Alistair T Pagnamenta

Pierre Heemeryck

Hilary C Martin

Christophe Bosc

Leticia Peris

Ivy Uszynski

Sylvie Gory-Fauré

Simon Couly

Charu Deshpande

Ata Siddiqui

Alaa A Elmonairy

Sandeep Jayawant

Sarada Murthy

Ian Walker

Lucy Loong

Peter Bauer

Frédérique Vossier

Eric Denarier

Tangui Maurice

Emmanuel L Barbier

Jean-Christophe Deloulme

Jenny C Taylor

Edward M Blair
email: ed.blair@ouh.nhs.uk

Annie Andrieux

Marie-Jo Moutin
email: moutinm@univ-grenoble-alpes.fr

Defective tubulin detyrosination causes structural brain abnormalities with cognitive deficiency in humans and mice

Reversible detyrosination of tubulin, the building block of microtubules, is crucial for neuronal physiology. Enzymes responsible for detyrosination were recently identified as complexes of vasohibins 1 or 2 with small vasohibin-binding protein (SVBP). Here we report three consanguineous families, each containing multiple individuals with biallelic inactivation of SVBP caused by truncating variants (p.Q28* and p.K13Nfs*18). Affected individuals show brain abnormalities with microcephaly, intellectual disability and delayed gross motor and speech development. Immunoblot testing in cells with pathogenic SVBP variants demonstrated that the encoded proteins were unstable and non-functional, resulting in a complete loss of vasohibin detyrosination activity. SVBP knock-out mice exhibit drastic accumulation of tyrosinated tubulin and a reduction of detyrosinated tubulin in brain tissue. Similar alterations in tubulin tyrosination levels were observed in cultured neurons and associated with defects in axonal differentiation and architecture. Morphological analysis of the SVBP knockout mouse brains by anatomical MRI showed a broad impact of SVBP loss, with a 7% brain volume decrease, numerous structural defects and a 30% reduction of some white matter tracts. SVBP knockout mice display behavioral defects, including mild hyperactivity, lower anxiety and impaired social behavior. They do not, however, show prominent memory defects. Thus, SVBP deficient mice recapitulate several features observed in human patients. Altogether, our data demonstrate that deleterious variants in SVBP cause this neurodevelopmental pathology, by leading to a major change in brain tubulin tyrosination and alteration of microtubule dynamics and neuron physiology.

Introduction

Microtubules are dynamic, polarized polymers composed of α/β tubulin dimers which have a wide range of cellular functions. They are crucial to cell division, cell shape and motility, and intracellular organization and transport. Thus, microtubules are key to normal development of the central nervous system. In recent years an increasing number of human brain malformations and neurological disorders have been associated with mutations in α and β tubulin genes [START_REF] Taylor | Factors influencing success of clinical genome sequencing across a broad spectrum of disorders[END_REF][START_REF] Yau | OncoSNP-SEQ: a statistical approach for the identification of somatic copy number alterations from next-generation sequencing of cancer genomes[END_REF].

Examples of genes associated with these "tubulinopathies" include TUB1A and TUBB2B where mutations in these genes result in a range of cortical malformations such as lissencephaly and polymicrogyria [START_REF] Plagnol | A robust model for read count data in exome sequencing experiments and implications for copy number variant calling[END_REF][START_REF] Handsaker | Discovery and genotyping of genome structural polymorphism by sequencing on a population scale[END_REF][START_REF] Rimmer | Integrating mapping-, assembly-and haplotype-based approaches for calling variants in clinical sequencing applications[END_REF][START_REF] Siepel | Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes[END_REF]. Moreover, deficiency of some microtubule binding proteins such as DCX, LIS1 or MAP1B are also well known to cause severe neurodevelopmental disorders [START_REF] Mclaren | The Ensembl Variant Effect Predictor[END_REF][START_REF] Popitsch | ReliableGenome: annotation of genomic regions with high/low variant calling concordance[END_REF].

Microtubules display functional specialization by not only having several tubulin isoforms but also through diverse post-translational modifications occurring generally at their C-termini which are located on the microtubule surface. These modifications control interactions with the many microtubule-associated proteins including molecular motors and stabilizing/destabilizing proteins. Among them is the reversible detyrosination of α-tubulin. Detyrosinated tubulin is generally thought to be associated with longer-lived microtubules, whereas more dynamic microtubules are mostly tyrosinated (9,10). In the cycle of detyrosination/tyrosination, the C-terminal tyrosine of α-tubulin is removed by a carboxypeptidase and re-added by a ligase. The enzyme catalyzing the tyrosination, the tubulin tyrosine ligase (TTL), was identified over 25 years ago (11) and mice lacking this enzyme have been shown to die perinatally, with poorly developed neuronal networks (12). In contrast, enzymes catalyzing the detyrosination (tubulin carboxypeptidases, TCPs) were discovered only very recently [START_REF] Aillaud | Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation[END_REF][START_REF] Nieuwenhuis | Vasohibins encode tubulin detyrosinating activity[END_REF]. Vasohibins (VASHs) were shown to be the major tubulin detyrosinating enzymes, and their partner SVBP (small vasohibin binding protein) to be a prominent regulator of their stability and activity [START_REF] Adamopoulos | Crystal structure of the tubulin tyrosine carboxypeptidase complex VASH1-SVBP[END_REF][START_REF] Li | Structural basis of tubulin detyrosination by vasohibins[END_REF][START_REF] Wang | Structural basis of tubulin detyrosination by the vasohibin-SVBP enzyme complex[END_REF]. SVBP binds to vasohibins with high affinity [START_REF] Wang | Structural basis of tubulin detyrosination by the vasohibin-SVBP enzyme complex[END_REF][START_REF] Suzuki | Isolation of a small vasohibin-binding protein (SVBP) and its role in vasohibin secretion[END_REF] and the tubulin carboxypeptidase function is achieved by the VASH-SVBP complexes.

