causative gene will be of interest for better follow-up and therapy (Arginase 1 deficiency)

Interestingly, following WES, I have potentially identified the causative gene in two families for which the effects of multiple variants need to be distinguished. The screening of additional cases of the SPATAX cohort to add new mutations and therefore genetic arguments, and the modeling in Zebrafish by knock down, including rescue experiments, will be crucial steps before definite conclusions. The study of fibroblasts of patients may also reveal cytological abnormalities that could help to define the causative genes among the candidates. In three other families, a single variant with convincing evidence of pathogenicity in at least two (one is a paralog of another gene involved in neurodegeneration, one is a stop mutation), were identified. Similar experiments are in progress to determine the frequency of these new genetic entities and the functional consequences in vitro.

Finally, WES is to be performed to the remaining families in which known genes have been excluded. There are also two families still under WES analysis, potentially with new causative genes to come. In case of negative results in WES, a genome analysis will be considered first to analyze the exome data better covered in that case than in WES, and intronic/structural regions.

The need for new nosology: time has come.

As illustrated in the introduction and discussion, the spasticity world is huge and overlaps with many disorders. Many genes with HSP gene name and number are more frequently accounting for other diseases when mutated and the reverse is true. My results also highlight this fact with HSP patients found mutated in genes mutated in classical HSP families and not fulfilling perfectly the typical HSP presentation and the other way around. A new nosology may emerge in the near future thanks to WES data. Figure [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF] Correlation between the modes of inheritance and the clinical phenotype (pure or complex).

6

Figure [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF] Regrouping of the age at onset of various HSP subtypes. 8

Figure [START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF] Schematic illustration of the phenomenon of genes presenting with closely related allelic phenotypes throughout the spectrum of the neurodegenerative disorders. Brain MRI abnormal signs encountered in the cohort. Figure Pedigree of family F21.

Figure Pedigree of family F14.

Figure [START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF] Pedigree of family F16.

Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF] Clinical data of two patients from family FM7 branch2

Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF] Clinical summary of 23 families with unidentified genetic cause (family 27 is presented in a separate table ).

Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF][START_REF] Novarino | Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders[END_REF] Clinical summary of family F27 with unidentified genetic cause Table (4-1) Detailed clinical signs complicating the 41 families included in the cohort.

Table Table summarizing the 88 HSP clinico-genetic entities with special focus on the functions of their proteins and our suggested primary and secondary functional categories.

Paraplégies spastiques héréditaires : exploration clinique au Soudan, études des origines moléculaires des formes autosomiques récessives et identification de nouveaux gènes en cause.

Résumé :

Les paraplégies spastiques héréditaires (PSH) font partie d'un groupe plus large de pathologies neurodégénératives associant une spasticité. J'ai exploré la variabilité clinique et moléculaire de ces pathologies à l'aide d'une cohorte de familles soudanaises.

Nous avons recruté 41 familles soudanaises [337 individus/106 atteints de PSH]. J'ai extrait l'ADN génomique et constituer une banque. Le criblage de gènes candidats a été réalisé dans 4 familles en fonction du phénotype des patients. La technologie de séquençage de nouvelle génération (SNG) appliquée à 74 gènes de PSH a ensuite été appliquée aux 37 cas restants. Enfin, le séquençage de l'exome a permis de rechercher les gènes en cause dans les cas négatifs. Dans certains cas, des études fonctionnelles ont été utilisées afin de valider l'effet biologique des mutations. J'ai pu identifier la cause génétique dans 17 familles. Pour 12 familles, la mutation concernait un gène de PSH connu. Dans 3 familles, un nouveau gène a pu être incriminé tandis que dans 2 autres, 5 gènes candidats restent à départager. Il est à noter que parfois, de multiple mutations ou maladies génétiques ségrégaient dans nos familles soit dans la même ou dans des branches séparées. La complexité de ces familles fortement consanguines a rendu l'analyse des données du SNG difficile. Une autre particularité a été l'hétérogénéité clinique associée à des mutations du même gène entre patients de la même famille ou en comparaison avec la littérature.

Ce travail est la première étude à grande échelle de patients soudanais avec PSH et rapporte de nouveaux gènes en cause, prérequis pour mieux comprendre dans le futur les mécanismes sous-jacents.

Mots clés : [paraplégies spastiques héréditaires; famille Soudanaise ; consanguinité ; séquençage de nouvelle génération ; le séquençage de l'exome ; nouveaux gènes]

(

  SPG13), FA2H (SPG35), GJC2 (SPG44), RNASEH2B, ADAR1, IFIH1]. The same applies for juvenile Parkinson disease [SPG11], HA [SPG7, SPG39], spastic ataxia [SPG39, SPG46, SPG58] and epilepsy [MT-ATP6]. These observations following the explosion of results from NGS techniques suggest that clinico-genetic classifications are imperfect. Along the same line, recently characterized novel clinico-genetic forms implicating lipidic or amino acid metabolism now raises a question of whether or not metabolic spastic disorders (previously IX classified as non HSP conditions [eg. ARG1] should now be reclassified as HSP or if a larger rebuilt of the nosology of neurological conditions has to be performed.

Figure 1 - 6 :

 16 Figure 1-6: Schematic illustration of the phenomenon of genes presenting with closely related allelic phenotypes throughout the spectrum of the neurodegenerative disorders.

Figure 1 - 7 :

 17 Figure 1-7: Figure showing the overlap zone between spastic neurogenetic disorders: evidence for the need of new nosology and objective case definition. HSP: hereditary spastic

  advanced substantially in the late 2000s. The latter includes all genetic-manipulationbased techniques (cell lines, animal models), protein modeling, biochemical techniques, electrophysiological cellular studies, cellular and organ imaging and counting. This is illustrated by the immense knowledge gained about HSP and other spastic neurogenetic disorders in recent years.Several cellular pathways have been associated with HSP. Multiple attempts have been done to regroup these pathways according to their shared functional role[START_REF] Blackstone | Cellular Pathways of Hereditary Spastic Paraplegia *[END_REF] (Noreau et al. in 2014). Blackstone has identified seven principal pathogenic themes including axon-path finding, myelination, endoplasmic reticulum network morphology, lipid synthesis and metabolism, endosomal dynamics, motor based transport and mitochondrial functions defects, in addition to three minor themes [anterograde transport, Golgi apparatus and microtubules related functions] (Blackstone, 2012) [Figure (1-8)]. Noreau et al. later have adopted slightly different functional localization themes including principally morphogenesis of endoplasmic reticulum (ER), disturbances of lipid metabolism (fatty acid, cholesterol and phospholipid), endosomal trafficking and mitochondrial regulation abnormalities in addition to five more categories of DNA repair, gap junctions, autophagy and lysosomal metabolism and myelination

  Variable disability scores are also used for assessment of the patient's disability stage. Additional radiological, electrophysiological and laboratory testing will give further evidence on the nature of the disease and help to exclude important differential diagnoses. The principal radiological tests used are magnetic resonance imaging (MRI) of the brain and spinal cord. While MRI of the spine does not show significant abnormalities, the MRI of the brain (plus or minus CT scan of the brain) can give essential information that can aid in exclusion of many of the differential diagnoses (eg. leukodystrophies and leukoencephalopathies, NBIA, MND/ALS, infectious and immune causes) and give further clue to the category of HSP (eg. TCC-HSP) and probable genes beyond it (eg. SPG11 is the most probable gene in TCC HSP). Electrophysiological testing includes electromyogram/ nerve conduction studies (EMG/NCS), evoked potentials: somatosensory evoked potential (SEP) and other relevant evoked potentials as determined by clinical presentation of individual cases [visual evoked potential (VEP) and auditory evoked potential (AEP)].

Figure ( 1 - 8 ):

 18 Figure (1-8):Common pathogenic themes in the HSPs[START_REF] Blackstone | Cellular Pathways of Hereditary Spastic Paraplegia *[END_REF].

Figure ( 1 - 9 ):

 19 Figure[START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF]: Illustration of the pathogenic mechanisms in hereditary spastic paraplegia (HSP) regrouped in nine functional and cellular localization categories(Noreau et al., 2014)

  The Qubit® fluorometer is a ThermoFisher SCIENTIFIC Life Technologies Invitrogen® product (Carlsbad, California, USA) that was used to measure dsDNA precise concentration. It offered two types of assays; the Qubit dsDNA HS (for High Sensitivity) Assay and the Qubit ®dsDNA BR (for Broad Range) Assay. The quantification method uses a fluorescent dye (Qubit reagent) which binds specifically to dsDNA and not to ssDNA. The intensity of the resulting fluorescence is directly proportionate to the quantity of dsDNA. The assessment of sample concentration is based on plotting the relationship between the two dsDNA Standards used in calibration (standard #1 at 0 µg/mL and standard #2 at 100 µg/mL). The Qubit® fluorometer produced final concentration values in either µg/mL (ng/µL). This concentration corresponded to the concentration calculated from the Qubit® fluorometer value (QF) and the DNA dilution factor we used (practically 1/200). It used the equation: Concentration of your sample = QF value × (200)/X where X is the number of microliters of sample we added to the assay tube.  Assay protocol was provided by Invitrogen® as summarized below: i. A working solution is prepared from 199µL Qubit® buffer per sample/standard plus 1 µL Qubit® reagent per sample/standard. ii. The working solution is then distributed into special Qubit® tubes provided from the manufacturer (190 µL/standard tube and 199 µL/sample ( 180-199 µL based on the dilution factor chosen) ) iii. 10µL of Standard #1 and Standard #2 are added and 1µL (1-20µL optional) of each to their corresponding tubes vortexed slightly incubated for two minutes. iv. Measurement is then done by choosing the specific assay (HS vs BR), calibration using Standard #1 then #2. Samples measurement is done by following a wizard indicated by the device.

5 .

 5 Hybridization to BeadChip (Post-Amp): At this step the samples are applied to a BeadChip and separated by an IntelliHyb seal (or gasket). The amplified and fragmented DNA samples anneal (hybridized) to locus-specific 50-mers [in contrast to the traditional oligomers (~20-mers) used in PCR]. The loaded BeadChip is incubated in the Illumina Hybridization Oven for an overnight.

  washed and dried flow cell and the PR2 bottle are loaded into the Miseq machine and the sequencing process is launched after assuring that the waste bottle is empty. Illumina sequencing is based on the concept of bridge amplification of the captured DNA fragments from the denatured library [Figure (2-3)]. The DNA fragments bind to the surface of the flow cell through annealing of the P5 and P7 sequences of the adapters to complementary sequences on the flow cell. The bridge amplification technique is considered a solid phase sequencing method. DNA polymerase then produces clusters of 1 million copies of clonal DNA fragments originating from each single DNA molecule. As sequencing is carried out from both end it is called a paired end sequencing and the reads obtained from each end are called R1 and R2. The index sequence of each adapter allows the machine to affiliate each sequence to the correct sample. In principle, Illumina sequencing technology is sequencing by synthesis (SBS). In this technology, a fluorescently labeled reversible terminator is imaged as each dNTP is added, and then cleaved to allow incorporation of the next base. Since all 4 reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias.

Figure ( 2 - 1 )

 21 Figure (2-1): Sequence Capture Protocol: Basic illustration of the protocol for library preparation for next generation sequencing (NGS) using capture enrichment technique

Figure ( 2 - 3 ):

 23 Figure (2-3): Illustration of the bridge amplification sequencing technique (Illumina Sequencig technology) (http://nextgen.mgh.harvard.edu/IlluminaChemistry.html)

  . Multiplexing in one tube: They are mixed together at equal amounts (by mass) of each of the amplified DNA sample libraries to obtain a single pool with a combined mass of at least 1.2 μg ("Multiplex DNA Sample Library Pool"). 1 μg of the multiplex pool will be used in the sequence capture hybridization step. The remaining 250 ng of the Multiplex Pool was stored at -15 to -25°C. iv. Hybridization of the biotinylated specific target probes (oligos that are complementary to the sequencing primer site inside the paired end adapter sequence of Kit A and B). After addition of the Multiplex Hybridization Enhancing Oligo pool the multiplex DNA samples are dried using DNA vacuum concentrater then resuspended using a mix of the hybridization component A and hybridization buffer. The sample for 10 min at 95° is incubated in a heat block to allow denaturation of the double stranded DNA multiplex. Then transferred the denatured sample to an aliquot of COT Human (contained in the SeqCap EZ® Accessory Kit v2).

Figure ( 2 - 4 ):

 24 Figure (2-4): Nextseq 500 emission.

5 . 7 . 2 . 11 . 2 . 3 .

 5721123 Day 2 : 0.5 mL of medium is added 6. Day 8/9: the fibroblast are observed under microscope and culture medium is changed Day 10: the flask are observed daily and trypsanization done when the fibroblast are dense and confluent (passages are started) Passages and Trypsanization: Trypsanization and new passages is done when fibroblasts are condensed to avoid cellular death due to accumulation of waste products and change in pH. The protocol is the same for all passage and the only unique criterion of the first trypsanization is that the fibroblasts are transterred from T25 to the larger T75 flask and all passages that follow are into T75 flasks. Summary of trypsanization protocol: 1. Culture medium is eliminated and the cellular layer is washed by 3 mL of Dulbecco's phosphate buffered saline (DPBS) ) (ThermoFisher SCIENTIFIC Life Technologies Invitrogen® product (Carlsbad, California, USA)) 2. 2 mL of Trypsin EDTA are added and the flask is incubated in 37 degrees for 3-5 min (to detach the cells from the walls of the flask.3. The trypsin is inactivated by addition of 8 mL of culture medium and the suspension is homogenized and 2 mL is transferred into a new T75 (labeled a new passage number: n+1) flask and 8mL of medium are added.

Figure ( 3 - 3 ):

 33 Figure (3-3): Patterns of inheritance in 41 families.

Figure ( 3 - 5 ):

 35 Figure (3-5): Distribution of age at onset in 41 extended families (44 nuclear families: 3 branches were treated as separate families).

3. 2 . 1 . 1 .

 211 Identification of a case (sporadic family) with Parkinson disease associated with pyramidal signs due to DNAJC6 mutation a) Summary: Because of the specific association of pyramidal signs (deep tendon reflexes, spasticity) in lower limbs and Parkinson's disease (PD) in a sporadic patient in family FM4, we screened nine autosomal recessive (AR) PD genes (PARK2, PINK1, DJ-1/PARK7, ATP13A2, PLA2G6, SYNJ1, DNAJC6, FBXO7, VPS13C) using a targeted next generation sequencing gene panel. The technique utilized a specific capture enrichment strategy (Roche NimbleGen®/SeqCap Ez®, USA) followed by massive parallel sequencing using the MiSeq platform (Illumina®, San Diego, USA). A nonsense mutation in DNAJC6 (exon 16: c.2365C>T, p.(Gln789*)) was found in a homozygous state in the patient, representing the sixth family so far reported. b) Manuscript o Article 1: Elsayed LE, Drouet V, Usenko T, Mohammed IN, Hamed AA, Elseed MA, Salih MA, Koko ME, Mohamed AY Siddig RA, Elbashir MI, Ibrahim ME, Durr A, Stevanin G, Lesage S, Ahmed AE, Brice A. A novel nonsense mutation in DNAJC6 expands the phenotype of autosomal recessive juvenile-onset Parkinson disease. Ann Neurol 2016 (advance online 2015 dec 24).

  variant was found shared by patients from all three branches, remained after filtering out the common and non-coding variants. This was a novel missense variant in the ARG1 gene. The mutation [Exon 4: g. 131902487T>A, c.434T>A, p.(Val145Glu)] co-segregated with the disease distribution [Figure (3-6)]. It was a transversion variation of a highly conserved nucleotide. The mutation was in a homozygous state in five patients from three branches of this extended family. All other seven sampled healthy related individuals were homozygous reference or heterozygous carriers.This mutation was predicted pathogenic by four algorithms [SIFT, Polyphen2, mutation taster and align GVGD] [Table[START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF]]. The variation resulted in a moderate physiochemical difference [Grantham distance of 121 (0-215)] from the highly conserved (12 species) hydrophobic amino acid valine to the acidic amino acid glutamate. The variant was located in the protein domains: Ureohydrolase, Arginase subgroup.Arginases(EC 3.5.3.1) are proteins involved in last reaction of the urea cycle in which they catalyze the hydrolysis reaction of L-Arginine to form the urea and ornithine. There are two forms of the arginase enzyme. Arginase 1 which predominates in the liver whereas arginase 2 is the main arginase in the kidneys.

Figure ( 3 - 6 ):

 36 Figure (3-6): Pedigree of family F15 caused by missense mutation in ARG1 segregating with the disease distribution in whole family presenting with spastic tetraplegia and mental retardation. Whole exome sequencing was performed for five affected individuals (138, 142, 143, 148, and 149) and the mother (137) of patient 138. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; yellow square indicates the two index patients; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

(

  Figure (3-7): Pedigree and MRI of the index patient of family F25 caused by splice donor mutation in PLA2G6 segregating with the disease distribution in whole family presenting with pyramidal signs and features associated with infantile neuroaxonal dystrophy (INAD). MRI shows no evidence of brain iron accumulation but neurodegeneration with white matter hyperintensities (WMH) in periventricular areas, basal ganglia including globus pallidus and to lesser degree head of caudate nucleus and Putamen. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

  )]. It resulted in a small physiochemical change [Grantham distance of 64 (0-215)] of the highly conserved hydrophobic amino acid alanine (12 species) (located in the protein domains: Armadillo-type fold) to another hydrophobic amino acid valine. The clinical presentation of the patients was in accordance with what had been reported in the majority of Marinesco-Sjogren syndrome (MSS) cases due to mutations in the SIL1 gene (*608005). Marinesco-Sjogren syndrome (OMIM # 248800) is an AR disorder characterized by cerebellar ataxia, progressive myopathy, and cataract (Anttonen et al., 2005). Hypogonadism had been described

Figure ( 3 - 8 ):

 38 Figure (3-8): Pedigree and MRI of the index patient of family F30 caused by missense mutation in SIL1 segregating with the disease distribution in whole family presenting with pyramidal signs associated with Marinesco Sjogren Syndrome (MSS). MRI shows marked cerebellar atrophy. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: sampled individual are numbered; Phenotype symbols: black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

Figure ( 3 - 9 ):

 39 Figure (3-9): Pedigree and colour image of the retina of the index patient of family F26 caused by pathogenic CAG repeat in ATXN7 family presenting with pyramidal signs associated with spinocerebellar ataxia type 7 (SCA7). Colour image of the retina shows macular degeneration and pale optic disc. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Others are standard medical pedigree symbols.

  Figure (3-10): Screening Panel success rate in classical HSP (2 3 famil ie s): numb er s and p er centag es are b ased o n fami li e s .

3 - 4 -

 34 Delayed psychomotor development followed by further regression of milestones Pyramidal signs (LL: spasticity, hyperreflexia and bilateral extensor plantar response) 5-Mental retardation 6-Sterotypic behavior (clapping and head slapping) 7-Extrapyramidal signs (choreoathetotic movement)

Figure ( 3 -

 3 Figure (3-12): Pedigree and MRI of the index patient of family F37 with a nonsense mutation in ABHD16A segregating with the disease distribution in the whole family. MRI shows thin corpus callosum (TCC). Example sanger sequencing of the mutation in patient (homozygous mutant) and in a healthy heterozygous carrier. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: M+ Heterozygous genotype; MM Homozygous mutant genotype.Others are standard medical pedigree symbols.

2 -

 2 Age at onset: birth 3-Trunkal hypotonia and UL / LL wasting. 4-Delayed psychomotor development 5-Pyramidal signs (vary between patients): spasticity, hyperreflexia 6-Cerebellar signs: UL, Gait, trunk and eyes 7

Figure ( 3 - 13 ):

 313 Figure (3-13): Pedigree of family F41 showing segregation of missense mutation in CAMSAP3 with the disease distribution in the whole family. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

Figure ( 3 - 14 ):

 314 Figure (3-14): Pedigree of family F41 showing segregation of missense mutation in MINK1 with the disease distribution in the whole family. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

  )]. There is a small physicochemical difference [Grantham distance of 46 (0-215)] between both hydrophilic amino acid serine and asparagine. The amino acid is a moderately conserved amino acid in mammals and vertebrates (considering 11 species : not conserved in pigs).

Figure ( 3 -

 3 Figure (3-15): Pedigree of family F41 showing segregation of missense mutation in ZNF433 with the disease distribution in the whole family. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino

2 -( 11 -

 211 Age at onset of two sibs: 7 months and two years 3-Pyramidal signs: Tetraspastic, hyperreflexia 4-Cerebellar ataxia: one patient showed UL, LL, speech, gait and eye Drooling of saliva 12-MRI of the brain: Cerebellar atrophy, hypomyelination, TCC and mild prominence of internal and external CSF space.

[exon 4

 4 : g. 76212781C>G, c.327C>G, p.Phe109Leu] (based on the longest transcript: NM_001012271.1); [exon 4: c.258C>G, p.Phe86Leu] in the transcript (NM_001168.2) which codes for the canonical isoform 1 of the survivin protein). The gene BIRC5 or baculoviral (IAP) repeat containing 5 (OMIM *603352) encodes for the apoptosis inhibitor 4 (API4), survivin and EPR-1.

  19169133C>G, c.430G>C, p.Ala144Pro] (based on the longest transcript NM_001100420.1) cosegregated with the disease distribution in the family [Figure[START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF]].C21ORF91 (chromosome 21 open reading frame 91) is also known as EURL,C21orf14, CSSG1, YG81, C21orf38. It is located on the long arm of chromosome21 (21q21.1) [Ensembl: 21: 17,788,819,386 (GRCh38);

Figure ( 3 - 16 ):

 316 Figure (3-16): Pedigree and MRI of two patients of family F50 showing missense mutations in two putative candidate genes BIRC5 and C21ORF91 segregating with the disease distribution in whole family. MRI of patient 402 shows cerebellar atrophy and hypomylination. MRI of patient 403 (lower two images) shows hypomyelination, thin corpus callosum (TCC) and mild prominence of internal and external CSF space. Example sanger sequencing of the BIRC5 mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

  The affected individuals were three sibs(2020, 2021, 2022) presented to the clinic at the ages of 5 years, 4 years 4 months and 2 yrs 3 months. They were born to first degree consanguineous parents. The two girls(2021, 2022) were born after an uneventful pregnancy and delivery but for the boy (2020) the mother noted decreased fetal movement at the end of the third trimester, which implied the possibility of prenatal onset. The two affected girls (2021 and 2022) looked healthy for few months after birth. Over all the three sibs showed homogenous disease course. The parents then noted delayed psychomotor development of their milestones as all kids could not sit till the age of presentation to the clinic and could only turn in bed. They also observed abnormal movement in all three kids.

2 -

 2 Age at onset of three sibs: prenatal (end of last trimester: boy), There was 15 homozygous rare variant identified upon analysis of the WES data of patients (2020, 2022). Only one known missense variant rs116335992 (ExAC frequency of 0.09498) in exon 1 of the gene ST7L [g.113161534C>A, c.202G>T, p.Ala68Ser] (based on the longest transcript NM_017744.4) cosegregated with the disease distribution in the family. ST7L (Suppression Of Tumorigenicity 7 Like) is also known as ST7R, FAM4B, STLR, FLJ20284.

  )]. There is moderate physicochemical difference [Grantham distance of 99 (0-215)] between the hydrophobic amino acid Alanine that changed to the polar uncharged amino acid Serine. The affected amino acid (located in the protein domain: ST7) is a moderately conserved amino acid (considering 12 species) but highly conserved in mammals].

Figure ( 3 -

 3 Figure (3-17): Pedigree and MRI of the index patient of family FM3 with missense mutation in ST7L segregating with the disease distribution in the whole family. MRI of the brain shows cortical and cerebellar vermial atrophy, periventricular subcortical white matter hyperintensities (WMH). Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

  ) all individuals were floppy at birth and had delayed psychomotor development. The five sibs had homogenous clinical picture but with marked variability of the disease severity. Two affected boys (2050, 2051) and one girl (2049) had the most aggressive disease manifestations whereas their sister (2048) had the mildest form. The patients developed clumsiness indicating cerebellar involvement at 15 to 18 months. Psychological symptoms were present in a severe form in three of them (2049, 2050, 2051) (nightmares, aggressive attacks, emotional lability) and less prominent in patient (2047, 2048).

  The mutation resulted in a change from valine to methionine. The two are hydrophobic amino acids. There is thus a small physicochemical difference[Grantham distance is 21 (0-215)].The amino acid is located in the domain of unknown function DUF89 (Pantothenate kinase, acetyl-CoA regulated, two-domain type). This change was predicted pathogenic by SIFT, Polyphen2 and Mutation taster. The nucleotide where the variant is located is conserved and has a PhastCons conservation score of 0.99 (0-1) and a PhyloP score of4.73 (-14.1-6.4)]. The amino acid is moderately conserved in 12 species but highly conserved in mammals.

Figure ( 3 -

 3 Figure (3-18): Pedigree and MRI of the two index patients from the two branches of family FM7. Branch 1 of the family was associated with a missense mutation with full segregation with the disease distribution in branch 1 (blue rectangles) but not segregating in branch 2 (red rectangles). MRI of the index of branch 1 (T1 sagittal and T2 FLAIR axial) shows cortical atrophy more prominent in the frontal lobe in addition to cerebellar atrophy. MRI of the index of branch 2 (T1 sagittal and T2 FLAIR axial) shows mild cortical and significant cerebellar atrophy Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

Figure ( 3 - 20 ):

 320 Figure (3-20): Pedigree of family F27. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals, others are standard medical pedigree symbols.

Figure ( 3 -

 3 Figure (3-22): WES success rate for known and new genes in the Sudanese cohort

  F1 branch C and 162 D) (Elsayed et al., under revision). In three families (F14, F16, F21) [Figure (4-4)] [Figure (4-5)] [Figure (4-3)], respectively) (Elsayed et al., under revision) the other coexisting phenotype is non-neurological and was based on clinical diagnosis and we have not explored the genetic background of these disorders as they are out of the scope of the study.

Figure ( 4 - 1 ):

 41 Figure (4-1): Main clinical signs complicating the 41 families included in the cohort.

Figure ( 4 - 2 )

 42 Figure (4-2): Brain MRI abnormal signs encountered in the cohort.

Figure ( 4 - 3 ):

 43 Figure (4-3): Pedigree of family F21. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: spastic ataxia individual, other filling patterns correspond to various clinical phenotypes in the family. Others are standard medical pedigree symbols.

Figure ( 4 - 4 ):

 44 Figure (4-4): Pedigree of family F14. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: spastic ataxia/SACS individuals, other filling patterns correspond to various clinical phenotypes in the family. Others are standard medical pedigree symbols.

Figure ( 4 - 5 ):

 45 Figure (4-5): Pedigree of family F16. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: HSP/SPG11 individuals, other filling patterns correspond to various clinical phenotypes in the family. Others are standard medical pedigree symbols.

  Figure[START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF]: Suggested flow chart designed to assist in the variant/gene prioritization process of gene panel data (based on inheritance and the most frequent clinical presentations). P: pure, C: complex, AR: autosomal recessive, AD: autosomal dominant, PN: peripheral neuropathy, Amyo: amyotrophy, TCC:thin corpus callosum, CI:cognitive impairment, OA: optic atrophy, RD: retinal degeneration, MD: macular degeneration.

Figure ( 4 - 8 ):

 48 Figure (4-8): Schematic representation of the ABHD12/ABHD16A axis (Kim, 2015). Nonsense mutation causing TCC HSP in family F37 mimics a knock out of ABHD16A (added to the illustration).

  ), and lysophosphatidylinositol (LPI). It strictly requires the serine residue of LysoPS. It also strictly requires the structure of the serine residue of LysoPS. Modification of the serine residue entirely abolishes the degranulation-stimulating activity of the mast cells[START_REF] Makide | Novel lysophosphoplipid receptors: their structure and function[END_REF]. Whether this is part of the phenotype in patient remain to be explored either by the study of macrophages from patients or by knock down experiments in macrophage strains in vitro.

Figure ( 4 - 9 ):

 49 Figure (4-9): Biological functions of lysophosphatidyl serine (LysoPS) (Makide et al, 2014).

  Zheng et al. identified it as marshalin. The protein was shown to have multiple protein interacting domains including a C-terminal CKK domain (DUF1781) and an N-terminal calponin homology (CH) domain, and three coiled-coil (CC) and two proline-rich (PR) domains in the middle[START_REF] Zheng | Marshalin, a microtubule minus-end binding protein, regulates cytoskeletal structure in the organ of Corti[END_REF]

  PanK2 is expressed ubiquitously, with high levels in retinal and infant basal ganglia PanK1 is found predominantly in heart, liver and kidney; PanK3 is limited to the liver, but expressed at a high level. The expression of PanK4 is ubiquitous, with a high level in muscles [Figure (4-1)] (Zhou B et al., 2001) PanK1, PanK3 and PanK4 isoforms are cytosolic whereas PanK2 is mitochondrial. Human PanK2 is also detected in the nucleus (Alfonso-Pecchio et al., 2012). The pantothenate kinases (PanK) catalyze a rate limiting step (the first committed step) in coenzyme A (CoA) biosynthesis. Coenzyme A (CoA) is a cofactor that plays a critical role in intermediary metabolism in all organisms. It functions as a carrier for carboxylic acid substrates. It supports multiple essential biochemical pathways which

  It was shown in Drosophila that human cytosolic PanK3 and PanK4 could mostly, but not fully, rescue Pank2/Fbl lack. PanK2 is more potent than the two cytosolic PanK4 and PanK3 in vivo but the ability of either PanK3 or PanK4 to significantly complement Pank2/fbl mutation indicated they were both functional pantothenate kinases. Furthermore, itwas shown that the human PANK4 gene can complement mutant of E.coli deficient in pantothenate kinase. This reflects the high degree of conservation of the pantothenate kinase and thus provides evidence for a probable compensatory mechanism in PKAN patients(Hӧrtnagel et al., 2003).

Figure ( 4 -

 4 Figure (4-10): Expression of pantothenate kinases (PanK 1, PanK2, PanK3 and PanK4)[START_REF] Zhou | A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome[END_REF]

  Figure (4-12): Statistical correlations identified using regression test. Filled boxes represent pathogenic themes while white boxes are clinical phenotypes/signs.

4. 7 .

 7 A new nosology and objective case definition of HSP: a question raising itself ?As highlighted in the introduction, HSPs share multiple overlap zones with other spastic neurogenetic disorders. Neurodegeneration is now forcefully imposing the concept of being a continuum of disorders through the huge amount of knowledge gained in the last ten years and the rapidly expanding numbers of HSP and overlapping disorders subtypes.The question about the urging need for a new nosology with a wider umbrella that includes all closely related neurological disorders is raised by the factors we stated earlier: the large number of HSP subtypes associated with other neurological signs that can be the major clinical feature of overlapping disoders, the phenomenon of multiplicity of allelic phenotypes of SPG genes together with the recently characterized novel HSP forms implicating lipid or amino acid metabolic pathways.The second point raised is the need for improved and more objective case definition to minimize the subjectivity in diagnosis especially with regards to the overlap zones and to decrease the technical diagnostic difficulties due to the lack of sufficient expertise in the rapidly growing field of neurogenetics. Difficulties are mainly encountered by less experienced clinical practioners or even by high clinicians and neurologists with less expertise in the field of neurogenetics. The problem is further augmented where the setting does not provide the necessary multisystemic medical neurogenetic centers with equipped facilities and required expertise for neurogenetic workup. A clear example for the latter situation is the practice in developing countries where minimal is known about neurogenetic disorders. If we consider that many of these communities are highly inbred with a predominant AR patterns of inheritance and with high chances of new genes discovery, then the importance of guideline and classification to facilitate proper phenotypic stratification with minimal cost would unveil. This point has been highlighted

3 .

 3 National collaborations with experts in related fields: To further ameliorate the process of phenotyping, we created further collaborations with experts in the fields of neuroradiology (Departments of Radiology in: Alamal Hospital, Police hospital, Antalia Medical center, Dar Alelaj Hospital), neurophysiology (Department of physiology: University of Khartoum, Soba University hospital) , ophthalmology (Department of Ophthalmology: University of Khartoum, Mekka Eye Charity Hospital) and psychiatry (Department of Psychiatry: University of Khartoum, Taha Baashar Hospital for Psychiatic disorders).

( 3 )( 5 )

 35 Patient is asked to walk as fast as possible a 10 meter distance including one turn 0: Normal 1: Mild stiffness, running still possible 2: Clearly spastic gait, interfering with running 3: Spastic gait requiring use of canes/walker 4: Unable to walk for a 10 meter distance even with maximal support Maximum gait speed Time for a 10 meter distance including one turn, taken by stop watch Timing: 0: Normal 1: Slightly reduced (10m: ≥ s) sec 2: Moderately reduced (10m: ≥ 10s) 3: Severely reduced (10m: ≥ 20s) 4: Unable to walk for a 10m distance or time ≥ 40s (4) Climbing stairs 5 steps upstairs -turn -5 steps downstairs 0: Normal: needs no support of the banister 1: Mild impairment: needs intermittent support of the banister 2: Moderate impairment: needs permanent support of the banister 3: Severe impairment: needs support of another person or additional walking aid to perform task 4: Unable to climb stairs Speed of stair climbing Time for 5 steps upstairs -turn -5 steps downstairs, taken by stopwatch Timing: 0: Normal 1: Slightly reduced (≥ s to perform task) sec 2: Moderately reduced (≥ 10s to perform task) 3: Severely reduced (≥ 20s to perform task) 4: Unable to climb stairs (6) Arising from chair Patient attempts to arise from a straight-back wood or metal chair with arms folded across chest 0: Normal 1: Slow, or may need more than one attempt. 2: Pushes self up from arms of seat. 3: Tends to fall back and may have to try more than one time but can get up without help. 4: Unable to arise without help.

( 7 )( 11 ) 2 :( 12 )( 13 )Figure

 71121213 Figure Relative Frequencies of AD HSPs in European Populations: based on SPATAX network statistics

Figure ( 1 - 7 )FigureFigure

 17 Figure[START_REF] Tesson | Delving into the Complexity of Hereditary Spastic Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are Revolutionizing Their Nosology[END_REF][START_REF] Fink | Hereditary Spastic Paraplegia Overview[END_REF][START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF] Figureshowingthe overlap zone between spastic neurogenetic disorders: evidence for the need of new nosology and objective case definition

Figure

  Figure Consanguinity rate in 41 families

Figure ( 3

 3 Figure[START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF] Pedigree and MRI of the index patient of family F30 caused by missense mutation in SIL1 segregating with the disease distribution in whole family presenting with pyramidal signs associated with Marinesco Sjogren Syndrome (MSS).

Figure ( 3 - 9 )

 39 Figure[START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF] Pedigree and colour image of the retina of the index patient of family F26 caused by pathogenic CAG repeat in ATXN7 family presenting with pyramidal signs associated with spinocerebellar ataxia type 7 (SCA7).