Tyrosination is an important regulatory signal for neuronal physiology. It is involved in brain development and neuron functioning, including axonal guidance, neurite extension and retrograde transport (12,[START_REF] Gumy | The kinesin-2 family member KIF3C regulates microtubule dynamics and is required for axon growth and regeneration[END_REF][START_REF] Hammond | Posttranslational modifications of tubulin and the polarized transport of kinesin-1 in neurons[END_REF][START_REF] Kahn | Effects of kinesin-5 inhibition on dendritic architecture and microtubule organization[END_REF][START_REF] Konishi | Tubulin tyrosination navigates the kinesin-1 motor domain to axons[END_REF][START_REF] Marcos | Tubulin tyrosination is required for the proper organization and pathfinding of the growth cone[END_REF][START_REF] Nirschl | alpha-Tubulin Tyrosination and CLIP-170 Phosphorylation Regulate the Initiation of Dynein-Driven Transport in Neurons[END_REF]. Moreover, knock-down experiments for each of the proteins in the detyrosinating VASH-SVBP complex were recently shown to cause severe neuronal differentiation defects and to alter neuronal migration in the developing mouse brain [START_REF] Aillaud | Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation[END_REF].

Interaction of VASH with SVBP and association of the complex to microtubules was shown to regulate axon specification of neurons [START_REF] Wang | Structural basis of tubulin detyrosination by the vasohibin-SVBP enzyme complex[END_REF].

Using a combination of whole-genome sequencing (WGS) and whole-exome sequencing (WES) we now show that biallelic inactivating SVBP variants in humans cause a syndrome involving brain anomalies, intellectual disability and delayed gross motor and speech development. We demonstrate that the Svbp knockout mouse model recapitulates several aspects of the neuropathology observed in the human subjects.

Results

Clinical descriptions of patients with SVBP mutations Family 1

The proband (individual V-6) is the 6th child in a sibship of 7 surviving children (Figure 1a) born to first-cousin parents of South Asian ancestry. She showed early signs of gross motor developmental delay, sitting at 9 months, standing at 19 months and was over 2 years of age before she walked independently. She has gained a few single words of expressive language.

She is relatively microcephalic (Table S1). She developed lower limb spasticity with brisk tendon reflexes. She received a number of botulinum toxin injections to relieve Achilles contractures. She has marked mirror movements of her fingers. Cranial nerve examination is normal. She has very short 3rd and 4th toes on both feet. An MRI scan of the brain showed irregular ventricular margins and a thin corpus callosum. Individual V-7 is the younger sister of the proband. She presented in a similar fashion to her sister with severe global developmental delay and an evolving spastic paraparesis. She also sat at 9 months, stood at 13 months and walked at 24 months. She has developed only a few single words of speech. She had a single episode of status epilepticus as a two-year-old and had an ongoing seizure disorder which resolved in adolescence. She also displays mirror movements of the fingers. Her head circumference was noted to be below the 0.4th centile for her age. Her height is between the 9th and 25th centiles. An MRI brain scan performed at age 4 years showed a small corpus callosum with dysmorphic ventricles. There was slight prominence of CSF spaces in keeping with a degree of volume loss, including the lateral cerebellar hemispheres. The cisterna magna was normal. There was completed myelination with no white matter abnormality and normal deep grey structures.

Individual IV-3 is the maternal aunt of the proband. Prior to her niece being referred to our service, she had been diagnosed with learning disability and short stature. She was noted to have short 3rd, 4th and 5th metacarpals which resulted in her being investigated for pseudohypoparathyroidism. However, all biochemical and metabolic investigations, including sequence analysis of GNAS1 identified no specific underlying cause. At 43 years of age she has very little speech. She had marked wasting of the intrinsic muscles of her hands with clawing of the fingers. She has delayed gross motor development and walks with the use of a frame. No cranial MRI was available.

Individual IV-4 is the maternal uncle of the proband. He was 37 years of age when first seen in our clinic. He also has learning disability with a spastic paraparesis requiring several operations to release contractures at his hips and ankles. He had seizures as an infant and child, resolving in adolescence. He walked at 5 years and has attained only a few single words of expressive language. He has hypothyroidism. He has coarse facial features similar to those seen in his affected sister and nieces. His thumbs are short and he has marked muscle wasting of the small muscles of his hands with clawing of his fingers. He has a spastic paraparesis with brisk lower limb reflexes and extensor plantar responses. No cranial MRI was available.

Family 2

The proband (II-3) is a 6-year-old female referred to the Kuwait medical centre aged 2 years due to developmental delay. She is the third of four offspring (Figure 1b), born weighing 2.9 kg to phenotypically normal 1st-cousin-once-removed parents of Kuwaiti ancestry. Increased muscle tone was observed, especially in both lower limbs. She has ankle clonus, easily elicited reflexes and an unsteady spastic gait with toe walking. Occipital frontal circumference (OFC) was <0.4th centile. At age 6 she could speak only a few words. MRI of her brain showed a thin corpus callosum with dilated ventricles and poor white matter volume. Cranial nerves examinations, electromyography, plasma amino acids and urinary organic acids were all normal.

Her elder brother (II-2) is similarly affected with an OFC of 49.7 cm aged 7 years (0.4th centile). He has generalized hypertonia predominantly in the lower limbs, brisk reflexes, an unsteady spastic gait, mirror hand movement and spoke using only a few words.