Figure ( 3 -

 3 Figure[START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF][START_REF] Novarino | Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders[END_REF]:Pedigree and MRI of the index patient of family FM3 with missense mutation in ST7L segregating with the disease distribution in the whole family

FigureFigure ( 3 -

 3 Figure[START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Spastic Paraplegia with Thin Corpus Callosum: Description of 20 New Families, Refinement of the SPG11 Locus, Candidate Gene Analysis and Evidence of Genetic Heterogeneity[END_REF][START_REF] Sch Le | Frequency and Phenotype of SPG11 and SPG15 in Complicated Hereditary Spastic Paraplegia[END_REF][START_REF] Novarino | Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders[END_REF][START_REF] Beetz | Inhibition of TFG Function Causes Hereditary Axon Degeneration by Impairing Endoplasmic Reticulum Structure[END_REF][START_REF] Ishiura | The TRK-Fused Gene Is Mutated in Hereditary Motor and Sensory Neuropathy with Proximal Dominant Involvement[END_REF] Clinical Summary of the 24 families with unidentified genetic cause

Figure

  Figure Main clinical signs complicating the 41 families included in the cohort

3.2.3. Investigations:

	4. Detailed natural history of the disease since the age at onset till the date of
	examination (Patients motor disability using a Seven-Stage Disability Score
	also obtained) (https://spatax.wordpress.com/) (Table 2-1)
	5. In pediatric cases or adult patients with early onset, disease pregnancy,
	delivery, early development (gross motor and intellectual disability)
	2.3.2.2. Examination:
	General and neurological assessment included the following:
	1. Centiles especially head perimetry in pediatric patients or when
	microcephaly was suspected
	2. UMN features (pyramidal syndrome)
	2. Clinical Assessment:
	Magnetic resonance imaging (MRI) of the brain and spine was performed in
	2.3.2.1. History: at least one patient per family in most families. Electrophysiological studies
	Points covered in history include: including nerve conduction studies (NCS), electromyography (EMG),
	electroencephalography (EEG) were performed when indicated and
	1. Personal data including origin and contact details feasible. Other laboratory tests were done based on individualized
	2. Family history of medical disorders including similar conditions or other differential diagnoses.
	neurological or non-neurological disorders
	3. Consanguinity (pedigree obtained)

1. Questionnaire and auditing

o Patients were clinically examined and diagnosed by the referring consultant neurologist/neuro-pediatrician. We performed a second scrutinized phenotyping and filled a standard questionnaire [the standard questionnaire of the SPATAX network (https://spatax.wordpress.com/)]. Later a version of the questionnaires with minor modifications was created and adopted. This was in particular, because our cohort contained mainly pediatric cases and consequently we had to adapt the clinical history and examination to this category [

Annex 12]

.

o Professional video-taping was performed to enable auditing and documentation of the physical examination and clinical signs. We examined all healthy related controls to exclude subtle disorders. 3. LMN feature (sensory and motor peripheral neuropathy features) 4. Cerebellar syndrome (UL, LL, eye movements, speech, gait ataxia, trunk and head tremor and neck ataxia) 5. Extrapyramidal signs (bradykinesia, rigidity, postural instability, dystonia, chorea, athetosis and related features depending on the preliminary findings) 6. Eye examination (including fundoscopy) and visual acuity assessment. 7. Skeletal deformities, dysmorphic features and any skin or hair changes. 8. Any additional clinical sign that was necessary to elaborate based on the provisional history and examination even if not directly related to the above points 2.

1. General description

	An automated Infinium high throughput screening (HTS) based assay protocol
	provided by the manufacturer was used. The assay was performed in a specialized
	Illumina platform in a local facility. A special MSA3 plate based protocol was used
	with a starting DNA quantity of 200 ng (4µL at 50ng/µL). The maneuver was
	conducted in two separate laboratory sections (Pre-Amp and Post-Amp) as indicated
	in the Illumina guidelines.
	HumanCore-24 BeadChip kit v1-0® (WG-330-2001) (Illumina®, San Diego,
	USA) was used for genome-wide genotyping. It is a silicon-based array device. The
	Beadchip provided contains 306,670 genome-wide tag SNPs of which over 20,000
	high-value markers. The exome-focused SNPs are 41,518. The mutations tested
	include 9,800 nonsense, 6,707 missense, 12,286 indels and 160 mitochondrial plus
	other types of variations spaced at a mean distance of 9.4 Kb and a median distance
	of 5.7 Kb.

Parkinson genes using targeted Next Generation Sequencing 1. General description:

	1042 with a total length of 220095 bps. The PARK kit targets all exons of six genes
	known to be involved in Parkinson disease (PARK2, PINK1, DJ-1/PARK7, ATP13A2,
	PLA2G6, SYNJ1, DNAJC6, FBXO7, VPS13C). Each region corresponds to exons plus
	20 base pairs of the up-and down-stream flanking non-coding or intronic regions. A
	capture enrichment strategy is used (Roche NimbleGen®/SeqCap Ez®, USA).
	Sequencing is performed using MiSeq platform (Illumina®, San Diego, USA) on 250 bp
	paired-end reads. In this maneuver, 24 DNA samples are processed (multiplexed) at a
	time after proper sample indexing is done for each sample. Base calling and quality
	control are performed using Genomics Workbench (CLC Bio®, Denmark). Variants are
	detected using probability-and quality-based algorithms.
	I used 2 multi-gene targeted next generation sequencing panels to screen index
	patients from the 41 families (at least 1-2 patients/family). The HSP kit targets all
	exonic regions of 67 genes known to be related to a variety of spastic disorders and 7
	unpublished spastic candidate genes (Annex 14). The total number of target regions is

2. Laboratory protocol: 2.9.2.1. Library preparation

	An illustration of the basic steps of library preparation protocol used for the NGS
	is provided in figures [Figure (2-1)] and [Figure (2-2)].
	1. Genomic (DNA) preparation and fragmentation:
	A starting quantity of DNA of 1µg (50 µL at a concentration of 20ng/µL)
	is used as recommended by Roche NimbleGen. DNA quality is tested
	using nanodrop® and gel electrophoresis. As the process includes many
	PCR dependent steps, samples are also passed in a test PCR before the start
	to exclude the presence of any PCR inhibitors. The genomic DNA
	fragmentation is performed using a Covaris shearing instrument. It is a
	focused ultrasonicator that utilizes ultrasonic acoustic energy to
	mechanically fragment the DNA. It employs an AFA technology [Adaptive
	Focused Acoustics™]. The fragmentation is achieved by directing bursts of
	ultrasonic waves towards discrete zones within the sample vessel immersed
	in a water bath. The two conditions required for successful fragmentation
	are the use of special Covaris tubes (52.5 µL) and conducting the maneuver
	at isothermal condition.

2.9.3.2. Variant annotation (detection):

	Is achieved through two algorithms: quality-based variant detection
	(QBVD) and probability-based variant detection (PBVD): this results in
	production of two excel sheets named as corresponding variant analysis
	files.
	2.9

.3.3. Variant prioritization

	Analysis was done through excel sheet filter tools. For Gene/Variant
	Prioritization a minimal threshold depth of 30x was chosen. To ensure
	inclusion of all valid variants, we first set a relaxed minor allele frequency
	(MAF) filter of <1.5% at the offset of our analysis. Indeed, allele frequency
	can vary greatly from one population to another and we preferred filtering on
	this criteria on later steps, including the frequency in our local database. The
	filter was tightened at a downstream step to <0.1% for more stringency.
	Functional consequences of variants considered were stop codons, frameshift,
	splice site variants and missense changes. Regarding missense alterations, five
	pathogenicity prediction algorithms were used (SIFT, Polyphen-2, Mutation
	Taster, Align GVGD, KD4v). The conservation score was calculated by
	PhastCons and PhyloP. Moreover, for the prediction of splicing sites, we used
	five tools (Human Splicing Finder, SpliceSiteFinder-Like, Gene Splicer,
	NNSPLICE, MaxEntScan). All tools were collectively provided by
	AlamutVisual ® software (Interactive Biosoftware, France).

2.10. Whole Exome Sequencing (WES) 1. General description

	the Hiseq property of the NextSeq-500 sequencer (Illumina®, San Diego, USA)
	available in our local facility, on 150 bp paired-end reads.
	In this maneuver, 10 DNA samples are processed (multiplexed) at a time after
	proper sample indexing is done for each sample. Sequencing is performed using

2. Laboratory protocol: 2.10.2.1. Library preparation:

	Library preparation is basically the same as for targeted NGS [Figure (2-1)] and
	[Figure (2-2)].
	The exception is that there is only one capture instead of two implying that
	there is a single hybridization, one capture and no middle-captured PCR. The
	hybridization is carried over longer period of 64-72 hours instead of overnight
	[Figure (2-2)].
	1. Genomic (DNA) preparation and fragmentation:
	A starting quantity of DNA of 1µg (50 µL at a concentration of
	20ng/µL) is used as recommended by Roche NimbleGen. DNA quality is
	tested using nanodrop® and gel electrophoresis. As with the Miseq library
	preparation, samples are also subjected to a test PCR before library
	preparation in order to exclude the presence of any PCR inhibitors. The
	genomic DNA fragmentation was performed using a Covaris shearing
	instrument.

3. Bioinformatic Analysis 2.10.3.1. Read mapping and variant detection

	data) file. The files are uploaded into the interface based software Polyweb (an
	unpublished software developed by Paris Descartes University, France).
	The NextSeq-500 output data is in form of eight FastQ files per sample
	(four for Read 1 and four for Read 2: Nextseq 500 flow cell contains four
	physical lanes). Base calling read mapping and quality control are performed
	using a locally developed bioinformatic pipeline. The reference sequence for
	read mapping and alignment is based on GRCh37 assembly. The output is a
	variant calling format (VCF) and a BAM (for alignment, reads and coverage

2.10.3.2. Variant selection and prioritization

		Supporting literature about gene and protein (OMIM, Genecards,
		Uniprot, PDB, Pubmed..)
	
	2.
	1. Polyweb software
	Analysis was carried out with the use of the interface based polyweb
	tool. For Gene/Variant Prioritization a minimal threshold depth of 20x was
	selected. To ensure inclusion of all valid variants, we set a relaxed minor
	allele frequency (MAF) filter of <1% at the offset of our analysis, in public
	and also in our local database. The filter was tightened at a downstream
	step to <0.1% for more stringency. Functional consequences of variants
	selected were stop codons, frameshift, splice site variants and missense
	changes.
	2. Alamut
	Regarding missense alterations, five pathogenicity prediction
	algorithms were used (SIFT, Polyphen-2, Mutation Taster, Align GVGD,
	KD4v). The conservation score was calculated by PhastCons and PhyloP.
	Moreover, for the prediction of splicing sites, we used five tools (Human
	Splicing Finder, SpliceSiteFinder-Like, Gene Splicer, NNSPLICE,
	MaxEntScan). All tools were collectively provided by AlamutVisual ®
	software (Interactive Biosoftware, France).
	3. Further Tools to enhance the search
		Gene prioritization software
		Genome Browsers (ensemble, UCSC, NCBI, ExAC)
		Databases (EVS, dbSNP, ExAC, ClinVar, 1000 genome)
		Genic intolerance assessment (RVIS, ExAC)
		Expression level (Allen Brain, Ensembl link)
		Mouse model (MGI)

10.3.3. Validation of Variants

	1. Primer design (See section 2.7.1)
	2. Polymerase chain reaction (PCR) (See section 2.7.2)
	3. ExoSAP purification:
	ExoSap purification is an enzymatic purification method to clean the
	PCR product from primers and dNTPs. After mix of specific buffer and
	two enzymes: the mix was added to the PCR product to obtain a final
	concentration of the Exonuclease I (EI) (20 U/µl) and the Alkaline
	phosphatase (AP) (1 U/µl), the product was incubated for 15 min at
	37°C (activate enzyme) then at 80°C for 5 min (inactivate the enzyme).

Table ( 2-2):

 ( Components of culture medium used for collection of skin biopsy and harvest of fibroblasts.

	Principal

Reagent Original Concentartion Final Concentration Dulbecco's Modified Eagle Medium (DMEM) (ThermoFisher)

	2.
		1X	1X
	Fetal Bovine Serum (FBS)	100%	15%
	Penicillin/Streptomycin	10 000 U/mL (10	50 U/mL (50
		mg/mL)	μg/mL)
	L-glutamine	200 mM	2 mM

11.2.2. Fibroblasts culture protocol summary:

Table ( 3-1):

 ( Clinical categories in the 41 families. HSP: hereditary spastic paraplegia, HA: hereditary ataxia, PD:Parkinson disease, INAD: infantile neuroaxonal dystrophy.

	Clinical Summary	No. Families /41	%	Families
	CLASSICAL HSP with predominant pyramidal syndrome
	Pure HSP	4	10%	F2, F7, F17, F48
	Complicated HSP (minimal	2	5%	F34, F35
	complexity: one additional feature
	only)
	Complicated HSP (more than one	17	41.5%	F1, F6, F14, F16, F19, F23,
	complicating features)			F28, F36, F37, F42, F44, F49,
				F50, FM1, FM3, FM5, FM6
	ATAXIA AND HSP (equal weight)
	Spastic Ataxia (equal burden)	2	5%	F21, F41
	ATAXIC syndromes with pyramidal features
	Complicated HA	10	24%	F12, F24, F26, F30, F45, F46,
				F47, F31, F38, FM7

Spasticity as a complicating feature of ANOTHER syndrome HSP (secondary to Metabolic disorder)

		1	2%	F15
	PD with pyramidal signs	1	2%	FM4
	Complex presentation with pyramidal	2	5%	F11, F22
	signs
	INAD with pyramidal signs	1	2%	F25
	Peculiar Syndromes (probably new)	1	2%	F27

Table (

 (

	Category	Age at onset	No of	%	Family codes
			families/44
			(additional
			three branches)
	1	Birth-1 yr	9	20%	F1 branchD, F12, F37, F41, F44, F45, FM3, FM6, FM7
	2	1 yr-5 yrs	11	25%	F1 branchC, F7, F11, F15, F19, F22, F23, F25, F27, F30, F42
	3	5 yrs-18 yrs	11	25%	F1 branchB, F2, F6, F14, F16, F24, F28, F36, F38, F48, FM4
	4	18 yrs -40 yrs	6	14%	F1 branch A, F17, F35, F47, F49, FM1
	5	>= 40 yrs	1	2%	F21
	Variable: category 1-2	Birth-5yrs	2	5%	F50, FM5
	Variable: category 2-3	1 -18 yrs	2	5%	F34, F46
	Variable: category 3-4	5-40 yrs	1	2%	F26
	Variable: category 2-4	1-40 yrs	1	2%	F31

3-2): Distribution of age at onset in 41 extended families (44 nuclear famiies:3 branches were treated as separate families)

  and ALS2. All mutations were novel except one in SPG11 previously reported with earlier onset compared to our patients. Interestingly, the TFG missense mutation (exon 2: c.64C>T p.(Arg22Trp) / PB1 domain) is the third

identified worldwide, and we demonstrated its effect on TFG oligomerization in vitro. Patients did not present visual impairment, as observed in the previously reported SPG57 family (p.(Arg106Cys) / coiled coil domain), suggesting unique contributions of the PB1 and coiled coil domains in TFG complex formation function. Further genetic heterogeneity is expected in HSP Sudanese families. b) Manuscript Article 2: Liena Elbaghir Omer Elsayed, Inaam N. Mohammed, Ahlam Abd Alrahman Ahmed Hamed, Maha Abdelmoneim Elseed, Adam Johnson, Mathilde Mairey, Hassab Elrasoul Siddig Ali Mohamed, Mohamed Nagib Idris, Mustafa A.M. Salih, Sarah Misbah El-sadig, Mahmoud E. Koko, Ashraf Yahia Osman Mohamed, Laure Raymond, Marie Coutelier, Frédéric Darios, Rayan Abubaker Siddig, Ahmed Khalid Mohamed Albashir Ahmed, Arwa Mohammed Abbker Babai, Hiba Mohamed Osman Malik, Zulfa Mohammed Babikir Mohammed Omer, Eman Osama Eldirdiri Mohamed, Hanan Babikir Eltahir, Nasr Aldin Ali Magboul, Elfatih Elfadl Bushara, Abdelrahman Elnour, Salah Mohamed Abdel Rahim, Abdelmoneim Alattaya, Mustafa Idris Elbashir, Muntaser Eltayeb Ibrahim, Alexandra Durr, Anjon Audhya, Alexis Brice, Ammar Eltahir Ahmed, and Giovanni Stevanin. Hereditary spastic paraplegias:

  Both patients had absent fundal striations in accordance with previous reports of Italian patients[START_REF] Criscuolo | A Novel Mutation in SACS Gene in a Family from Southern Italy[END_REF][START_REF] Grieco | Novel SACS Mutations in Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay Type[END_REF] and Japanese sibship.36 Overall, patients from the three families with mutations in SPG11 presented with a complex HSP frequently associated with cognitive deficit. MRI of the brain showed atrophy of the corpus callosum in all six tested cases often associated with periventricular white matter abnormalities (Figure (1)) and signs of motor demyelination frequently mixed with axonal degeneration at electrophysiological examination. This agrees with previous reports.37,38 It is worth noting that of the 18 families (72%) in which the genetic diagnosis is not yet established, 2 families only presented as pure HSP. Six families showed HSP complicated with stereotypic

movement

(2/18)

, bulbar signs and sensory motor polyneuropathy (1/18), cognitive decline (1/18), dystonia

(1/18)

or severe cortical atrophy

(1/18)

. Ten families presented with spastic ataxia, complicated in six families with dysmorphic features

(2/18)

, chorea (1/18) and visual impairment

(3/18)

. Discussion:

  Although both mutations resulted in perturbed oligomerization of the protein, the mechanism and consequent functional effect are distinct based on our biochemical evidence. Structural studies indicate that full length TFG self-assembles into octameric ring-like complexes in solution.18 Our analysis of the PB1 domain indicates that it exhibits a similar propensity to form octamers. Thus, the coiled coil domain may play a distinct role in promoting TFG complex assembly, potentially facilitating the assembly of the ring-like organization, which is likely under considerable strain. The relation between phenotypic spectrum and mutation localization is well known in HSP genes including PNPLA6 reported by Schabh ttl et al.39 and TFG/SPG57 reported byBeetz et al.18 Mutations in the proline and glutamine-rich domain (carboxyl terminus) of TFG/SPG57 result in AD HSMN-P.[START_REF] Ishiura | The TRK-Fused Gene Is Mutated in Hereditary Motor and Sensory Neuropathy with Proximal Dominant Involvement[END_REF] On the contrary, mutations in the amino terminus result in AR HSP as shown byBeetz et al.18 and in this study. Conclusions: We were able to identify the first Sudanese families carrying novel mutations in SPG11, ATL1, ALS2, SACS and TFG/SPG57 genes and establish their relative frequencies. However, the difficulty to reach a genetic diagnosis in the majority of studied families suggests the possibility of new genes or noncoding variations underlying spinocerebellar degeneration. The phenotypes of the undiagnosed families and the high frequency of spastic ataxic phenotypes imply a high possibility to expand the phenotypes and molecular basis of HSP. Genetic screening using exome or genome sequencing is therefore highly recommended to explore these families. TFG novel mutation resulting in p.Arg22Trp change was found to disrupt the ability of the PB1 domain to oligomerize highlighting a mechanistic basis for HSP. doi:10.1016/j.ejmg.2010.10.006. 3 . Schabh ttl M, Wieland T, Senderek J, et al. Whole-Exome Sequencing in Patients with Inherited Neuropathies: Outcome and Challenges. J Neurol 2014; 261 (5): 970-82. doi:10.1007/s00415-014-7289-8.

	Titles and legends to figures:
	Figure (1): Three families (F1, F6, and F16) with autosomal recessive hereditary
	spastic paraplegia (HSP) with thin corpus callosum (TCC) caused by SPG11. (A I)
	Pedigree of Family F1. Subfamily II (branch C and D) presents with two different HSP
	phenotypes. (A II) MRI of the brain (Sagittal section T1) of index individual (arrow)
	showing severe TCC. (B I) Pedigree of Family F6. (B II) MRI of the brain (Sagittal
	section T1) of index individual showing severe TCC and cortical atrophy. (C I)
	Pedigree of Family F16. (C II) MRI of the brain (Axial section T2 FLAIR) of index
	individual showing TCC and periventricular hypersignal intensity lesions. In all
	families, example of Sanger sequencing results of the mutation site in a patient and a
	heterozygous carrier is shown. HTZG: heterozygous, * sampled individual, black
	shading indicates affected individuals, ++ homozygous reference, M+ heterozygous
	genotype, MM homozygous mutant.
	Figure (3): Three families (F14, F34, and FM5) with spastic ataxia and hereditary
	spastic paraplegia (HSP) caused by SACS, SPG3A and ALS2, respectively. (A I)
	Pedigree of Family F14 with AR spastic ataxia of Charlevoix Saguenay (ARSACS)
	caused by nonsense mutation of SACS gene. Example Sanger sequencing of the
	mutation in a patient and in a heterozygous carrier is shown. (A II) MRI of the brain
	(Sagittal section T1) of individual 14133 showing thinning of the posterior half of the
	body of corpus callosum and moderate thinning of cerebellum. (B) Pedigree of Family
	F34 with Autosomal Dominant HSP caused by SPG3A/ATL1 gene. Example Sanger
	sequencing of the mutation site in a patient and in a control (homozygous reference) is
	shown. (C) Pedigree of Family FM5 with Autosomal Recessive HSP caused by
	missense mutation of ALS2 gene. Example Sanger sequencing of the mutation site in a
	patient and in a heterozygous carrier is shown. In all pedigrees: HTZG: heterozygous,

The previously identified p.Arg106Cys mutation is located in the coiled coil (CC) domain of TFG. The novel p.Arg22Trp mutation lies in the PB1 domain. Figure (2): One family (F19) with autosomal recessive hereditary spastic paraplegia (AR HSP) caused by TFG/SPG57. (A) Pedigree of Family F19. Example Sanger sequencing of the mutation site in a patient, a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown along with aminoacid conservation. HTZG: heterozygous, * sampled individual, black shading: individuals with HSP, ++ homozygous reference genotype, M+ heterozygous, MM homozygous mutant genotype. (B) Purified, untagged forms of TFG (amino acids 1-96; wild-type or mutated to include the p.R22W substitution), were separated over a gel filtration column (Wyatt TWC-030S5) that was coupled to a multi-angle light scattering device. Representative light scattering profiles (wild type TFG , blue; TFG p.R22W, red) are plotted (B I) and eluted fractions were separated by SDS-PAGE and stained using Coomassie to hig hlight the elution profiles of the both forms of TFG (B II). Based on 3 independent experiments for each protein, wild type TFG (amino acids 1-96) exhibits a molecular mass of 92.4 kD +/-3.7 kD, and p.R22W TFG (amino acids 1-96) exhibits a molecular mass of 24.4 kD +/-1.0 kD. (C) MRI of the brain (Sagittal section T1) of individual 19172 showing thinning of the body of the corpus callosum (C I) and MRI of the brain (Axial section T2 FLAIR) of individual 19171 showing thin corpus callosum and very mild periventricular hypersignal intensity lesions at the occipital pole (C II). * sampled individual, black shading: affected individuals, ++ Homozygous reference genotype, M+ Heterozygous, MM Homozygous mutant.

Table ( 3

 ( -3): Clinical data of five patients from family F15 with hyperargininemia due to a mutation in ARG1 gene. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: -Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition.

	Individual Code	138	142	143	148	149
	Sensory Loss	-	-		-	-		-
	Optic Atrophy	-	-		-	-		-
	Extrapyramidal	-	-		-	-		-
	Signs
	Other Signs	Severe	Severe		Anal	Pes		Pes
	Family Code	F 15 urinary/ anal incontinence	F 15 urinary/ anal incontinence	F 15 incontinence	F15 cavus/ Scoliosis	cavus/ F15 Scoliosis
	Mutated Gene	Pes cavus/	Epilepsy	ARG1
	Individual Code	138 Scoliosis	142		143	148	149
	Gender	F Epilepsy	M		M	M	F
	Clinical Diagnosis	Rt. Hearing impairment		Spastic quadriplegia
	Origin Disability Score	5	6	North of Sudan, Gaalia tribe 5	6	4
	Age at Onset of Motor Symptoms MRI of the brain	1 year NAD	3 years NAD	2 years/ 9 months NAD	3 years NAD	2 years 8 months NAD
	Age at Initial Examination Studies (NCS) Electrophysiological	8 year ND	10 year ND		6 year ND	10 year ND	ND	8 year
	Spasticity UL/LL Plasma amino acids level	++/+++ ↑Arginine in one	++/++ ↑Arginine in one	+++/+++ ↑Arginine in one	++/+++ ↑Arginine in one	↑Arginine +++/+++ in one
	Motor Deficit PUL/DUL	+/+ occasion, normal in a second test	+/+ occasion, normal in a second test	+/+ occasion, normal in a second test	+/-occasion, normal in a second	occasion, -/-normal in a second
	Motor Deficit	++/++	+/+		++/++	+++/+++ test	test	-/-
	PLL/DLL
	Tendon Reflexes	↑/↑	↑/↑		↑/↑	↑/↑	↑/↑
	UL/LL Patellar
	Ankle Reflex/ Planter	↑/↑↑	↑/↑↑		↑/↔	↑/↑↑	↑/↑↑
	Response
	Ataxia Eye/UL/LL/	-/-/-/-	-/-/-/-		-/-/-/-	-/-/-/-	-/-/-/-
	Gait
	Dysarthria	+++/-	+/-		+/ -	+++/-	+++/-
	Spastic/Cerebellar
	Muscle Atrophy	+/+	+/+		-/+	-/+	-/-
	DUL/DLL
	Facial Atrophy	-	-		-		-	-
	Cog/ Psych signs	+/+	+/+		+/+	-/-	+/+

Table ( 3

 ( -4): Clinical summary of family F15. HSP:hereditary spastic paraplega, UL: upper limbs, LL: lower limbs, ND: not done, CT: computerized tomography scan, MRI: magnetic resonance imaging, EEG: electroencephalogram, EMG: electromyogram, NCS: nerve conduction studies.The two patients presented with homogenous disease presentation with the mean age at onset at 22 months (18, 24 months) and history of regression of milestones.

		 Family F25
	Fam.	Origin/	Inherita	Phenotype	Age at	Electrophys	Brain Imaging	Clinical features summary
	code	Consan Examination revealed marked hypotonia, pyramidal signs (hyperreflexia in both nce/ summary onset/Exam. iological
		guinity individuals and extensor planter response in one patient). Additional features Gene studies EMG/NCS,
		EEG) included dysphagia and aphasia, mental impairment and psychiatric manifestation
	F15-BRANC H-1	North Gaalia/ (blunt mood). The older sib (proband) had optic atrophy. There was no AR/ ARG1 Complicated HSP (metabolic) Two sibs (one died) : 1yr/8 yrs ND MRI: Normal 1-Pyramidal signs(LL &/> UL: spasticity, hyperreflexia and extensor planter response) 2-Sterotypical behaviour 3-Mental retardation 4-Epilepsy (atonic initiated by laughter) 5-Skeletal deformities (pes cavus, scoliosis) 6-Rt extrapyramidal signs or evidence of PN in both sibs. MRI of the brain was also homogenous but with more overt changes in the older sib. T2 Weighted FLAIR consanguineous conductive deafness 7-Psychiatric symptoms (hyperactivity, distractability) 7-Sphincter disturbance 8-images showed hyperintense signals in periventricular areas, basal ganglia
									Regression of milestone (aggravation at 2.5 yrs age) 9-
									Abnormal way of looking at objects
	F15-BRANC H-2	consanguineous	North Gaalia/	AR/ ARG1	Complicated HSP (metabolic)	Two sibs : 2yrs rs 9months,3yrs/6,10y	ND	CT scan: Normal MRI: Normal	1-Pyramidal signs(LL &/> UL: spasticity, hyperreflexia and extensor planter response) 2-Mental retardation 3-Epilepsy (generalized tonic clonic epilepsy) 4-Sphincter disorder 5-Abnormal way of looking at objects
	F15-BRANC H-3	consanguineous	North Gaalia/	AR/ ARG1	Complicated HSP (metabolic)	Two sibs : 2yrs 8months, 3 yrs/ 8,10yrs	ND	CT scan: Normal	1-Pyramidal signs(LL &/> UL: spasticity, hyperreflexia and extensor planter response) 2-Mental retardation 3-Psychiatric symptoms 4-Sphincter disturbance 5-Regression of milestones 6-Skeletal deformities (pes cavus, scoliosis) 7-Abnormal way of looking at objects

including globus pallidus and to lesser degree head of caudate nucleus and putamen as well as the cerebellum. Mild generalized cerebellar atrophy was found in both patients but there was no evidence of brain iron accumulation (hypointense signals in T2 and hyperintense signals in T1 weighted images) [Figure

[START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF]

] [Table

[START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF]

].

Table ( 3

 ( -5): Clinical data of two patients from family F25 with infantile neuroaxonal dystrophy (INAD) due to a mutation in PLA2G6 gene. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: -Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition. WMH: white matter hyperintensities.

	Family Code	F25	F25
	Mutated Gene	PLA2G6
	Individual Code	209	210
	Gender	M	F
	Clinical Diagnosis	INAD
	Origin	White Nile/ Kwahla tribe
	Age at Onset of Motor Symptoms	2 years	1 year 8 months
	Age at Initial Examination	2.5 years	3 years 8months
	Spasticity UL/LL	-/-	-/-
	Motor Deficit PUL/DUL	++/++	++/++
	Motor Deficit PLL/DLL	NA/NA	++/++
	Tendon Reflexes UL/LL Patellar	↑/↑	↑/↑
	Ankle Reflex/ Planter Response	N/↑↑	N /↓
		-/++/NA/NA	-/NA/NA/NA

Ataxia Eye/UL/LL/ Gait

	Dysarthria Spastic/Cerebellar	Aphasic	Aphasic
	Muscle Atrophy DUL/DLL	-/-	+/+
	Facial Atrophy	-	-
	Cog/ Psych signs	+/+	+/+
	Optic Atrophy	-	+
	Extrapyramidal Signs	-	-
	Sensory Loss	NA	NA
	Other Signs	Dysphagia	Dysphagia

WMHS in periventricular areas, basal ganglia including globus pallidus and to lesser degree head of caudate nucleus and Putamen plus the cerebellum. Mild Cerebellar atrophy

	5 by the two algorithms Polyphen2 and mutation taster [Table Disability Score	7
	MRI of the brain	WMHS in periventricular
		areas, basal ganglia
		including globus pallidus
		and to lesser degree head of
		caudate nucleus and
		Putamen plus the
		cerebellum. Mild Cerebellar
		atrophy.
	Electrophysiological Studies	Normal	Normal
	(NCS)
	CT scan of the brain	ND	ND
				98

Table ( 3-6):

 ( Clinical data of four patients from family F30 with Marinesco Sjogren Syndrome due to a mutation in SIL1 gene. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: -Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not applicable due to patients condition.

	Family Code		F30				F30	F30	F30
	Individual Code		257				258	259	260
	Dysarthria		++/++			++/++	-/-	++/++
	Spastic/Cerebellar
	Muscle Atrophy		-/-				-/-		-/-	-/-
	Family Code DUL/DLL		F30				F30	F30	F30
	Mutated Gene Facial Atrophy		-				-		SIL1	-	-
	Individual Code Cog/ Psych signs		257 +/-				258 -/-	259 +/-	260 +/-
	Gender Cataract	F Bilateral Cataract at 4 yrs	F Bilateral Cataract (operated on the	F Bilateral Cataract at 6 yrs (	Bilateral Cataract F at 4 yrs
		Clinical Diagnosis					right side)	operated on the Complex HA left side)
	Origin Extrapyramidal		-				-		Gazira, Almanagil -	-
	Signs	Age at Onset of Motor Symptoms			2 Yrs			2 Yrs	2 Yrs	2 Yrs
	Age at Initial Sensory Loss		-	37 Yrs		-	25 Yrs	-	15Yrs	-	13 Yrs
	Examination Other Signs of Eye	↓Visual acuity,		↓Visual acuity,	↓Visual acuity,	↓Visual acuity
		Motor Development ophthalmoplegia Delayed vertical		Delayed vertical ophthalmoplegia	Delayed vertical ophthalmoplegia	vertical Delayed ophthalmoplegia
		Intellectual development	N upgaze), saccadic (Restricted pursuit	N upgaze), saccadic (Restricted pursuit	N upgaze), saccadic (Restricted pursuit ,Slow	(Restricted upgaze), saccadic N pursuit
		Spasticity				+/++			-/++	saccades	-/+	-/+
		UL/LL
	Motor Deficit hypogonadism PUL/DUL Signs of	15yrs menopause -/-Menarche at		menstrual cycles -/-Never had	12yrs menopause -/-Menarche at	NA	-/-
		Motor Deficit		at 16 yrs	-/-			-/-	at 15 yrs	-/-	-/-
	PLL/DLL Other signs		Urinary			Short stature	Short stature	Epilepsy at age
		Tendon Reflexes	↑/↑ Incontinence			N/ N	N/ N	N/ ↑ of 7 months
		UL/LLPatellar	Dysphagia to				Short stature
		Ankle Reflex/ Planter liquids (mild) ↑/↓			N/ ↓	N/ ↓	↑/↓
		Response	Short stature
	Ataxia Eye/UL/LL/ Gait Disability Score	5	+/-/-/+++	5	+/++/++/++	+/++/++/+++ 5	+/++/++/+++ 6
	MRI of the brain	Global cerebellar		Global cerebellar	Global cerebellar	Global cerebellar
				atrophy			atrophy	atrophy	atrophy
	Electrophysiological		ND				ND	ND	ND
	Studies (NCS)

Table ( 3

 ( -7): Clinical data of three patients from family F26 with spinocerebellar ataxia (SCA7) due to a pathologic CAG repeat in ATXN7. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: -Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition.

	Family Code	F26	F26	F26
	Individual Code	216	218	220
	Cog/ Psych signs	+/-	-/-	+/-
	Optic Atrophy	+	+	-
	Family Code Extrapyramidal Signs	F26 Dystonia,	F26 -	F26 Marked bradykinesia
		Hypokinesia
	Mutated Gene		SCA7
	Sensory Loss	-	-	-
	Individual Code	216	218	220
	Other Signs and	Eye: optic atrophy.	Eye: optic	Eye summary: optic
	Gender specifications	F Ankle clonus,	F atrophy and	M atrophy, macular
	Clinical Diagnosis Origin	urinary incontinence /urgency	Complicated HA Khartoum -Alilfoon macular degeneration. Mild pes cavus, mild hearing impairment,	degeneration, diminished visual acuity, slow saccades, mild
	Age at Onset of Motor	34 yrs	17Yrs Urinary	14 Yrs horizontal & vertical
	Symptoms		incontinence	ophthalmoplegia
				(limitation)
	Age at Initial	49 Yrs	26 Yrs Myoclonus	17 Yrs
	Examination Disability Score	7	3	3
	Spasticity Brain MRI	++/++ ND	-/-Mild diffuse	-/+ ND
	UL/LL		brain atrophy
			(cortical,
	Motor Deficit	+/+	-/-cerebellar)	-/-
	PUL/DUL
	Motor Deficit Eye colour Image	+/+ ND	-/-Pale optic disc	-/-Pale optic disc with
	PLL/DLL		with vessel	vessel attenuation,
			attenuation.	salt and pepper
	Tendon Reflexes	↑/↑	↑/↑ Maccular	↑/↑ appearance of the
	UL/LL Patellar		degeneration.	retina wity thinning
	Ankle Reflex/ Planter Response	↑/↔	N /↔	(Retinitis pigmentosa ↑/↓ sine pigmentosa), macular
	Ataxia Eye/UL/LL/	NA	+/ /-/+	+/+++/+++/+++ degeneration.
	Gait	(blind)/+++/+++/NA
	Dysarthria	++/++	+/+	-/++
	Spastic/Cerebellar
	Muscle Atrophy	++/++	-/-	++/++
	DUL/DLL
	Facial Atrophy	-	-	-
				105

Table ( 3-8):

 ( Summary of mutations in known genes found in three families with pyramidal features. N.S.: Nonsense, M.S: Missense, Hom: homozygous, Het: heterozygous, Deleter: Deleterious, Prob Damag: Probably Damaging, NA: Not Applicable. NF not found. PhastCons score ranges from zero (low conservation) to 1 (high conservation), Phylop score from -14.1 (low conservation) to 6.4 (high conservation).

	Family Code	Gene	Involved	Exon/Intron	Predicted Change	Protein Change	Consequence	Zygosity	Conservation PhastCons PhyloP	Predicted Pathogenicity SIFT Polyphen2 Mutation Taster	Align GVGD	dbSNP	MAF EVS	Genomes	1000	EXAC Browser	Novelty/Reference
	F15	ARG1		Exon 4	c.434T>A	p.(Val145Glu )	M.S.	Hom	1.0	4.64	Deleter.	Damag.	Prob.	Causing	Disease	C35	NF	NF	NF	NF	Novel
	F25	PLA2G6	10 site Intron	Donor Splice	c.1427+2T> C	NMD possible Skip of exon 10 is very likely	S.D	Hom	1.0	2.38	NA	NA	NA	NA	NF	NF	NF	NF	Novel
	F30	SIL1		Exon 8	c.740C>T	p.(Ala247Val)	M.S.	Hom	0.99	4.64	Tolerated	Damag.	Prob.	Causing	Disease	C0	NF	NF	NF	0.00082%	Heterozygous	Databases
													108

Table ( 3-9):

 ( Summary of mutations in putative candidate new genes found in five families with spastic neurogenetic disorder. N.S.: Nonsense, M.S: Missense, Hom: homozygous, Het: heterozygous, Deleter: Deleterious, Prob Damag: Probably Damaging, NA: Not Applicable. NF not found. PhastCons score ranges from zero (low conservation) to 1 (high conservation), Phylop score from -14.1 (low conservation) to 6.4 (high conservation).