Family 3

The proband (II-3, Figure 1c) is the third child of first-cousin parents from a multiply consanguineous South Asian family. She was referred to the genetics clinic at 3 years of age with progressive microcephaly, progressive difficulty in walking and global developmental delay. The antenatal scans were reportedly normal and her birth weight was 2.7 kg at term (10th centile). Head circumference at birth was 31.5 cm (<0.4th centile). There were no concerns in early infancy and she was walking independently at 14 months of age but remained unsteady on her feet and would trip easily. Upon examination she was seen to be of proportionate short stature and a failure to thrive had been noted. She also demonstrated spasticity with delayed gross and fine motor development. She has profound intellectual disability with delayed speech and language development and demonstrated autistic behavior. Neurometabolic investigations were normal. Hyperpigmentation of the skin in the form of small café au lait macules was noted.

In view of the microcephaly and the café au lait macules, a DNA repair disorder was considered as a possible differential diagnosis.

Her older sibling (II-1) was initially assessed at 3 years of age because of microcephaly, progressive difficulty in walking and developmental delay. His birth weight and head circumference were reported on the 3rd centile although no measurements were available. At 3 years of age his head circumference was 46 cm (<0.4th centile). He was subsequently lost to follow-up and was seen with his younger sister when he was 21 years old. At that time, he had only 10 words. He had spastic diplegia and was non-ambulant. Additional features included 2-4 toe cutaneous syndactyly and coarse facial features.

Genetic findings

Family 1

We first identified a consanguineous family of Pakistani ancestry in which four individuals, two sisters and their uncle and aunt, had a syndrome characterized by learning disability, spastic paraparesis, a thin corpus callosum and decreased cerebral white matter volume (Table S1).

Individual V-6 underwent WGS as part of the WGS500 project. We carried out linkage analysis under a recessive model, combining the WGS data with SNP array data from other relatives (Figure 1a). This revealed a single significant linkage peak (Figure S1): a 12.7 Mb region on chr1:40,921,145-53,713,336 (GRCh37/hg19) that had a LOD score of 3.01 and was homozygous in all affected individuals. This region contained 145 protein-coding genes, and was interrogated for rare simple recessive variants in the WGS data.

There was only one coding variant that was sufficiently rare and that disrupted a well conserved position. This was a homozygous transition, chr1: [START_REF] Bahlo | Generating linkage mapping files from Affymetrix SNP chip data[END_REF] LoF variants in either of these cohorts. We also searched for potentially causal variants under other inheritance models and outside the coding region in this family (see Supplemental Methods), but found none more likely to be pathogenic than the LoF variant in SVBP.

Family 2

In the hope of replicating the results from Family 1, we searched the CentoMD mutation database [START_REF] Trujillano | A comprehensive global genotype-phenotype database for rare diseases[END_REF] 1c). Using SNP array data, Families 1 and 3 were shown to share a 3.9 Mb region of identity by descent on chromosome 1, between rs9659165 and rs7515271. Using WGS, this shared region was refined to 39,550,261 -43,442,698 (rs76104968 and rs1770796).

The chr1:43,282,134G>A (p.Q28*) variant is thus likely to be an ancestral variant rather than a recurrent mutation.

Clinical comparison across families

Clinical details for affected individuals are reported in Table S1. Most individuals exhibited microcephaly (on or below the 0.4 th centile, where data were available), moderate to severe learning disability and speech delay. A detailed comparison of the MRI data available in these families demonstrated a subtle but consistent pattern comprising reduced brain volume, a thin corpus callosum and abnormal ventricles in all three families (Figure 1 d,e and data not shown).

Affected individuals also exhibited spasticity and in most cases this involved predominantly the lower limbs. More variable features included mirror movements observed in 4/8, coarse facial features in 5/8, seizures in 3/8, short stature in 3/8 and digital abnormalities in 4/8.

Consequences of SVBP pathogenic variants on VASH enzymatic activity

We obtained whole-blood RNA from individuals from Family 1, and found no evidence that the p.Q28* variant resulted in nonsense mediated decay (Figures S2 andS3). Hypothesizing that these protein-truncating variants would nonetheless impair the function of SVBP, we examined the ability of mouse VASH1 enzyme to detyrosinate α-tubulin in the presence of SVBP proteins either in their native form, or with the p.Q28* and p.K13Nfs*18 variants observed in human patients (Figure 2a). Expression of VASH in HEK293T cells alone resulted in a slight increase of detyrosinated tubulin (due to the presence of endogenous SVBP in cells), whereas expression with native SVBP resulted in a substantial increase in detyrosinated tubulin and a large loss of tyrosinated tubulin, thus revealing the detyrosination activity due to exogenous VASH-SVBP complex. When VASH was expressed with the SVBP mutants, the levels of modified tubulin did not differ from the conditions observed in the absence of SVBP (Figure 2a). This is mostly due to instability of the SVBP mutants: whereas native SVBP was easily detected, in similar experimental conditions p.Q28* was hardly detected and p.K13Nfs*18 could not be detected at all (see Flag signals in Figure 2a). Moreover, even the expression of the VASH was affected by the expression of the SVBP mutants (GFP signals), as expected from the known instability of the VASH in the absence of its chaperone SVBP [START_REF] Aillaud | Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation[END_REF][START_REF] Nieuwenhuis | Vasohibins encode tubulin detyrosinating activity[END_REF]. Thus, the p.Q28* and p.K13Nfs*18 variants effectively result in a functional knockout of SVBP.

To investigate the function of Svbp in vivo, KO mice were produced by CRISPR/Cas9 technique. Homozygous mice, having a deletion after the third residue of SVBP generating a frameshift adding only four chimeric residues (Figure S4), were obtained by selective breeding.