	Code	Family	Gene	Involved	Exon	Change	c DNA	Change	Protein	e	Consequenc	Zygosity	PhastCons	PhyloP	SIFT	Polyphen2	Taster	Mutation	GVGD	Align	Freq./Count	dbSNP	(AA/EA)	EVS	Genomes	1000	Browser	EXAC		Novelty
		Family Code F50 F37 F50 F41 FM3 FM7	Gene BIRC5 C21ORF91 ABHD16A CAMSAP3 ST7L PANK4		Exon Involved 4 5 3 12 1 15	c DNA Change c.340C>T c.327C>G c.430G>C c.1264C>T c.202G>T c.1798G>A p.(Val600Met) Protein Change p.(Phe109Leu) p.(Arg114*) p.(Ala144Pro) p.(Arg422Trp) p.(Ala68Ser)		Consequence M.S. M.S. N.S. M.S. M.S. M.S.	Zygosity Hom Hom Hom. Hom Hom Hom	Conservation 1.0 2.06 PhastCons PhyloP 1.0 2.71 2.63 0.94 1.42 1.0 2.95 0.99 4.73	SIFT Deleter. Deleter NA Deleter. Tolerated Deleter	Predicted Pathogenicity Polyphen2 Mutation Taster Prob. Damag. Disease Causing Benign Benign Disease Causing Benign NA NA Prob. Damag. Disease Causing Benign Benign Disease Causing Benign Prob. Damag. Disease Causing Benign	Align GVGD C 0 C 0 NA C 0 C 0 C 0	Freq./Count	dbSNP NF 0.002/0 NF <0.001/0 0.002/0 NF	MAF EVS (AA/EA) NF NF 0.03/0.08% NF 0.00/0.01%	Genomes	1000 NF NF NF NF NF		EXAC Browser NF 0.06295 % NF 0.098% 0.095 % s 0.00083 % s	Heterozygou Heterozygous Heterozygou	Novelty Novel Databases Novel Databases Databases Databases
		F41	MINK1		12	c.1207G>C p.(Glu403Gln)		M.S.	Hom		3.35	Tolerated	Benign Disease Causing	Causing	Disease	Benign	C 0		NF		NF		NF		NF		Novel
		F41	ZNF433		4	c.707G>A	p.(Ser236Asn)		M.S.	Hom		1.01	Tolerated	Poss.. Damag.		NA	Benign	C 0		NF		NF		NF		NF		Novel

Table ( 3-10):

 ( Clinical data of two patients from family F37 with a nonsense mutation in ABHD16A. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: -Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition. PVWMH: periventricular white matter hyperintensities.Three Sudanese sibs (one boy and two girls) were born to first degree consanguineous parents. They were born after uneventful pregnancy and delivery. Since birth they were noted to be abnormally developing. They were hypotonic at first and the parents noted delayed development. The proband (349) sat without support and sustained head control at 8 months. He could stand up at 2 years and two months. He had delayed speech; he said two words at 1.5 years then few more words at 3 years but till now no full sentence.

	Family Code		F37	F37
	Mutated Gene	ABHD16A
	Individual Code 3.3.2. Candidate genes in family in F41	314	315
	Gender	3.3.2.1.Case report	M	M
	Clinical Diagnosis	HSP with TCC
	Origin		Kosti/ Sudan
	Age at Onset of Motor Symptoms	6 months	6 months
	Age at Initial Examination	10 years	7 years
	Spasticity UL/LL	-/+++	-/+++
	Motor Deficit PUL/DUL	-/-	-/-
	Motor Deficit PLL/DLL	-/NA	-/+++
	Tendon Reflexes UL/LLPatellar	N/↑	N/↑
	Ankle Reflex/ Planter Response	NA/NA	N/↑↑
	Ataxia Eye/UL/LL/ Gait	-/-/-/-	-/-/-/-
	Dysarthria Spastic/Cerebellar	-/-	-/-
	Muscle Atrophy DUL/DLL	-/-	-/ ++
	Facial Atrophy	-	-
	MR/ Psych signs	+/+	+/ +
	Optic Atrophy	NA	NA
	Family Code		F37	F37
	Individual Code	314	315
	Extrapyramidal Signs	+ Choreoathetosis	+ Choreoathetosis
	Sensory Loss	NA	NA
	Other Signs		Sterotypic	Sterotypic behavior :

  novel mutation in exon 12 of the gene MINK1 [g.4791062G>C,

	c.1207G>C, p.Glu403Gln] (based on the longest transcript NM_153827.4)
	cosegregated with the disease distribution in family F41. MINK1 (misshapen-
	like kinase 1) is also known as MAP4K6,GN MINK, YSK2, hMINK, ZC3,
	MINK, MAP4K6, B55 and hMINKbeta.

MINK1 is located on the short arm of chromosome

17 (17p13.2)

[Ensembl:

Table ( 3

 ( -11): Clinical data of two patients from family F41. UL: upper limbs. LL: lower limbs.

	PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych:
	psychological. Clinical signs severity: -Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes:
	↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N:
	Normal. NA: Not Applicable due to Patients Condition.
	Family Code	F41	F41
	Mutated Gene	CAMSAP3 or MINK1 or ZNF433
	Individual Code	349	351
	Gender	M	F
	Clinical Diagnosis	Complex spastic ataxia
	Origin	White Nile State
	Age at Onset of Motor Symptoms	Birth	Birth
	Age at development of cataract	2 Yrs	20 months
	Age at Initial Examination	11 yrs 1 month	20 months
	Spasticity	+/++	-/-
	UL/LL
	3.3.3.
	Motor Deficit PUL/DUL	++/+	-/-
	Motor Deficit PLL/DLL	++/+	-/-
	Tendon Reflexes UL/LL Patellar	N/↑	↑/↑
	Ankle Reflex/ Planter Response	↑/↓	↑/↓
	Ataxia Eye/UL/LL/ Gait	+ sacc.pur/-/-/+	N/-/-/NA
	Dysarthria Spastic/Cerebellar	-/-	NA
	Muscle Atrophy DUL/DLL	+++/+++	-/-
	Facial Atrophy	+	-
	Cog/ Psych signs	+/+	NA/+
	Optic Atrophy	-	-
	Extrapyramidal	Tetubation & Resting Tremor	-
	Signs	Choreoathetosis Neck dystonia	123
		(retrocolis)

Exploration of family F50 3.3.3.1.Case report:

	Family Code	F41	F41
	LMN signs	Trunkal hypotonia Moderate	Trunkal hypotonia
		UL/LL muscle wasting (distal and
		proximal)
	Sensory Loss	-	-
	Dysmorphic features	Elongated face Malocclusion of	Mild Protrusion of upper and
		mouth	lower jaws
			Malocclusion of the mouth
	Skin	Café au lait spots Pustules	Café au lait spots
	Skeletal deformities	Exaggerated lumber lordosis ,	Exaggerated lumber lordosis
		Scoliosis
	Other Signs	Scoliosis	Lt divergent squint
		Nocturia
	Disability Score	5	3
	The affected individuals were two girls (402, 403) who were born to first degree
	consanguineous parents after an uneventful pregnancy and delivery. They looked
	healthy for few months after birth. The disease started with epilepsy. The index
	(older, 402) had earlier onset at 7 months of age whereas the younger had it at 2
	years. They had myoclonic epilepsy with myoclonus occurring mainly when
	awake. Delayed psychomotor development was observed by the family. This was
	evident in the delayed social smile, speech, apparent decreased intellect and gross
	motor development. The younger sib (403) showed regression of speech and she
	became aphasic after she could say a few words. They couldn't walk until the age

of 4-5 years. They acquired sphincter control just around the age of examination

(4-5 years)

. The parents noticed that the index developed cerebellar symptoms (eg.

Table (

 (

	Family Code	F50	F50
	Mutated Gene	BIRC5 or C21orf91
	Individual Code	402	403
	Gender	F	F
	Clinical Diagnosis	Complicated HSP
	Origin	Neyala, Western sudan
	Age at Onset	7 months	2 Yrs
	Age at Initial Examination	5 Yrs	4 Yrs
	Spasticity	++/	+++/+++
	UL/LL	++ (Rt)+++ (Lt)
	Motor Deficit PUL/DUL	+/+	-/-
	Motor Deficit PLL/DLL	+/+	-/-
	Tendon Reflexes UL/LLPatellar	↑/↑	↑/↑
	Ankle Reflex/ Planter Response	↑/↓	NA/ ↑↑
	Ataxia Eye/UL/LL/ Gait	+(saccadic pursuit)/++/+/++	-/NA/NA/NA
	Dysarthria Spastic/Cerebellar	NA/NA	NA/NA

3-12): Clinical data of two patients from family F50. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: -Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition. TCC: thin corpus allosum. CSF: cerebrospinal fluid.

Table ( 3-13):

 ( Clinical data of three patients from family FM3. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: -Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition. PVWMH: periventricular white matter hyperintensities. WMH: white matter hyperintensities.

	Dysarthria	Aphasic	Aphasic		Aphasic
	Spastic/Cerebellar
	Muscle Atrophy	-/-	++/++		/+
	DUL/DLL
	Facial Atrophy	-	-		-
	Family Code MR/ Psych signs	FM3 +/-	FM3 +/-		FM3 +/-
	Mutated Gene Optic Atrophy	-	ST7L -		-
	Individual Code Extrapyramidal	2022 Severe generalized dystonia	2020 Severe generalized dystonia		Severe generalized 2021 dystonia
	Gender Signs	F	M		F
	Clinical Diagnosis Skeletal deformities	Pes cavus	Complicated HSP (Dystonia) Pes cavus	Kyphoscoliosis
	Origin Sensory Loss	NA	Gezira/ Awamra tribe NA		NA
	Age at Onset of Motor Other	4 months Scarce movement	Before birth Scarce movement		4month Scarce movement
	Symptoms Disability Score	7	(decreased movement 7		7
			in late pregnancy)
	MRI of the brain	MRI of the brain:	ND		MRI of the brain:
	Age at Initial	2 Yrs mild increase of	7 Yrs		4 Years cortical and
	Examination	internal CSF spaces			cerebellar vermial
		(mild subcortical			atrophy, PVWMH
	Spasticity	+++/+++ atrophy?) and	+++/+++		+++/+++ and subcortical
		PVWMH			WMH.
	UL/LL
	Electrophysiological
	Motor Deficit Studies (NCS)	NA	NA		NA
	PUL/DUL
	Motor Deficit PLL/DLL	NA	NA		NA
	Tendon Reflexes UL/LL	↑/↑	↑/↑		↑/↑
	Patellar
	Ankle Reflex/ Planter	N/↑↑	↑/↑↑		↑/↑↑
	Response
	Ataxia Eye/UL/LL/ Gait	-/-/-/-	-/-/-/-	+(Slow saccades)/-/-
					/-
					135

Table ( 3-14):

 ( Clinical data of five patients from family FM7 branch 1. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: -Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition.

	Facial Atrophy	-		-		-		-		-
	Cog/ Psych	+/+		+/+		+++/+		+++/ +		+++/+
	signs	Nightmares,								Nightmares.
		emotional	Blunt mood	Nightmares		Nightmares,		emotional
		lability and				emotional		temper,		lability and
		euphoria				lability and		aggression		euphoria
						euphoria		episodes and
								euphoria		aggressive
	Family Code					FM7 branch 1				episodes
	Mutated Gene Optic Atrophy	-		-		PANK4 -		-		-
	Individual Extrapyramidal	2047 -		2048 -		2049 -		2050 -		2051 Hypokinesia
	Code Signs
	Gender Sensory Loss	M NA		F -		F NA		M NA		M NA
	Clinical				Complicated HA
	Diagnosis
	Other Signs	Hearing		Hearing	Khartoum Gamooia tribe Microcephaly Microcephaly	Microcephaly
		impairment	impairment
	Age at Onset	and mutism	and mutism		Birth Hearing		Urinary		Urinary
					impairment	incontinence,	incontinence,
	Age at Initial	29yrs		26yrs	20yrs and mutism		13yrs Dysphagia,		18yrs Progressive
	Examination							Hearing		Hearing
							impairment and	impairment and
	Spasticity	-/-		-/-		-/+ Rt.		+/+/+++ mutism		++/++ mutism
	UL/LL/ Gait Disability Score	2		1		2		7		6
	Motor Deficit MRI of Brain	-/-ND		-/-ND	3+ (difficult to Severe cortical	3+ (difficult to Severe cortical	3+ (difficult to Severe cortical
	PUL/DUL				assess) atrophy	assess) atrophy	assess) atrophy
	Motor Deficit PLL/DLL	-/-		-/-	3 (difficult to especially the frontal region assess) Cerebellar	3 (difficult to assess) especially the frontal region Cerebellar	(difficult to assess) especially the frontal region Cerebellar
	Tendon	↑/↑		↑/↓	↑/↑ atrophy TCC	↑/↑ atrophy TCC	↑/↑ atrophy TCC
	Reflexes UL/LL				Hypomyelinati	Hypomyelinatio	Hypomyelinatio
	Patellar					on WMH		n WMH	n WMH
	Ankle Reflex/ Electrophysiolo	↑Rt. /↔ N	N	↓/↔	N	N /↔	N	↑/Rt.↔ Lt. ↓	N	↑/↓Lt. ↔Rt.
	Planter gical Studies
	Response (NCS)
	Ataxia	-/++/-/++		-/++/-/++		Interrupted		+/+/+/+	Nystagmus/+/+/
	Eye/UL/LL/					saccadic				+
	Gait				pursuit /++/-
						/++
	Dysarthria	NA/NA		NA/NA		NA/NA		NA/NA		NA/NA
	Spastic/Cerebel
	lar
	Muscle	-/-		-/-		-/-		-/-		+/+	141
	Atrophy
	DUL/DLL

Table ( 3-15):

 ( Clinical data of two patients from family FM7 branch2. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: -Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition.

	Family code	FM7 branch 2
	Gene		?
	Individual code	2056	2059
	Gender	M	F
	Clinical Diagnosis	Complicated HA
		Khartoum Gamooia tribe
	Age at onset		Birth
	Age at Initial	29yrs	9yrs
	Examination
	Spasticity	-/-	-/-
	UL/LL
	Motor Deficit	-/-	-/-
	PUL/DUL
	Motor Deficit	-/-	-/-
	PLL/DLL
	Tendon Reflexes	N/N	N/↓
	UL/LL Patellar
	Ankle Reflex/	↑/↓Rt. ↔Lt.	N /↔
	Planter Response
	Ataxia	Saccadic	Slow saccades /+/-
	Eye/UL/LL/ Gait	pursuit /+/-/+	/+
	Dysarthria	NA/NA	NA/NA
	Spastic/Cerebella
	r
	Muscle Atrophy	-/-	-/-
	DUL/DLL
	Facial Atrophy	-	-	143
	Cog/ Psych signs	-	-

Table ( 3-16):

 ( Clinical summary of 23 families with unidentified genetic cause (family 27 is presented in a separate table). HSP:hereditary spastic paraplega, UL: upper limbs, LL: lower limbs, ND: not done, CT: computerized tomography scan, MRI: magnetic resonance imaging, EEG: electroencephalogram, EMG: electromyogram, NCS: nerve conduction studies.