PCR and Sanger sequencing results confirmed the deletion of targeted region. These Svbp KO mice are viable, fertile, exhibited normal general development and do not exhibit weight loss compared to wild-type mice.

Defects in tubulin tyrosination in brain of SVBP knockout mice

We analyzed the levels of tyrosinated, detyrosinated and Δ2 α-tubulin (a modification that follows, and thus depends on, detyrosination) in the brain of Svbp KO mice (Figure 2 b-e).

Detyrosinated and Δ2 tubulin drastically decreased (-39.9 ± 2.3 % and -33.0 ± 2.3 %, respectively), and a 3-fold increase in tyrosinated tubulin was observed (+233.4 ± 7.9%). Thus the absence of the VASH-stabilizing SVBP chaperone leads to drastic changes of α-tubulin modifications, with a large reduction of tubulin detyrosination. The remaining pool of detyrosinated α-tubulin could be due to α4-tubulin (which genetically lacks the C-terminal tyrosine) or to other detyrosinases [START_REF] Aillaud | Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation[END_REF][START_REF] Nieuwenhuis | Vasohibins encode tubulin detyrosinating activity[END_REF]. The accumulation of tyrosinated tubulin could be the result of neosynthesis of tyrosinated tubulin that cannot be detyrosinated at specific stage of brain development.

SVBP-null neurons show anomalies of their differentiation and morphology.

We then examined the three pools of α-tubulin (tyrosinated, detyrosinated and Δ2) in hippocampal neurons prepared from wild-type and Svbp KO mice brains embryos (Figure S5) cultured 2, 7 and 17 days in vitro (2, 7 and 17 DIV). In young neurons, the absence of SVBP leads to a drastic decrease of detyrosinated tubulin (-63.6 ± 1.8 % and -73.8 ± 1.5 % at day 2 and day 7 of DIV, respectively) and Δ2 tubulin almost disappeared. In mature Svbp KO neurons (17 DIV), the decrease of detyrosinated tubulin is maintained but less pronounced (-37.7 ± 11.7 %) and Δ2 tubulin present (36 % ± 9.1 % of the controls). On the other hand, tyrosinated tubulin accrued extensively in the KO neurons, with a 1.5-fold increase observed at 2 DIV and a 3-fold increase observed after 7 DIV.

As in brain, the remaining pools of detyrosinated α-tubulin in neurons missing SVBP (and thus vasohibin-SVBP detyrosinating activity) could be due to α4-tubulin or to other detyrosinases 13,14 . Yet, the notable accumulation of tyrosinated tubulin in neurons observed before 7 DIV strongly suggest that, at these early stages of neuronal differentiation, the detyrosination of large pools of α-tubulin cannot be performed by other enzymes than vasohibin-SVBP complexes.

Confocal images of Svbp KO neurons cultured 2 DIV showed a large decrease in the amount of detyrosinated tubulin (Figure 3a) compared to wild type neurons. Remaining detyrosinated pools are concentrated in axons, whereas the other neurites exhibit tyrosinated tubulin. Moreover, SVBP deficiency led to a large decrease of neurons bearing an axon at 2 DIV (from 55.8 ± 4.0 % to 29.7 ± 3.3 %), revealing a clear delay in axonal differentiation (Figure 3b). Svbp KO neurons at 2 DIV developed an increased number of primary neurites and extra branches whereas the axon length was significantly reduced (-26.5 % ± 4.4 %) (Figure 3c-e). At 17 DIV, after complete neuronal maturation, Svbp KO neurons still show a significant increase in their dendritic branching compared to wild-type neurons within the first 150 µm close to the soma, as measured by Sholl analysis (Figure 3f).

Altogether, in neurons, SVBP deficiency resulted in marked changes in the pools of modified α-tubulin and in a delay of axon differentiation associated with severe morphological defects.

Brain morphological defects associated with Svbp deletion in mice

We performed brain imaging and morphometric assessments of wild-type and Svbp KO mice using anatomical MRI (Figure 4). Svbp KO mice show a significant decrease of the whole brain volume (7.3 %) affecting a large number of the brain regions including the cortex (7.8 %), the striatum (10.5 %), the hypothalamus (10.0 %), the thalamus (10.4 %) and the brainstem (10.0 %). We also performed measurements of white matter structures and found a strong significant reduction of several structures, for example the corpus callosum (33.7 %), the fimbria (31.7 %)

and the anterior commissure (38.9 %). Thus, Svbp deletion in mice results in a microcephaly affecting more severely the white matter, revealing deficiencies of axonal tracts.

Behavioral defects associated with Svbp deletion in mice

A comprehensive behavioral analysis of Svbp KO mice as compared to wild-type mice was performed. Firstly, we assayed locomotor activity in an open field. The total distance traveled by a mouse cumulated over 30 min was recorded. The responses exhibited by Svbp KO mice were enhanced compared with the wild-type (Figure 5a; WT: 7306 ± 464cm, Svbp KO: 9397 ± 595cm), indicating that the KO mice were more active than their wild-type counterparts.

We next evaluated cognitive abilities by investigating memory and social behavior. We monitored spontaneous alternation in a Y-maze, a test of spatial learning and memory. We found no difference between wild-type and Svbp KO mice in the alternation frequency (Figure S6a) indicating that the memory and willingness of KO mice to enter new environments was not affected. Social behavior was then assayed using the Resident-Intruder Test. Male mice are territorial and will react to an unfamiliar male placed in their home cage. We analyzed the behavior of a resident male, isolated either 1 or 3 weeks, when exposed to an intruder by measuring the time spent in active investigation. As shown in Figure 5b, Svbp KO mice exhibited a significant reduction of social investigation, compared to wild-type mice (WT:

150.1 ± 9.2 s and 142.6 ± 12.7 s, Svbp KO: 105.3 ± 12.6 s and 84.3 ± 3.1 s, respectively for 1 and 3 weeks of isolation). In addition, we investigated the exploratory behavior of mice in the Hamlet test [START_REF] Crouzier | Topographical memory analyzed in mice using the Hamlet test, a novel complex maze[END_REF]. Animals were repeatedly trained in the apparatus and analyses of the time spent in each houses during training days showed a significant increase in the presence of Svbp KO mice in the Hide house, a trend to decreased presence in the Interact house and no change in the 3 other houses, Run, Eat and Drink (Figure 5d), as compared to the group of wild-type mice. Data analyzed in terms of number of entries failed to show any difference whatever the house.

In parallel, we monitored possible depressive/anxiety-like status of Svbp KO mice. For that we performed the Novelty Suppressed Feeding (NSF) test where the animal's motivation to eat competes with the aversive signal of bright illumination. We measured the latency to eat and found that Svbp KO mice behave as wild-type mice (Figure S6b). We next used the Elevated Plus Maze test, using a maze composed of two open and two enclosed arms. Mice exhibit aversion to open spaces and will remain in enclosed spaces. In this test, Svbp KO mice behave as wild-type mice (Figure S6c). Finally, we used the Marble Burying Test, a test based on the fact that mice will bury harmful objects in their bedding. Each mouse was introduced in a cage containing 24 marbles for 30 min, and the number of buried marbles was then counted. As shown in Figure 5c, Svbp KO mice buried significantly less marbles than wild-type mice (WT:

12.77 ± 1.2, Svbp KO: 8.00 ± 0.81) indicating a possible lessening of anxiety and of interaction with the environment.

In summary, Svbp KO mice were more active than wild-type mice, do not exhibit severe memory defects, and seem to be less anxious than wild-type mice. Interestingly, they exhibit an impaired social behavior, showed by reduced interaction with the stranger mouse in the resident intruder test and a trend in the Hamlet, together with a higher time spent in the safe place provided by the Hide house in this device.

Discussion

In this study, we initially assessed a large consanguineous Pakistani kindred (family 1) using a combination of linkage analysis and WGS, identifying a c.82C>T; p.Q28* variant in SVBP as the most likely candidate. Although genetic co-segregation (Figure 1a) was confirmed in June 2012, the gene was then known as CCDC23 and little was known about the function of the encoded 66 amino-acid protein. The variant could therefore not conclusively be confirmed to be causative for the intellectual disability, microcephaly and spasticity observed in affected family members. Although initially identified as a secretory chaperone for VASH1 and proposed to play a regulatory role in angiogenesis [START_REF] Suzuki | Isolation of a small vasohibin-binding protein (SVBP) and its role in vasohibin secretion[END_REF], it was the recent studies confirming SVBP's importance in tubulin detyrosination and neuron differentiation [START_REF] Aillaud | Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation[END_REF][START_REF] Nieuwenhuis | Vasohibins encode tubulin detyrosinating activity[END_REF]) that prompted the functional testing required to confirm this variant as the causative allele. The proteintruncating changes were associated with an absence of a catalytically active enzymatic complex VASH-SVBP, as shown by in cell-activity tests of the variants. Results from HEK293T cells were then recapitulated in the brains of KO mice, where reduced levels of detyrosinated tubulin were observed, together with concomitant increases in tyrosinated tubulin. This is the first demonstration of the link between the clinical characteristics observed in patients with SVBP mutations and the underlying molecular defect of tubulin detyrosination. This study also highlights the importance of reassessing clinical genomic data [START_REF] Eldomery | Lessons learned from additional research analyses of unsolved clinical exome cases[END_REF][START_REF] Wright | Making new genetic diagnoses with old data: iterative reanalysis and reporting from genome-wide data in 1,133 families with developmental disorders[END_REF].

Additional evidence supporting pathogenicity comes from our recent identification of SVBP mutations in two additional families, with affected individuals sharing significant phenotypic overlaps. Although we registered the phenotype and genes in the chromosome 1 linkage region from Family 1 on GeneMatcher (https://genematcher.org/), this genetic replication came through a more unconventional route; Family 2 was identified during a two-week trial of the CentoMD mutation database [START_REF] Trujillano | A comprehensive global genotype-phenotype database for rare diseases[END_REF]. Family 3 was ascertained through access to the 100K Genomes Project; this study represents one of the first novel genes to be reported from that program.

Whilst this manuscript was in preparation, Iqbal et al described four individuals with intellectual disability, microcephaly, hypotonia and ataxia who were homozygous for the same p.Q28* variant [START_REF] Iqbal | Loss of function of SVBP leads to autosomal recessive intellectual disability, microcephaly, ataxia, and hypotonia[END_REF]. Although their two kindreds were of Syrian and Pakistani ethnicity, a single ancestral disease haplotype of 1.8 Mb was shared across both families. Where a genotype-phenotype relationship is based on a single disease haplotype, caution should be exercised as it is possible that an unobserved pathogenic mutation may lie on the same haplotype as the candidate variant [START_REF] Karaca | Phenotypic expansion illuminates multilocus pathogenic variation[END_REF][START_REF] Macarthur | Guidelines for investigating causality of sequence variants in human disease[END_REF]. Without WGS data, it is difficult to exclude the possibility that a rare non-coding variant or a cryptic structural variant could have been missed.