	Fam. code Fam. code F12 code code code Fam. code code code code code Fam. Fam. Fam. Fam. Fam. Fam. Fam.	Origin/ Consan guinity Origin/ Consan guinity Elfashir , Darfur/ Consan guinity Consan guinity nsanguini ty Origin/ Consan guinity Consan guinity nsanguini ty onsangui nity Consan guinity consanguineous Origin/ Origin/ Origin/Co Origin/ Origin/Co Origin/C Origin/	Inheritan ce/ Gene Inheritanc e/ Gene e/ Gene e/ Gene e/ Gene Inheritanc e/ Gene e/ Gene e/ Gene ce/ Gene e/ Gene AR Inheritanc Inheritanc Inheritanc Inheritanc Inheritanc Inheritan Inheritanc	Phenotype summary Phenotype summary HA summary summary summary Phenotype summary summary summary summary summary Complicated Phenotype Phenotype Phenotype Phenotype Phenotype Phenotype Phenotype	Age at onset/E xam. Age at onset/E xam. sibs : Birth / 17 month, onset/E xam. onset/Exam. Electrophysiol ogical studies EMG/NCS ,EEG) Electrophysio logical studies EMG/NCS ,EEG) logical studies EMG/NCS ,EEG) siological studies EMG/NCS ,EEG) onset/E xam. logical studies EMG/NCS ,EEG) Age at onset/E xam. Electrophysio logical studies EMG/NCS onset/E xam. logical studies EMG/NCS ,EEG) onset/E xam. logical studies EMG/NCS ,EEG) onset/E xam. ological studies EMG/NCS ,EEG) onset/Exam. ysiologic Brain Imaging Brain Imaging brain: Cerebellar atrophy and PVWMH Brain Imaging al studies EMG/NC S ,EEG) Two ND MRI of the Age at Electrophysio Brain Imaging Age at Electrophy Brain Imaging Clinical features summary Clinical features summary Clinical features summary normal tone and flexor planter response) 2-Delayed psychomotor development 3-Cerebellar signs (severe: trunk, LL, UL, gait and eye 4-Mental retardation 5-Drooling of saliva Clinical features summary 1-Pyramidal signs (In one individual: LL and />UL:, hyperreflexia but Clinical features summary Age at Electrophysio Brain Imaging Clinical features summary Age at Electrophysio Brain Imaging Clinical features summary Age at Electrophysio Brain Imaging Clinical features summary Age at Electrophysi Brain Imaging Clinical features summary Age at Electroph Brain Imaging Clinical features summary
	F1-BRANC H-C F1-BRANC H-D F2 F7 F11-BRANC H-1 F11-BRANC H-2 F17 ous F21 F22 F23 F24 F28 F31 F35 F36 BRANC H-1 F38-BRANC H-1 F38-BRANC H-2 F42 F44 F45 F46 F47 F48 FM1 FM6 FM5 F49 FM7-BRANC H-2	Al Jazira/ Al Jazira / White Nile State Kurdufan/ consanguineous Al Jazira / consanguineous Al Jazira / Sennar North Danagla/ Nile River State(Atbara, Gaalia)/ White Nile State (Dwem Kawahla)/ North (Danagla)/ consanguineous Kurdufan/ Nile River State/ consanguineous Darfur Nyala/ consanguineous consanguineou consanguineous (Dwem)/ non-consanguineous Falata/ consanguineous consanguineous consanguineous Nyala Darfur Falata/ consanguineous consanguin s consanguineous consanguineous White Nile State/ consanguineous White Nile State/ consanguineous Al Jazira / consanguineous Al Jazira / consanguineous Al Jazira / consanguineous Khartoum Gaalia/ consanguineous Kurdufan / consanguineous North (Mahas)/ consanguineous White Nile ,Gezira consanguineous Gadarif states/ North Shaaygia/ (Elfashir)/ non-consanguineous Gaalia / consanguineous Darfur (Elfashir)/ non-consanguineous Darfur Khartoum Gamooia/ consanguineous	Sporadic Complicated HSP Sporadic Complicated HSP Sporadic Pure HSP AR/Pure HSP Pure HSP Sporadic Complex disorder with HSP Sporadic Complex disorder with HSP Sporadic/ Pure HSP Pure HSP Sporadic Spastic Ataxia AR Complex disorder with HSP AR Comlicated HSP (with Dementia) PLP1 like Identical twins : sporadic Complicated HA Sporadic Complicated HSP AD Complicate d HSP/HA spectrum Sporadic Complex HSP (minimal complexity ) AR Complex HSP AR Complex HA (minimal comlexity) AR Complicate d HSP/HA spectrum AR Complicate d HSP AR Complicate d HSP Sporadic Complicate d HA AR Complicate d HA AR Complicat ed HA AR/Pure HSP Pure HSP AR/MND like Complicate d HSP (ALS like) AR Complicated HSP AR/ALS2 Complicated HSP (Juvenile ALS) AD Complicate d HSP HA AD Complicated	1.5 yrs/ 15 yrs Birth/1 0.5 yrs 14yrs/ 23 yrs Three sibs(tw o of them identica l twins): 4yrs/ 34-40 yrs 1.4 yrs / 5 yrs 4 months 1.5 yrs/ 5 yrs 3 yrs 28yrs/3 8yrs 44yrs/4 7yrs Two sibs (one died) : 1.5 yrs / 8 yrs Two sibs (only one sampled 4.5 yrs /7 yrs Identical twins : 11 yrs/18 yrs 7 yrs/12.5yr ND ND ND ND EEG: evidence of generalized epilepsy ND ND ND EEG: active generalized epilepsy ND EMG/NC S: low motor amplitude in lower limb (axonal motor PN) EEG :phot osensitive generalize d epilepsy (1 Pt) ND Father and two daughte rs 38, 4, 2 yrs/ 38 yrs, 7m/7m ND 19 yrs/ 27 yrs ND One patient : 13 yrs/ 26 yrs ND ,EEG) Three sibs : 7, 7, 12 yrs/ 15, 18, 21 yrs ND Two sibs : 11, 13 yrs / 20, 17 yrs ND One patient (one died) : 2 yrs / 8 EMG/NCS: Normal Two male sibs : Birth/ 4,9 years EMG/NCS: prolonged distal motor latency in LL Birth/ 4 yrs ND Two sibs : 3, 13 yrs /12,25 yrs ND Boy and his materna l uncle : 35, 38 yrs / 38, 52 yrs EMG/NCS: Sensorimotor axonal neuropathy Two sibs : 15, 16yrs 7month ND One patient ND sampled : 39 yrs/ 40 yrs Two sibs : ND 4, 8 months/ Four patients (three sibs and a cousin): ND 15,18 yrs Gradual amelioration and disease progression plateau reached by 4, 5 yrs s / 15 /16 yrs 10 months 20 , 24 yrs /24, 33 yrs axonal neuropathy Two male sibs : EMG/NCS (older patient): Mild sensorimotor 3-18 months/ 11-13 yrs daughter : Birth / 29,9 years CS: Normal ND ND Normal CT scan of the brain ND MRI brain:Mild global cerebellar atrophy and mild TCC MRI brain:Mild global cerebellar atrophy and mild TCC Normal Mild cerebellar lobes and vermial atrophy with prominent folia. Few lacunar high signal intensities Brain MRI: generalized brain atrophy (cortical, cerebellar and TCC) PVWMH and cerebellar hyperintensity signals MRI Brain : mild cortical atrophy. Cerebellar atrophy. PVWMH signal lesions Normal MRI Cerebellar atrophy (prominent foli, vermial atrophy and mega cistern magna) ND ND ND MRI brain : Mild hypomyelination features Mild cerebellar atrophy ND MRI: ND ND ND ND MRI of the brain: Cerebellar atrophy (the younger patient) Spinal MRI : no spinal anomaly or compression MRI brain: ND Hypomyelination, TCC and mild prominence of internal and external CSF space for age MRI brain : Moderate TCC 1-Pyramidal signs(LL &/> UL: spasticity, hyperreflexia and extensor planter response) 2-Cerebellar signs (UL, dysarthria and eyes) 3-Congenital hip dislocation 4-Cognitive Impairment 5-Optic atrophy 6-Scoliosis 7-Distal muscle wasting (UL &LL) 1-Pyramidal signs (LL:spasticity, hyperreflexia ) 2-Cerebellar signs (LL & dysarthria) 3-Recurrent congenital cataract and postoperative retinal detachment (almost blind) 4-Cognitive Impairment 5-floppy after general anaesthesia 6-Generalized muscle wasting 1-Pyramidal signs (LL: spasticity, hyperreflexia and extensor planter response)2-Cerebellar signs (Mild:UL & nystagmus) 3-Vibration sense diminished 1-Pyramidal signs(LL: spasticity, hyperreflexia and extensor planter response) 1-Pyramidal signs (Moderate LL and UL: spasticity, hyperreflexia but flexor planter response) 2-Matching level of motor deficit (UL and LL) 3-Cerebellar signs (Moderate Gait, trunk: limbs and eye movement could not be assessed properly) 4-Regression of milestones 5-Cognitive deterioration 6-Mild urinary urgency 7-Epilepsy at 4 yrs 3 months 8-Dysphagia 9-Mild psychiatric symptoms 10-Optic atrophy,milde lid retraction and decreased visual acuity 11-Auditory test: mild to moderate sensory neural hearing loss 1-Pyramidal signs (mild:UL > LL: spasticity, hyperreflexia but flexor planter response) 2-Matching level of motor deficit (UL and LL) 3-Cerebellar signs (Moderate Gait, trunk: limbs and eye movement could not be assessed properly) 4-Regression of milestones (speech and motor) 5-Cognitive deterioration 6-Deterioration following a febrile illness then 1-Pyramidal signs(LL: spasticity, hyperreflexia) 1-Pyramidal signs(LL &/> UL: spasticity, hyperreflexia & indifferent planter response) 2-Cerebellar signs (UL, LL, Gait, trunk and eyes(nystagmus & saccadic pursuit)) 3-Vitiligo secondary to botulinum toxin injection as treatment for spasticity) 4-Urinary urgency 1-Pyramidal signs (LL and UL: spasticity, hyperreflexia and extensor planter response) 2-Cerebellar sigs (trunkal)(limbs could not be assessed) 3-Stereotypic behavior: head movements and teeth clenching 4-Extrapyramidal signs (dystonia) 5-UL and LL wasting (distal and proximal) 6-Mental retardation 7-Psychiatric symptoms: bouts of excessive crying 8-Epilepsy: generalized tonic clonic since 1.5 yrs 9-Pale optic disc, thin retinal background and hypermetropic fundi. 10-Skeletal deformities: talipes equina varus 11-Regression of milestones 1-Pyramidal signs (LL and UL: spasticity, hyperreflexia, speech and extensor planter response) (spasticity predominant and intellectual deterioration) 2-Intellectual deterioration (dementia like with loss of memory) 3-Cerebellar signs (gait, speech and eye:nystagmus, saccadic pursuit) 4-Regression of milestones 5-Mild horizontal eye movement limitation(ophthalmoplegia) 6-Generalized tonic clonic epilepsy 7-Sphincter disturbance 8-Generalised mild muscle wasting 1-Pyramidal signs(Mild to moderate: LL &/> UL spasticity, hyperreflexia) 2-Cerebellar signs (Moderate: UL, LL, Gait, trunk and eyes(saccadic pursuit)) 3-Epilepsy (seizures controlled by treatment) 4-Decreased visual acuity but normal fundus (myopia) 5-Minimal cognitive impairment 1-Pyramidal signs (moderate to severe: LL &>UL: spasticity and extensor planter response but absent other reflexes) 2-Cerebellar signs (moderate: UL, LL, Gait, trunk and eyes (saccadic pursuit) 3-Extrapyramidal signs (choreoathetotic movement) 4-Cognitive Impairment 5-Psychiatric symptoms (irritable mood) 6-Skeletal deformities and contractures (scoliosis, Lt talipes equinovarus, fixed flexion deformity of the hip joints Lt>Rt) 7-Dysphagia 1-Cerebellar signs (UL, LL, Gait, trunk) 2-Pyramidal signs (LL &/>UL: spasticity, hyperreflexia and extensor planter response) 3-Convergent squint (horizontal ophthalmoplegia) 4-Psychiatric symptoms (irritability: in one) 5-The father (previously not known to have a disease) only has horizontal opthalmoplegia/ mild UL ataxia / mild pes cavus 1-Pyramidal signs ( LL &/> UL: spasticity, hyperreflexia) 2-Hearing Impairment 1-Pyramidal signs(LL & UL: spasticity, hyperreflexia and extensor planter response) 2-Cerebellar signs (could not be examined appropriately) 3-Blindness 4-Severe proximal & disal muscle wasting (UL & LL) 5-Contracture(fixed flexion of knee) 6-Sphincter disturbance 7-Impaired sexual function 8-Hearing impairment 8-Dysphagia 8-Normal cognition 1-Cerebellar signs (UL, LL, Gait, trunk) 2-Pes cavus (one) 3-Seasonal skin rash (winter: one patient)  One sib(the younger): Cerebellar signs (UL, LL, Gait, trunk) 2-Pes cavus (one) 3-Seasonal skin rash (winter: one patient)  The other (older): 1-Pyramidal signs (LL(Lt>Rt): spasticity, hyperreflexia and unilateral Lt extensor planter response (indifferent in the other) 2-Intellectual impairment 3-ataxia, 4-Generalized tonic clonic epilepsy at 6 yrs 5-Scoliosis Lt sided pes cavus and wrist deformity 6-LL wasting (proximal and distal) 1-Pyramidal signs predominant(spasticity, hyperreflexia and flexor planter response) 2-Cerebellar signs (UL, LL, Gait, trunk and eyes:slow saccades)) 3-Regression of milestones 4-Sphincter disturbances 5-Epilepsy: myoclonic epilepsy (awake or asleep) 6-Skeletal deformity: Lumbar kyphosis 7-Dysphagia (drooling of saliva) 8-MR 9-Psychiatric symptoms: irritability 10-Skin: After he received omega3 fatty acids: 1-Pyramidal signs (LL & UL: spasticity, hyperreflexia but flexor planter response) 2-Cerebellar signs (UL, LL, Gait, neck, trunk and eyes) (spastisity predominant) 3-Delayed psychomotor development 4-Drooling of saliva 5-Eye:slow saccades, saccadic pursuit, nystagmus, horizontal ophthalmoplegis and occulomotor apraxia (one) 6-Sphincters intact 7-Normal cognition 8-Febrile convulsions once (one) 1-Floppy at birth 2-Cerebellar signs (UL, LL, Gait, speech, trunk and eyes:nystagmus) 3-Pyramidal signs (spasticity: UL and LL (proximal more than distal), hyperreflexia and flexor planter response) (ataxia predominant) 4-Delayed psychomotor development 5-Recurrent febrile convulsions since 3 months (mother noted eye movement increased with fever) 6-Sphincters: not yet dry by day 1-Cerebellar signs (Both: UL, LL, speech, gait, trunk and eyes: nystagmus) 2-Pyramidal signs (In the older the individual: spasticity, hyperreflexia and extensor planter response) 3-Scoliosis 4-Sphincter disturbance (the younger individual) 5-Normal cognition 6-Epilepsy (partial with secondary generalization: the younger individual) 1-Cerebellar ataxia (one patient: UL, LL, speech, gait and eye (saccadic pursuit)) 2-Mild Pyramidal signs (In the younger individual: spasticity, hyperreflexia and flexor planter response) (ataxia predominant) 3-Extrapyramidal signs in the older individual (mild dystonia and chorea) Pure Pyramidal signs (LL only in the older and mild UL and LL in the younger: spasticity, hyperreflexia and Rt unilateral extensor planter response in the older) 2-Mild scoliosis (older sib) 1-Pyramidal signs (LL &/> UL: spasticity, hyperreflexia and extensor planter response) 2-Mild cerebellar signs (UL, LL, Gait) 3-Pseudobulbar palsy (dysphagia to both liquid and solids, spastic anarthria, spastic tongue) 4-Lower motor neuron signs (generalized LL and UL muscle wasting: distal > proximal, fasciculations) 5-Subjective feeling or respiratory muscle weakness. 6-Facial muscle atrophy (temporalis) 7-Pes cavus 1-Pyramidal signs (LL: spasticity, hyperreflexia and extensor (posteriorly more) planter response) 2-Nystagmus (2 BEATS), saccadic pursuit and slow saccades 3-Epilepsy (one: febrile convulsions at 8 ND 1-Pyramidal signs (LL &/> UL: spasticity, hyperreflexia and extensor planter response) 2-Dysphagia 3-Involuntary laughter 4-Distal muscle wasting (variable in two individuals) Features of months and diagnosed and started treatment at 10 month) 4-hypomyelination Extrapyramidal signs (in one:hypokinesia) 5-Delayed motor development and speech. 6-Normal intelligence. atrophy and PVWMH signal lesions (spastisity predominant) 3-LL muscle wasting (distal and proximal) 4-Hypopigmented spots 1-2cm in diameter behind RT knee-and in LT axilla (one patient) 5-Dysphagia to fluids mainly (same individual) MRI Brain (older patient): Cerebellar 1-Pyramidal signs (UL and LL: severe spasticity, hyperreflexia and extensor planter response) 2-Cerebellar signs (UL, LL, Gait, trunk and eyes: slow saccades) MRI : Father and EMG/N CT scan : Normal 1-Deafness 2-Mutism 3-Mild ataxia
					yrs 4	improved gradually 7-Auditory test: mild to moderate conductive hearing hypopigmented areas non scaly maccules on the face,neck and UL
					months	loss associated with Eustachian tube obstruction (? Temporary)
						146 149

Table ( 3-17):

 ( Clinical summary of family F27 with unidentified genetic cause. HSP:hereditary spastic paraplega, UL: upper limbs, LL: lower limbs, ND: not done, CT: computerized tomography scan, MRI: magnetic resonance imaging, EEG: electroencephalogram, EMG: electromyogram, NCS: nerve conduction studies.

	Fam. code	sanguinity	Origin/Con	/ Gene	Inheritance	summary	Phenotype	/Exam.	onset	Age at	,EEG)	EMG/NCS	studies	siological	Electrophy	Imaging	Brain	Clinical features summary
	F27		Kurdufan/ non-consanguineous	AR		Syndromic features ( ?new syndrome)	Three sibs: 1.5-3 yrs / 5-15 yrs	EEG: Normal (one) EMG/NCS : ND		Severe generalized brain atrophy with PVWMH signals and hypomyelinatio n. features of	1-Pyramidal signs (LL &/> UL: spasticity, hyperreflexia and flexor planter response) 2-Cerebellar signs (UL = LL, Gait, trunk and eyes) 3-Extrapyramidal signs (hypokinesia) 4-Facial dysmorphism 5-Mental retardation 6-Short stature and severely delayed psychomotor development (general growth entrapment/arrest since around 1-3 yrs of age) 7-Psychiatric symptoms (irritability, emotional lability) 8-

Fundus: optic atrophy with retinitis punctata albescence (in all three sibs) (mottled retina and is a variant from retinitis pigmentosa); hypermetropia in the older sib with most advanced disease 9-Skin rash (scaly seasonal) 10-Hair lighter colour and smoother texture than sibs. 11-DM in the older sib with more advanced. 12-Impaired liver function in one (mild increased liver enzymes: ALT,AST,ALPand increased bilirubin)

/Amyo + CI SPG11, SPG15, SPG20, SPG22, SPG26 SPG35, SPG64 No SPG : EXOSC3 Ataxia + PN/Amyo -CI SPG28, SPG30, SPG60, SPG68 SPG75/ MAG No SPG: LYST ARSACS

			Autosomal Recessive
			C
	P
		C: +TCC HSP	C: +CI	C: +PN/Amyo	C: +Ataxia	EYE
	SPG7, SPG5 SPG24, SPG28 SPG30, SPG48 SPG51, SPG27 SPG52, SPG11 SPG62, SPG65	SPG11, SPG15 SPG18, SPG21 SPG32, SPG35 SPG46, SPG47 SPG3A, SPG48 SPG56/CYP2U1	SPG11, SPG15 SPG58, SPG5 SPG14, SPG18 SPG20, SPG21 SPG23, SPG26 SPG27, SPG32 SPG35, SPG44	PN + Amyo SPG3A, SPG9 SPG11, SPG39 SPG43, SPG55 SPG66, SPG68 SPG74	SPG7, ARSACS /SACS, SPG58, SPG5, SPG11, SPG15, SPG20 SPG21, SPG26,	OA SPG7 SPG35 SPG57 SPG45 SPG54
	SPG15, SPG71 SPG3, SPG72	SPG55, SPG54 SPG63, SPG65	SPG45, SPG46 SPG47, SPG48	PN -Amyo SPG14, SPG15	SPG27, SPG28, SPG30, SPG35,	SPG55 SPG68
	SPG73 No SPG: ADAR1	SPG66, SPG67 SPG71, SPG74	SPG51, SPG50 SPG49/SPG56	SPG26, SPG28 SPG23, SPG25,	SPG46, SPG54, SPG39, SPG44,	SPG74
	IFIH1	SPG49/ TECPR2	SPG52, SPG53	SPG30	SPG59, SPG60,
	RNASEH2B		SPG54, SPG59	SPG56/CYP2U1	SPG64, SPG68,
			SPG55, SPG64	SPG57, SPG60	SPG75/MAG
			SPG65, SPG69 No SPG: GAD1 EXOSC3 SPG49/TECPR	SPG61, NoSPG: CCT5 LYST ARSACS/SACS FAM134B	No SPG: EXOSC3, LYST, SPG49/TECPR	RD/MD SPG11 SPG15
			2	Amyo -PN	2
				SPG5, SPG20,
				SPG35 SPG47,
				SPG52 SPG51, SPG64 SPG65, SPG67 SPG70, No SPG: EXOSC3 BICD2		Cataract SPG9 SPG46 SPG69
				MAG
			Ataxia + PN

4.6.2. Correlations: 4.6.2.1. Microtubules dynamics, Development, Amino acid metabolism, Transport and Organelle shaping:

Table ( 4-2):

 ( Tablesummarizingthe 88 HSP clinico-genetic entities with special focus on the functions of their proteins and our suggested primary and secondary functional categories. P: pure, C: complex, AD: autosomal dominant, AR: autosomal recessive.

	SPG7 (AR/ SPG13 (AD) SPG36 (AD) SPG39 (AR) SPG49 (AR) SPG64 (AR) No SPG (AR) No SPG (Mito)	#607259 #605280 613096% #612020 #615031 #615683 / % #256840 No	SPG7 HSPD1 12q23-24 PNPLA6 TECPR2 ENTPD1 CCT5 MT-ATP6	4-42 17-68 14-33 Infancy, Infancy 1-4 Infancy 30-50	P/C P C C C C C P/C	Paraplegin Heat shoch protein 60 Kda Protein not identified Neuropathy target esterase Tectonin beta-propeller repeat-Ectonucleoside triphosphate protein T-complex protein 1 subunit ATP synthase subunit a	Component of the Mitochondrial chaperonin / Protein not identified Enzyme: Neuropathy Protein involved in Lipid Mitochondrial protein Enzyme: Hydrolyzes ATP Cytosolic chaperonin of Respiratory chain complex	Mitochondria Mitochondria Protein not Lipid Metabolism Endolysosome Nucleotide Metabolism Protein Folding? Mitochondria	Degradation	Casari et al., Hansen et al., Schüle et al., 2011 Rainier et al., Oz-Levi et al., Novarino et al., 2014 Bouhouche et Verny et al.,
	SPG code (Inheritance) ?AD) SPG20 (AR) SPG17 (AD) SPG37 (AD) SPG3A (AD/AR) SPG58 (AR/ ?AD) SPG38 (AD) SPG56 (AR) SPG74 (AR) No SPG (Mito) SPG50 (AR) SPG45 No SPG (AR) (SPG65) (AR) SPG31 (AD) SPG43 (AR) SPG9 (AD/AR) #601162 ALDH18A1 OMIM # / % Gene OMIM # #275900 KIAA0610 for HSP #270685 BSCL2 611945% 8p21.1-Spartin #606439 ATL1 No OMIM # for HSP KIF1C q13.3 612335% 4p16-p15 #615030 CYP2U1 #616451 IBA57 No MT-CO3 #612936 AP4M1 #613162 NT5C2 #614678 EXOSC3 OMIM # for HSP #610250 REEP1 #615043 C19orf12 SPG30 (AR/AD) #610357 KIF1A SPG72 (AR/AD) #615625 REEP2 No SPG (AR/AD) #615290 BICD2 SPG4 (AD) #182601 SPAST SPG6 (AD) #600363 NIPA1 SPG8 (AD) #603563 KIAA0196 SPG10 (AD) #604187 KIF5A SPG12 (AD) #604805 RTN2 SPG33 (AD) #610248 ZFYVE27/ Protrudin SPG42 (AD) #612539 SLC33A1 SPG73 (AD) #616282 CPT1C No SPG (AD) NO OMIM # for HSP VCP SPG19 (AD) 607152% 9q33-q34 SPG29 (AD) 609727% 1p31.1-21.1 SPG40 (AD) No OMIM # / % ? SPG41 (AD) 613364% 11p14.1-11p.2 SPG5/SPG5A (AR) #270800 CYP7B1 SPG11 (AR) #604360 KIAA1840 SPG15 (AR) #270700 ZFYVE26 SPG21 (AR) #248900 ACP33 SPG57 (AR) #615658 TFG No SPG (Mito) No MT-TI SPG51 (AR) #613744 AP4E1 SPG75 (AR) MAG OMIM # #616680 for HSP SPG44 (AR) #613206 GJC2 SPG66 (AR) NO ARSI SPG59 (AR) No OMIM # USP8 OMIM # for HSP SPG14 (AR) #605229 3q27-q28 SPG1 (X-#303350 L1CAM No SPG (AR) No FAM134B linked) OMIM # SPG26 (AR) #609195 B4GALNT1 SPG28 (AR) #609340 DDHD1 SPG46 (AR) #614409 GBA2 SPG47 (AR) #614066 AP4B1 SPG52 (AR) #614067 AP4S1 SPG53 (AR) #614898 VPS37A / % SPG60 (AR) No OMIM # / % WDR48 SPG67 (AR) No OMIM # / % PGAP1 SPG23 (AR) 270750% 1q24-q32 for HSP No SPG (AR) No IFIH1 SPG68 (AR) No OMIM # FLRT1 SPG24 (AR) 13q14 OMIM # %607584 for HSP SPG2 (X-#312920 PLP1 SPG61 (AR) #615685 ARL6IP1 / % SPG25 (AR) #608220 6q23-24.1 linked) No SPG (AR) #214500 LYST SPG54 (AR) #615033 DDHD2 SPG69 (AR) No OMIM # RAB3GAP2 SPG27 (AR) 609041% 10q22.1-q24.1 SPG22 (X-#300523 SLC16A2 linked) / % SPG62 (AR) No ERLIN1 SPG32 (AR) 611252% 14q12-q21 No SPG (AR) No RNASEH2B SPG16 (X-300266% Xq11.2 SPG18 (AR) #611225 ERLIN2 SPG35 (AR) #612319 FA2H SPG48 (AR) #613647 AP5Z1 OMIM # SPG55 (AR) #615035 C12orf65 SPG71 (AR) No ZFR for HSP for HSP OMIM # OMIM # / % SPG63 (AR) #615686 AMPD2 SPG70 (AR) No OMIM # MARS OMIM # linked) for HSP No SPG (AR) No ADAR1 SPG34 (X-300750% Xq24-q25 / % OMIM # No SPG (AR) No SACS linked)	Age at onset adolescence Infancy 2-60 8-60 <1 to 51 (mainly <10) 2-4 16-19 <1-8 3-12 Infancy Infancy Infancy Infancy Variable 7-12 1-30 10-39 3-4 Infancy 1-80 8-37 10-60 2-51 7-24 42-50 4-42 19-48 ? 36-55 Infancy adulthood Mean 17 ± 3 4-47 <1 to 33 4-19 Adulthood Infancy Adulthood Infancy Infancy 1st or 2nd decade Infancy Infancy ~30 Congenital 2-3 2-19 7-15 1-16 Birth Infancy 1-2 Infancy <1-4 Infancy 2 2-3 Infancy Variable Infancy 30-46 Late (48-58) <2 <1 P: 25-45 C: 2-7 Early infancy Infancy 6-7 18-21 Early infancy P/C P/C Protein protein 1/chaperonin containing protein 2 diphosphohydrolase 1 epsilon C Spartin C Seipin P Protein not identified P/C Atlasin GTPase1 P/C Kinesin family member 1C P Protein not identified P/C Cytochrome P450 2U1 C Putative transferase CAF17, C Mitochondrial Cytochrome c C AP-4 complex subunit mu-1 P/C Cytosolic purine 5'-mitochondrial C Exosome complex component oxidase subunit III nucleotidase RRP40 P/C Receptor expression-enhancing C Protein C19orf12 C Pyrroline-5-carboxylate synthase (P5CS) protein P/C Kinesin-like protein KIF1A P Receptor expression-enhancing protein 2 P/C Bicaudal D Homolog 2 P/C Spastin P/C NIPA1/ Non-imprinted in Prader Willi/ Angelman syndrome 1 P/C Strumpellin P/C Kinesin heavy chain isoform 5 A P Reticulon 2 protein 1 P Protrudin P Acetyl-coenzyme A transporter 1 P Carnitine palmitoyl-transferase C Valosin-Containing Protein P Protein not identified C Protein not identified P/C Protein not identified p Protein not identified P/ C 25-hydroxycholesterol 7-alpha-hydroxylase P/ C Spatacsin P/ C Zinc finger FYVE domain-containing protein 26 C Maspardin C Protein TFG P/C Mitochondrial tRNA for C AP-4 complex subunit epsilon-C Myelin-associated glycoprotein isoleucine 1 C Gap junction gamma-2 protein Oligodendrocyte connexin Function mitochondrial AAA protease Mitochondrial regulation target esterase (NTE) of metabolism and syntheis, uncertain and other nucleotides to the ER, mitochondria, V subunit Protein with multiple lipid metabolism, involved Autophagy Function regulate purinergic cytoskeleton, proteasome ER scaffolding protein for Protein not identified functions such as in membrane curvature, transmission and apoptosome Dynamin GTPase: ER shaping, ER and lipid Motor protein (retrograde Golgi to ER transport) lipid metabolism, lipid droplet biogenesis at ER Protein not identified Cytokinesis, BMP signaling, Lipid droplet maintenance, Enzyme of lipid Member of the iron-sulfur Respiratory chain complex axonal maintenance, Member of the trafficking metabolism Enzyme: Hydrolyses IMP cluster (ISC) assembly Core component of the IV subunit phospholipid homeostasis adaptor protein complex 4 in both purine/pyrimidine machinery in mitochondria RNA exosome complex ER-shaping protein, Mitochondrial Ca2+ Mitochondrial protein with nucleotide metabolism droplet fusion, Inhibit BMP signaling Enzyme: Pyrroline-5-carboxylate synthase with glutamate kinase (GK) and γ-glutamyl phosphate reductase activities (amino-acid metabolism) Motor protein, axonal anterograde transport ER membranous protein: ER shaping Adaptor protein of the dynein-dynactin motor complex AAA protein: Microtubule dynamics (Microtubule severing), inhibits BMP signaling ER morphogenesis, Endosomal trafficing (MIT domain), Cytokinesis(abscission) Mg2+ transporter: Inhibitor of BMP pathway, Endosomal trafficking Cytoskeleton/Actin remodeling, Endosomal traffic Motor protein: microtubule dependent ATPASE, axonal transport ER shaping protein mitochondrial-ER interface functions, ER-microtubule interaction ER morphology protein, regulates neurite outgrowth Acetyl-CoA transporter (role in glycolipid metabolism) Enzyme : neuronal isoform of Carnitine Palmitoyltransferase-1c AAA protein member; Role in the ubiquitin-proteasome system Protein not identified Protein not identified Protein not identified Protein not identified Enzyme: Hydroxylase, cholesterol and neurosteroïd Lysosome recycling protein, involved in endosomal traffic Lysosome recycling protein, involved in cytokinesis, autophagy, endosomal traffic homeostasis Protein associated predominantly with markers for the trans-Golgi and endocytic unknown functions Protein of the ER Mitochondria tRNA Member of the trafficking morphology, necessary for Myelin component isoleucine adaptor protein complex 4 vesicle transport between (intercellular gap junction ER and Golgi C Arylsulfatase I Enzyme: Arylsulfatase 1, C Ubiquitin carboxyl-terminal hydrolase 8 Deubiquitination enzyme Hydrolyses sulfate esters, hormone biosynthesis C Protein not identified Protein not identified C Neural cell adhesion molecule Cell adhesion and C Reticulophagy receptor Golgi protein L1 signaling protein involved FAM134B in axonal guidance, neurite compartments (Endosomal traffic ) C Beta-1,4 N-acetylgalactosaminyltransferase 1 Enzyme: GM2 synthase, Ganglioside metabolism P/C Phospholipase DDHD1 Enzyme: Phospholipase A1, lipid metabolism channel C Non-lysosomal glucosylceramidase Enzyme: Non-lysosomal b-glucosidase of the ganglioside metabolism C AP-4 complex subunit beta-1 Member of the trafficking adaptor protein complex 4 C AP-4 complex subunit sigma-1 Member of the trafficking adaptor protein complex 4 C Vacuolar protein sorting-associated protein 37A Member of the ESCRT-I complex involved in Endosomal trafficking C WD repeat-containing protein 48 Deubiquitination regulation protein C GPI inositol-deacylase GPI-Anchor synthesis pathway / GPI-AP sorting by ERES C Protein not identified Protein not identified outgrowth ,neuronal cell migration and survival P Interferon-induced helicase C RNA binding protein C Leucine-rich repeat transmembrane protein FLRT1 Transmembrane protein of the FGF pathway P Protein not identified Protein not identified domain-containing protein 1 P/C Proteolipid protein 1 Myelin component C ADP-ribosylation factor-like ER morphology protein C Protein not identified Protein not identified Oligodendrocyte C Lysosomal-trafficking Protein involved in progenitor cell migration C Phospholipase DDHD2 Enzyme: Phospholipase (lipid metabolism) protein 6-interacting protein 1 C Rab3 GTPase-activating protein non-catalytic subunit ER morphology protein P/C Protein not identified Protein not identified regulator Lysosome fusion/fission C Monocarboxylate transporter 8 Thyroid hormone regulation transporter MCT8 P Erlin-1 ER-associated degradation C Protein not identified Protein not identified P Ribonuclease H2 subunit B Enzyme of the metabolism Protein not identified Protein not identified <2 C Erlin-2 ER-associated degradation pathway (ERAD) 2-17 (1 family late onset) C Fatty acid 2-hydroxylase Enzyme: involved in Myelin stability, cell differentiation 2-50 P/C AP-5 complex subunit zeta-1 Member of the trafficking Adaptor protein complex 5, involved in DNA repair mitochondria response 2-7 C ribosome rescue system in nucleotide metabolism Infancy P Zinc finger RNA-binding RNA binding protein factor C12orf65, mitochondrial Member of the mediated to IMP in purine Probable peptide chain release Infancy C AMP deaminase 2 Enzyme: Deaminates AMP <1 C Methionine--tRNA synthetase, cytoplasmic Enzyme: Cytosolic methionyl-tRNAsynthetase months of ribonucleotides 2 P Double-stranded RNA-specific RNA binding protein 16-25 P Protein not identified Protein not identified adenosine deaminase Infancy C Sacsin Chaperone	Functional category identified Trafficking Metabolism Degradation? Mitochondria ER stress in HSP Protein not ER Dynamics Intracellular Trasnport cases identified Protein not Lipid Metabolism Mitochondria Mitochondria Endolysosome Nucleotide Nucleotide Trafficking Metabolism Metabolism ER dynamics identified Mitochondrial Metabolism Intracellular Trasnport ER dynamics Intracellular Trasnport ER Dynamics Endolysosome Trafficking Not clear Intracellular Trasnport ER dynamics ER dynamics Metabolism Oxidative metabolism endolysosome trafficking Protein not identified Protein not identified Protein not identified Protein not identified Lipid Metabolism Endolysosome trafficking Endolysosome Trafficking Endolysosome Trafficking Protein With Unknown ER-Golgi Mitochondria Endolysosome Trafficking Myelin Trafficking (Synthesis Functions Lipid Metabolism Pathway) Degradation Pathway Protein not identified Axonal Guidance Unclear Lipid Metabolism Lipid Metabolism Lipid Metabolism Endolysosome Trafficking Endolysosome Trafficking Endolysosome Trafficking Degradation Pathway Lipid Metabolism Protein not identified Nucleotide Signalling Pathways Protein not Metabolism identified Myelin ER Dynamics Protein not identified Endolysosome Lipid Metabolism ER Dynamics Protein not identified Trafficking Development ER Stress Protein not Nucleotide Protein Not ER Stress Lipid Metabolism Endolysosome Trafficking Nucleotide Mitochondria Metabolism Nucleotide Nucleotide Metabolism identified Metabolism Identified Nucleotide Protein Not Metabolism Mitochondria Identified	Second Pathway functional category Mitochondria Endolysosome Trafficking Mitochondria Degradation Pathway Degradation Pathway Degradation Pathway Nucleotide Development Metabolism ER (Biosynthesis Pathways) Development Saugier-Veber Reference 2002 2009 2008 2012 al., 2014 al., 2006 2011 1998 Patel et al., Windpassinger Hanein et al., 2002 Synofzik et Zhao et al., Novarino et al., 2014 et al., 2004 2007 Orlacchio et al., 2014 Tesson et al., Lossos et al., Tiranti et al., Verkerk et al., 2012 Dursun et al., 2015 Zanni et al., 2000 2009 2009 2013 2001 Dor et al., Züchner et al., al., 2008 Meilleur et Seri et al., 1999 Coutelier et al., 2015 Klebe et al., 2006 Erlich et al., 2011 Esteves et al., 2014 2014 Hazan et al., 1999 Rainier et al., 2003 Valdmanis et al., 2007 Reid et al., 2002 Montenegro at al., 2012 2006 Mannan et al., 2006 Lin et al., 2008 Rinaldi et al., 2015 De Bot et al., 2012 Valente et al., 2002 Orlacchio et al., 2005 Subramony et al., 2009 Zhao et al., 2008 Tsaousidou et al., 2008 Stevanin et al., 2007 Hughes et al., 2001 Simpson et al., 2003 Beetz et al., Novarino et Halevy et al., Corona et al., al., 2010 Landouré et Abou Jamra et 2013 al., 2014 Novarino et 2014 2002 al., 2011 al., 2014 al., 2013 Orthmann-Murphy et al., Novarino et Moreno-De-Luca et al., 2011 Novarino et al., 2014 al., 2014 Rosenthal et Vazza et al., Ilgaz Aydinlar al., 1992 2000 et al., 2014 Wilkinson et al., 2005 Boukhris et al., 2013 Bouslam et al., 2005 Tesson et al., 2012 2009 Boukhris et al., 2010 Martin et al., 2013 Abou Jamra et al., 2011 Novarino et al., 2014 Jouet et al., Blumen et al., 1995 2003 Crow et al., Novarino et al., 2014 Hodgkinson et 2014 Novarino et al., 2002 al., 2014 Zivony-Elboum et al., 2012 Novarino et et al., 1994 Zortea et al., 2002 Degradation Shimazaki et Abou Jamra et al., 2011 Bauer et al., Al-Yahyaee et al., 2014 Novarino et al., 2014 Pathway al., 2014 Signalling Schwartz et Meijer et al., Pathways al., 2005 2004 al., 2006 2012 Degradation Novarino et Stevanin et al., Crow et al., Steinmüller et Hanein et al., 2008 Alazami et al., 2011 Yıldırım et al., Dick et al., 2008 Dick et al., 2010 Degradation Pathway Słabicki et al., 2010 Schuurs-Hoeijmakers et al., 2012 al., 2012 Novarino et Shimazaki et al., 2014 2000 Pathway al., 2014 Novarino et 2007b 2014 al., 1997 Novarino et al., 2014 Crow et al., Macedo-Souza 2014 Engert et al., at al., 2008
										201 202 203 204 205 208 209

Table 1 a

 1 The development of WES or large gene : HSP forms complicated with clinical signs of cerebellar syndrome.

	Annexes
	Annex 1: 1-SPG 2-Inheritance 3-Gene	4-Comment
	5-SPG30	6-AD/AR	7-KIF1A	9-Cerebellar
			8-	atrophy
	10-SPG58 11-AR, AD?	13-KIF1C	15-Cerebellar
		12-	14-	atrophy
	16-SPG7	17-AR, AD?	19-SPG7	21-Cerebellar
		18-	20-	atrophy
	22-SPG4	24-AD	25-SPAST 26-No reported
	23-			cerebellar atrophy
	27-SPG5/SPG5A	29-AR	30-CYP7B1	32-No reported
	28-		31-	cerebellar atrophy
	33-SPG11	34-AR	35-KIAA1840 36-Cerebellar
				atrophy
	37-SPG15	38-AR	39-ZFYVE26 40-Cerebellar
				atrophy
	41-SPG20	42-AR	43-	45-No reported
			44-KIAA0610	cerebellar atrophy
	46-SPG21	47-AR	48-ACP33	49-Cerebellar
				atrophy
	50-SPG26	51-AR	52-B4GALNT1 53-No reported
				cerebellar
				atrophy
	54-SPG27	55-AR 56-10q22.1-q24.1 57-Cerebellar
				atrophy

Table 1 b

 1 : Cerebellar atrophy with no clinical signs of cerebellar syndrome

	SPG32	AR	14q12-q21	No cerebellar signs reported
	SPG50	AR	AP4M1	No cerebellar signs reported
	SPG66	AR	ARSI	No cerebellar signs reported
	SPG67	AR	PGAP1	No cerebellar signs reported
	SPG74	AR	IBA57	No cerebellar signs reported
	No SPG	Mito	MT-CO3	No cerebellar signs reported
	Annex 2:

Table 2 :

 2 HSP forms complicated with cognitive/mental function impairment.

	147-		SPG 148-	Inherit	149-	Gene 150-	Comment
				ance			151-
	152-	SPG5 153-	AR,	154-	KIF1C 155-	developmental delay
		8	AD?				or MR
	156-	SPG4 157-	AD 158-	SPAST 159-	cognitive impairment
	160-	SPG6 161-	AD 162-	NIPA1 163-	memory
							164-	impairment
	165-	SPG3 166-	AD 167-	4p16-	168-	cognitive impairment
		8				p15
	169-	SPG5 170-	AR 171-	CYP7B 172-	cognitive

Table 3a :

 3a HSP forms complicated with peripheral neuropathy without evidence of amyotrophy.