In highlighted consistent albeit subtle features including enlarged ventricles and a thin corpus callosum. Notably in KO mice this last brain region was also reduced by 33.7%. The most conspicuous difference between the families described here and those reported by Iqbal et al [START_REF] Iqbal | Loss of function of SVBP leads to autosomal recessive intellectual disability, microcephaly, ataxia, and hypotonia[END_REF] is that the previous group of patients were all reported to have hypotonia, whereas the majority of patients we identified had increased muscle tone, predominantly in the lower limbs.

Other variable features observed multiple times in our case series include digital abnormalities, mirror movements, coarse facial features and infantile seizures. Iqbal and colleagues reported chest abnormalities in 2 of their 4 patients but this feature was not noted in our families.

Behavioral characteristics may include aggression, described in two patients (29) and autism, described in one patient reported here. Although behavioral traits are difficult to model in rodents, we note that the Svbp KO mice in this study also exhibited a significant reduction of social investigation and possibly lower levels of anxiety associated with a low interest for their physical environment.

The tubulin tyrosination/detyrosination cycle is one of several post-translational modifications of neuronal tubulin. Other modifications include acetylation, polyglycylation and polyglutamylation, and collectively these are known as the "tubulin code". Over the last decade, studies in mice have linked tubulin acetylation and polyglutamylation to neurodegeneration [START_REF] Dompierre | Histone deacetylase 6 inhibition compensates for the transport deficit in Huntington's disease by increasing tubulin acetylation[END_REF][START_REF] Magiera | Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport[END_REF][START_REF] Outeiro | Sirtuin 2 inhibitors rescue alpha-synucleinmediated toxicity in models of Parkinson's disease[END_REF][START_REF] Rogowski | A family of protein-deglutamylating enzymes associated with neurodegeneration[END_REF]. More recently, a causal relationship between the CCP1 deglutamylase and neurodegeneration was found in humans [START_REF] Shashi | Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration[END_REF]. CCP1 deglutamylase is responsible for the successive removal of the two glutamate amino acids immediately preceding the C-terminal tyrosine, yielding Δ2-and Δ3-α-tubulin [START_REF] Aillaud | Evidence for new C-terminally truncated variants of alpha-and betatubulins[END_REF]. Detyrosinated α-tubulin is a substrate for the CCP1 enzyme and so it is not surprising that decreased levels Δ2-α-tubulin were observed in brains of the Svbp KO mice (Fig 2 b,e). Whilst the patients described in the present study did not show signs of cerebellar atrophy, shared features seen in both SVBP and CCP1 patient cohorts include microcephaly, hypotonia, spasticity, developmental delay and corpus callosum dysplasia.

The most frequent form of hereditary spastic paraplegia is caused by mutations in SPAST [START_REF] Tarrade | A mutation of spastin is responsible for swellings and impairment of transport in a region of axon characterized by changes in microtubule composition[END_REF]. SPAST encodes an enzyme that shows microtubule-severing activity, a process which in turn is regulated by tubulin glutamylation [START_REF] Valenstein | Graded Control of Microtubule Severing by Tubulin Glutamylation[END_REF], another type of post-translational modification that is common in mammalian brain tissue. It is also worth noting that detyrosinated microtubules are known to play a role in modulation of mechanotransduction in muscle [START_REF] Kerr | Detyrosinated microtubules modulate mechanotransduction in heart and skeletal muscle[END_REF]. It is therefore unsurprising that the abnormal phenotype seen in patients included muscular component and in most cases this was in the form of lower limb spastic paraparesis.

Interestingly, this feature was not recapitulated in the Svbp KO mouse model, where the open field locomotor activity was demonstrated to be increased.

The present study is the first detailed description of Svbp KO mice. Anatomical MRI indicates microcephaly and this whole brain volume reduction affects all brain structures analyzed. The brain size reduction is not associated with a general loss of body weight, indicating a specific alteration. Svbp deletion results in a severe reduction of the white matter with both intra-hemispheric (corpus callosum) and cortical tracts (fimbria, anterior commissure) defects.

The observed reduction of brain volume most possibly originates from an abnormal differentiation and maturation of deficient neurons. In agreement, Svbp KO cultured neurons showed a clear delay of their axon differentiation and severe morphological defects. Moreover, downregulation of SVBP was shown to disrupt neuronal migration in developing mouse neocortex [START_REF] Aillaud | Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation[END_REF]. Anomalies of progenitor proliferation might also occur since VASH-SVBP complexes were recently shown to be critical during mitosis [START_REF] Liao | Molecular basis of vasohibins-mediated detyrosination and its impact on spindle function and mitosis[END_REF]. Finally, the reduction of brain volume might also involve a reduction of synapse density, as suggested by shRNA knockdown experiments in rat hippocampal neurons [START_REF] Iqbal | Loss of function of SVBP leads to autosomal recessive intellectual disability, microcephaly, ataxia, and hypotonia[END_REF]. A whole in vivo analysis of neurons morphology and spines integrity will be needed to obtain a full picture of synaptic functioning in the absence of SVBP. Altogether our work clearly indicates that Svbp deletion results in brain defects more probably originating from neurodevelopmental anomalies. VASH-SVBP complexes are major regulators of detyrosination and thus of microtubule dynamics in neuronal cells [START_REF] Aillaud | Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation[END_REF][START_REF] Wang | Structural basis of tubulin detyrosination by the vasohibin-SVBP enzyme complex[END_REF]. The defects linked to Svbp deletion reported in this work highlight the importance of microtubules integrity for brain homeostasis and support the view that microtubule dysfunction can lead to neurodevelopmental disorders and neurodegeneration.

The comprehensive behavioral analysis of Svbp KO mice indicates several abnormal parameters. Svbp KO mice exhibit an enhanced locomotor sensitivity reaction when exposed to novelty. The possible anxious state of the mutant mice was investigated using the Marble Burying Test and the Elevated Plus Maze, however these results were difficult to interpret. Svbp KO mice showed no anxious behavior in the Elevated + Maze, whereas they buried a substantially lower number of marbles than wild-type mice in the Marble Test, possibly indicative of a low anxious state. This latter defect might rather be attributed to a lower interest for their environment. This result is in agreement with (a) the reduced interest of SVBP mice for their congener observed in the resident intruder test, and (b) the results of the Hamlet test.

Indeed, training of the animals in this complex environment providing distinct functionalized spots for eating, drinking, running, hiding and interacting identified only two differences in the Svbp KO mice behavior as compared with wild-type littermates, namely a trend to decrease interaction with the stranger mouse and a significant increase of time spent in the tunnel of the Hide house. Assessment of intellectual disability related defects in mice often relies on analysis of the working memory. Svbp KO mice do not exhibit deficit in Y-maze test which relies on measuring memory related to spatial exploration while factors such as motivational or emotional states are minimized and in which learning a rule is not involved. This result suggests that the working memory per se is not affected by Svbp deletion.

In summary, we have shown that rare biallelic variants in SVBP result in loss of tubulin detyrosination activity, causing anatomical brain deficits associated with intellectual disability.

The three families we describe have several features which are recapitulated in the KO mouse model, which is described here for the first time. This study thus adds to the growing list of diseases linked to aberrant microtubule cytoskeleton and point to the importance of microtubule dynamics.

Materials and methods

Whole-genome or whole-exome sequencing and variant filtering

Appropriate informed consent was obtained for all human subjects.

Family 1

Individual V-6 from family 1 underwent WGS in December 2011 using a HiSeq2000 machine (Illumina) and v3 chemistry, as part of the WGS500 project. Further details of the sequencing, read alignment and variant calling have been described previously [START_REF] Taylor | Factors influencing success of clinical genome sequencing across a broad spectrum of disorders[END_REF]. The proband's unaffected mother (IV-2), affected sister (V-7) and affected uncle and aunt (IV-3 and IV-4)

were genotyped on either the Illumina CytoSNP12 or the OmniExpress microarray (Figure 1a).

Since the parents of both pairs of affected siblings were first cousins, we considered the simple recessive model most likely, so focused on regions that were identical-by-descent for both alleles (IBD2) and homozygous in all the affected individuals. However, a compound recessive model would also fit the pedigree, and so we also considered regions that were IBD2 and heterozygous. To identify these regions, we selected SNV markers for linkage with linkdatagen [START_REF] Bahlo | Generating linkage mapping files from Affymetrix SNP chip data[END_REF] and then ran MERLIN (44) using the Kong and Cox exponential model [START_REF] Kong | Allele-sharing models: LOD scores and accurate linkage tests[END_REF], specifying a recessive model.

We then searched for rare coding variants within the linked regions. We excluded variants with a frequency greater than 0.5% in the 1000 Genomes project [START_REF] Genomes Project | A global reference for human genetic variation[END_REF], the NHLBI Exome Sequencing project (http://evs.gs.washington.edu/EVS/), gnomAD, or in our own WGS500 project [START_REF] Taylor | Factors influencing success of clinical genome sequencing across a broad spectrum of disorders[END_REF], and those that were seen as homozygotes in any of these projects (other than in individual V-6). We also prioritized variants in regions that were highly conserved across the 46 vertebrate species in the UCSC conservation track. Candidate variants were genotyped in the other unaffected siblings by Sanger sequencing to check segregation.

Since there was insufficient evidence based on the WGS500 project to implicate a pathogenic variant in these patients' condition the family were later recruited to the 100,000 Genomes Project (https://doi.org/10.6084/m9.figshare.4530893.v4), a national project that aims to establish the use of WGS in the National Health Service and which has been described in detail elsewhere [START_REF] Turnbull | The 100 000 Genomes Project: bringing whole genome sequencing to the NHS[END_REF]. Reads were mapped to GRCh37 using the Isaac aligner and variants were called using the Isaac variant caller (Illumina). Structural variants were called with Manta and Canvas (Illumina). As part of this study, samples were also genotyped using the InfiniumCoreExome-24v1 array (Illumina).

Families 2 and 3

The affected proband (II-3) from family 2 and her parents underwent WES analysis at CentoGene. WES workflows and results from the 1000 families were described previously [START_REF] Trujillano | Clinical exome sequencing: results from 2819 samples reflecting 1000 families[END_REF]. Results for family 2 were incorporated into the searchable CentoMD mutation database (CentoGene, www.centogene.com/pharma/mutation-database-centomd.html) [START_REF] Trujillano | A comprehensive global genotype-phenotype database for rare diseases[END_REF].

Family 3 were whole-genome sequenced as a quad, as part of the 100,000 Genomes Project (as described above). Samples were also genotyped using the InfiniumCoreExome-24v1 array (Illumina). Family 3 was identified from the rare disease subgroup of the 100,000

Genomes Project using a customized bcftools script designed to search through vcf.gz files specifically looking for loss of function (LoF) variants in SVBP. Genotypes from WGS and from the InfiniumCoreExome-24v1 arrays were used to assess identity by descent sharing across chromosome 1 by looking for extended regions sharing identical genotype calls.

In cellulo tubulin detyrosination assay

A plasmid encoding mouse SVBP (NM_024462) with C-terminal myc and Flag tags (SVBPmyc-Flag) was obtained from OriGene (MR200054). Using this plasmid we deleted residues 28-66 (mimicking p.Q28*) or replaced residues 13-66 by the 17 residues resulting from the frameshift p.K13Nfs*18. Mouse Vash1 cDNA (NM_177354) was PCR-amplified from plasmid Vash1-myc-DDK (Origin, MR222250) and inserted into pEGFP-N1 vector (Clontech), to generate vasohibin-1 with EGFP fused to its C-terminus (V1-GFP). All plasmids were verified for sequence integrity.