	SPG code	inheritance	Gene
	SPG30	AR/AD	KIF1A
	SPG4	AD	SPAST
	SPG6	AD	NIPA1
	SPG31	AD	REEP1
	SPG36	AD	12q23-24
	SPG14	AR	3q27-q28
	SPG23	AR	1q24-q32
	SPG25	AR	6q23-24.1
	SPG26	AR	B4GALNT1
	SPG28	AR	DDHD1
	SPG56	AR	CYP2U1
	SPG57	AR	TFG

Table 4c :

 4c Optic atrophy

	SPG	Inheritance	Gene
	SPG7	AR	SPG7
	SPG35	AR	FA2H
	SPG55	AR	C12orf65
	SPG57	AR	TFG
	SPG68	AR	FLRT1
	SPG74	AR	IBA57
	SPG45	AR	10q24.3-q25.1
	SPG54	AR	DDHD2
	Annex 5:

Table 5 :

 5 Characteristic syndromes of some HSP subtypes.

	SYNDROME	SIGNS and SYMPTOMS	GENES
			ASSOCIATED
			WITH IT
	MASA syndrome	Mental retardation Aphasia Shuffling gait and	SPG1
		Adducted thumb
	CRASH syndrome	Corpus callosum hypoplasia Retardation Adducted	SPG1
		thumb Spastic paraplegia and Hydrocephalus
	Silver syndrome	neuropathy, amyotrophy	SPG10, SPG17
	`Kjellin syndrome	Spastic paraplegia ,cognitive decline, neuropathy,	SPG11, SPG15
		retinopathy
	Troyer Syndrome	dysarthria, distal amyotrophy in	SPG20
		hands and feet, cerebellar signs, mild ID and skeletal
		abnormalities (short stature)

Table 6a :

 6a Skeletal deformities reported in HSP.

	SPG59	AR	USP8	pes equinovarus
	SPG65	AR	NT5C2	pes equinovarus
	SPG66	AR	ARSI	pes equinovarus
	SPG	Inheritance	Gene	Comment
	SPG18	AR	ERLIN2	Contractures,
				Troyer Syndrome
	SPG20	AR	SPG20/	short stature
			KIAA0610
	SPG23	AR	1q24-q32	skeletal
				deformities,
				Lison syndrome
	SPG27	AR	10q22.1-q24.1 skeletal
				abnormalities
	SPG47	AR	AP4B1	foot
				deformity
	SPG49	AR	TECPR2	dysmorphic
				features
	SPG51	AR	AP4E1	foot
				deformity
	SPG52	AR	AP4S1	foot
				deformity
	SPG53	AR	VPS37A	marked kyphosis

Table 6b :

 6b Microcephaly in HSP.

	SPG	Inheritance	Gene	Comment
	SPG47	AR	AP4B1
	SPG49	AR	TECPR2	brachycephalic
				microcephaly
	SPG51	AR	AP4E1
	SPG52	AR	AP4S1
	SPG64	AR	ENTPD1

Table 6c :

 6c Short stature in HSP.

	SPG	Inheritance	Gene
	SPG29	AD		1p31.1-21.1
	SPG10	AD		KIF5A
	SPG69	AR		RAB3GAP2
	No SPG	MITO		MT-TI
	Annex 10:
	SPG	Inheritance	Gene
	SPG58	AR, AD?	KIF1C
	SPG20	AR	KIAA0610
	SPG27	AR	10q22.1-q24.1
	SPG52	AR	AP4S1
	SPG54	AR	DDHD2
	SPG56	AR	CYP2U1
	SPG63	AR	AMPD2

Table 10 :

 10 Developmental delay in HSP.

	SPG	Inheritance	Gene
	SPG58	AR, AD?	KIF1C
	SPG47	AR	AP4B1
	SPG51	AR	AP4E1
	SPG52	AR	AP4S1
	SPG53	AR	VPS37A
	SPG54	AR	DDHD2
	Annex 11:

Table 11 :

 11 Hypogonadism and infertility in HSP

	SPG	Inheritance	Gene	Comment
	SPG46	AR	GBA2	infertility in males
	SPG64	AR	ENTPD1	delayed puberty
	SPG5	AR	CYP7B1	27-hydroxy-cholesterol
				accumulation in blood and
				CSF.(decreased libido)
	No SPG	Mito	MT-ATP6	hypogonadism

Normal Delayed dsoeseesR At age Specify:

									-OTHER CLINICAL SIGNS H-CLINICAL DIAGNOSTIC CONCLUSION
	1. Muscle wasting	Cerebellar ataxia			2. Facial dysmorphia Spastic paraparesis
	Autosomal	None Pure form	Mild	Moderate Definitely affected Severe	Autosomal	Pure form	Definitely affected
	dominant • Proximal UL Autosomal		Complicated	Probably affected	dominant Autosomal	No Complicated	Yes Probably affected (enhanced or very brisk Describe:
	• Distal UL recessive		form				(only dysarthria)	recessive	form	LL reflexes +/-Babinski)
	• Proximal LL Isolated case						___________________ Possibly affected (only	________________________________ Isolated case	________________________________ Possibly affected (enhanced LL
							mild gait ataxia)	reflexes)
	• Distal LL X-linked									X-linked
	3. Fasciculations or Myokymias				4. Skeletal abnormalities
	PPP(Facial contraction fasciculations) please circle	None	Mild	Moderate	Severe
				No			Yes	Localisation:	• Scoliosis
											• Pes cavus
								• Other: __________________ __________________________________________________ J-COMPLEMENTARY INVESTIGATIONS
	5. Centiles EXAMINATION	NOT DONE	NOR-MAL	AB-NORMAL		SPECIFY	6	P.	gisP	E. lonteoNPcnenger
	Head 1. Cerebral MRI		esoetesrsin	CsinnNs	los	yfsHnpr	Signs at onset Hypotonic eglnroNPrP	lg	sse lgeroN	los yfoennH	yfsHnpr dnonR
	Circumferance esnornPpgePlos									nenoNPrP • rgcgegeeec eglnroNPPP
	P los	cnornPpge									• yPcemvree nenoNPPP
											• roeevnvoe llnoN
	7. Motor deficit									• yaPrgoeoevree 8. Reflexes
				None		Mild 4/5	Moderat 2-3/5	Severe <2/5	• Dcggrg	Normal Increased Diffused Decreased Absent Clonus
	• Facial palsy/ atrophy							• Pain and/or cramps • Jaw jerk
	• Proximal UL • Distal UL Motor Development • Proximal LL Intellectual Development • Distal LL									• NcvegcaP • Biceps • NancecPcemvree • Finger flexor RPamoccgve rervee • • Patellar
	Mile Stones									• Others :
	9. Extra-pyramidal symptoms					• Adductor
		None Mild Moderate Severe	F. PREDOMINANT SIGNS at examination • Ankle Specify:
	• Resting tremor						SPASTICITY	CEREBELLAR ATAXIA
	• Postural tremor		None		Mild	egRseons Severe Not Applicable	None Absent Present Mild egRseons Severe Not Applicable
	• Chorea • Upper limbs • Lower limbs* • Dystonia • Myoclonus • Gait • Hypokinesia • Dysarthria • Rigidity									•Hoffmann's sign • Plantar reflex	Flexor Indifferent	Unilat. 	Bilat. 
	* at rest for spasticity and knee-heel for ataxia
	11. Sensory deficit
				None	G-DISABILITY STAGE Mild Moderate Severe Abolished
	• Vibration sense  (ankles)									At age	At age
				(8/8) (> 5/8) (2-5/8) (<2/8)	(0/8)
	• Superficial sensory loss		No	Touch	Prick		Cold

0: no functional handicap 4: severe, walking with one stick 1: no functional handicap but signs at examination 5: walking with two sticks 2: mild, able to run, walking unlimited 6: unable to walk, requiring wheelchair 3: moderate, unable to run, limited walking without aid 7: confined to bed

None Mild Moder. Severe

Novarino et al., 2014

Acknowledgment: We thank the members of the DNA and cell bank of the ICM, Federica Barreca, Emeline Mundwiller, Khalid El Hachimi and Delphine Bouteiller for their valuable contribution. This study was supported by the Agence Nationale de la Recherche SPATAX-QUEST project (to GS), Verum Foundation, Roger de Spoelberg Foundation (to AB), European Union Neuromics projects -OMICS call (to AB, AD, GS), NIH (grant GM110 67 to AA) and the Programme d'Investissement d'Avenir IHU-A-ICM. LE received Campus France and University of Khartoum fellowships.

Funding and support: This study was supported financially by the Agence Nationale de la Recherche (SPATAX-QUEST project, to Giovanni Stevanin), the Verum Foundation and Roger de Spoelberg Foundation (to Alexis Brice), the European Union (Neuromics projects, OMICS call, to Alexis Brice, Alexandra Durr and Giovanni Stevanin) and the National Institutes of Health (GM110567 to Anjon Audhya) and benefited from the Programme d'Investissement d'Avenir IHU-A-ICM. Liena Elsayed was the recipient of a Campus France and University of Khartoum fellowships.

Conflicts of Interests:

The authors declare no conflicts of interests. Supplementary information is available at European Journal of Human Genetics's website.  There are very few connections between signs, or groups of signs and certain functions: cerebellar ataxia with degradation;

myelination/development and lipid metabolism; ER shaping and nucleotide metabolism with pure forms or endomembrane traffic and CI.

Understanding these links and coupling these data with expression levels and profiles could be useful for better deciphering of the mechanisms involved in theses diseases. panels covering multiple pathologies will allow better assessment of the full clinical spectrum of neurological conditions.

SPG61

AR ARL6IP1

SPG60 AR WDR48

No SPG AR SACS

No SPG AR FAM134B

No SPG AR LYST

No SPG AR CCT5

No SPG MITO MT-ATP6

Nine-hole Pegboard testdominant hand

The patient is seated and holds nine dowels (9mm in diameter and 32-mm long) in one hand and places them randomly, one by one, with the other hand in a board with nine holes. Timing begins when the first peg is placed in a hole and ends when the last peg is placed. The examiner holds the board steady on the table during the test. The trial is performed once only with the dominant hand. If the patient drops a peg the examiner stops the timer and the patient starts the test again once from the beginning.

Timing dominant hand: sec

Click testdominant hand

The patient is seated facing the examiner across a table on which is placed a device composed of two mechanical counters fixed on a wooden board 39 cm apart. The patient uses his index finger to press the buttons on the counters alternately 10 times. Timing begins when the first button is pressed and stops when the second counter reaches 10. The trial is performed once only with the dominant hand.

43.

Could subject engage in gainful employment in his/her accustomed work? 0 1

44.

Could subject engage in any kind of gainful employment? 0 1 258

45.

Could subject engage in any kind of volunteer or non gainful work? 0

46.

Could subject manage his/her finances (monthly) without any help? 0

47.

Could subject shop for groceries without help? 0

48.

Could subject handle money as a purchaser in a simple cash (store) transaction? 0

49.

Could subject supervise children without help? 0

50.

Could subject operate an automobile safely and independently? 0

51.

Could subject do his/her own housework without help? 0

52.

Could subject do his/her own laundry (wash/dry) without help? 0

53.

Could subject prepare his/her own meals without help? 0

54.

Could subject use the telephone without help? 0

55.

Could subject take his/her own medications without help? 0

56.

Could subject feed himself/herself without help? 0

57.

Could subject dress himself/herself without help? 0

58.

Could subject bathe himself/herself without help? 0

59.

Could subject use public transportation to get places without help? 0

60.

Could subject walk to places in his/her neighbourhood without help? 0

61.

Could subject walk without falling? 0

62.

Could subject walk without help? 0

63.

Could subject comb hair without help? 0

64.

Could subject transfer between chairs without help? 0

65.

Could subject get in and out of bed without help? 0

66.

Could subject use toilet/commode without help? 0

67.

Could subject's care still be provided at home? 0

Functional Assessment Score: _____/25

Information Sources

Was the Functional Assessment information obtained from:

170-171

Figure [START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF] Comparison of the success rates of all approaches that were utilized in genetic investigation. Clinical data of three patients from family F26 with spinocerebellar ataxia (SCA7) due to a pathologic CAG repeat in ATXN7.

List of Tables

Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF] Summary of mutations in known genes found in three families with pyramidal features.

Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF] Summary of mutations in putative candidate new genes found in five families with spastic neurogenetic disorder.

Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF] Clinical data of two patients from family F37 with a nonsense mutation in ABHD16A.

Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF] Clinical data of two patients from family F41

Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF] Clinical data of two patients from family F50.

Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF] Clinical data of three patients from family FM3

Table [START_REF] Schmit -H Bsch | An Update on Inherited Ataxias[END_REF][START_REF] Finsterer | Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance[END_REF][START_REF] Coutelier | Genetic Landscape Remodelling in Spinocerebellar Ataxias: The Influence of next-Generation Sequencing[END_REF][START_REF] Noreau | Molecular Aspects of Hereditary Spastic Paraplegia[END_REF][START_REF] Ruano | The Global Epidemiology of Hereditary Ataxia and Spastic Paraplegia: A Systematic Review of Prevalence Studies[END_REF][START_REF] Finsterer | Hereditary Spastic Paraplegias with Autosomal Dominant, Recessive, X-Linked, or Maternal Trait of Inheritance[END_REF][START_REF] Coutinho | Clinical Heterogeneity of Autosomal Recessive Spastic Paraplegias: Analysis of 106 Patients in 46 Families[END_REF][START_REF] Boukhris | Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum in Tunisia: SPG11, SPG15, and Further Genetic Heterogeneity[END_REF][START_REF] França | Prospective Neuroimaging Study in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Stevanin | Mutations in SPG11, Encoding Spatacsin, Are a Major Cause of Spastic Paraplegia with Thin Corpus Callosum[END_REF][START_REF] Shibasaki | Linkage of Autosomal Recessive Hereditary Spastic Paraplegia with Mental Impairment and Thin Corpus Callosum to Chromosome 15A13-15[END_REF][START_REF] Casali | Clinical and Genetic Studies in Hereditary Spastic Paraplegia with Thin Corpus Callosum[END_REF] Clinical data of five patients from family FM7 branch 1

Hereditary spastic paraplegias: clinical spectrum in Sudan, further deciphering of the molecular bases of autosomal recessive forms and new genes emerging Abstract:

Hereditary spastic paraplegias (HSP), a heterogeneous group of spastic neurodegenerative disorders which impose diagnostic challenges. I explored the clinical varieties and genetic pathways of spastic neurodegeneration in a familial Sudanese cohort.

We recruited 41 Sudanese families [337 individuals/106 HSP patients]. I have established a genomic DNA bank and when necessary, skin biopsies and fibroblasts were also obtained. A phenotype-based candidate gene approach was followed in 4 families. A targeted next generation sequencing (NGS) for 74 HSP-related genes was the main screening strategy in all remaining 37 families. Whole exome sequencing (WES) was done in search for novel mutations in new genes in families with negative screening results. Occasionlly, functional studies were conducted when feasible and relevant.

I identified the genetic cause in 17/41 families. In 12 families, the mutated genes were known HSP genes. In 3 families, novel genes were identified mutated. 5 candidate genes segregated with disease in 2 other families with more experiments needed to conclude. Analysis of the NGS screening panel and of WES data imposed certain challenges as multiple genetic disorders were sometimes found running in parallel in the same/ different branches of highly inbred families. We could expand the phenotypic heterogeneity of these disorders due to clinical differences observed between Sudanese patients and patients of other origins even when caused by mutations by the same gene/variant. This is the first genetic screening in a large set of HSP families in Sudan. It describes new causative genes, paving the way for further deciphering of the underlying mechanisms. Key Words: [Hereditary spastic paraplegias; Sudanese families; consanguinity; next generation sequencing (NGS); Whole exome sequencing; novel genes]