HEK293T cells were maintained under standard conditions and were transfected using JetPEI transfection reagent (Polyplus-Transfection). A ratio of 1:1 was used for cDNA co-transfections (for VASH1 with SVBP, and with variants). Protein extracts from HEK293T cells were prepared directly by scraping the cell layer in Laemmli buffer.

Generation of Svbp KO mice

The Svbp mutation was generated by CRISPR/Cas9 editing. Guide selection and off-target predictions were made with CRISPOR software [START_REF] Haeussler | Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR[END_REF]. Exon 2 target sequence (GATTTTTCTTTCCGGGCAGG) was unique in the genome. Predicted off-target sequences on the same chromosome were all non exonic, and with at least 4 mismatches (partly localized close to the PAM) with on-target sequences. A dual guide RNA was prepared by combining synthetic tracrRNA (TriLink BioTechnologies) and crRNA (Eurogentec, Angers, France).

Sequences are given in Table S2. A cloning free procedure (50) was used to modify the Svbp locus, by microinjection of Cas9/RNA complexes into in vitro fertilized B6D2F1xFVB embryos. F0 mosaic animals born after reimplantation of microinjected embryos were genotyped by PCR and Sanger sequencing, and then mated to C57BL/6 mice to provide F1 founder mice. Among these, a mouse was selected for the frameshift introducing as few extra amino acids as possible after the third amino-acid of SVBP. The allele was designated SvbpP4Rfs*6 and the corresponding protein sequence is MDPRKIQS (extra amino-acids in italics). At least 8 additional back-crosses were performed with C57BL/6 mice before phenotyping in order to avoid possible confounding effects due to off-target mutations. The study protocol was approved by the local animal welfare committee (Comité Local Grenoble Institute Neurosciences, C2EA-04) and complied with EU guidelines (directive 2010/63/EU).

Every precaution was taken to minimize stress and the number of animals used in each experiment. All experiments were conducted on wild-type and Svbp KO adult (3-6 months) littermates with a C57BL/6 genetic background.

Antibodies

Tyrosinated, detyrosinated and Δ2 α-tubulin were detected in cells and tissues by immunoblots using specific antibodies, as described previously [START_REF] Aillaud | Evidence for new C-terminally truncated variants of alpha-and betatubulins[END_REF]. The other primary antibodies used were as follows: rabbit anti-Flag from Molecular Probes (1:10,000), rabbit anti-GFP from Chromotek

(1:5,000), rabbit anti-GAPDH from Sigma (1:10,000).

Immunoblot analysis of α-tubulin variants in neuron and brain protein extracts

For neurons protein extracts analysis, cells were collected after 2, 7 or 17 DIV. After washing with phosphate-buffered saline (PBS) medium at 37°C, neurons were directly lysed in Laemmli buffer.

For analysis of mice tissues, crude protein extracts were prepared using a FastPrep Instrument (MP Biomedicals, Illkirch, France). Brain proteins were extracted in 100 mM 1,4piperazinediethanesulfonic acid at pH 6.7, 1 mM EGTA, 1 mM MgCl2, and protease inhibitors (Complete Mini EDTA-free; Roche Diagnostics, Meylan,France). Cell remnants were eliminated by a 10 min centrifugation at 10,000 × g, and Laemmli buffer was added to the supernatant.

Protein extracts were then loaded on 10% acrylamide gels (Mini-PROTEAN® TGX Stain-Free™, Biorad) and transferred with Trans-Blot® Turbo (BioRad). Membranes were incubated with primary antibodies, with secondary antibodies conjugated with HRP (1:10 000, from Jackson ImmunoResearch), and finally revealed with Chemidoc camera (Biorad). For analysis and graphical representations of immunoblots (Fig. 3b,c and S6c), protein bands were quantified from triplicate blots of 3 different experiments using Image Lab software (Bio-Rad).

Tyrosinated, detyrosinated or 2 α-tubulin signals were normalized to the protein content of the sample estimated from its GAPDH signal.

Neuronal cultures and morphometric analysis

Hippocampal neurons were cultured from E17.5 WT or Svbp KO embryos as described (12).

For axonal and neurite morphometric analysis, neurons were fixed after 2 DIV, immunolabeled and analyzed as previously described [START_REF] Aillaud | Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation[END_REF]. 20 neurons per embryo, from 6 different WT or Svbp KO embryos were analyzed.

For Sholl analysis, 1% of cultured hippocampal neurons were infected with 30 MOI of a lentivirus containing GFP (pWPT-GFP addgene.org/12255) and plated onto a monolayer of non-infected neurons. After 17 DIV, neurons were fixed and images were taken with an 20x/0.5 objective with an inverted Nikon microscope equipped with a camera EMCCD (Coolsnap, photometrics) piloted by Metaview software. Images were processed by the Tubeness filter, thresholded and converted to mask. Final masks were analyzed using the Sholl Analysis plugin of ImageJ. 15 to 20 neurons per embryo, from 4 WT and 3 Svbp KO embryos were analyzed.

Brain preparation for ex vivo MRI acquisitions

Brains were prepared as previously described [START_REF] Gimenez | 3D imaging of the brain morphology and connectivity defects in a model of psychiatric disorders: MAP6-KO mice[END_REF]. Briefly, brains were fixed before harvesting from animals by a transcardiac perfusion of a 4% paraformaldehyde solution in phosphate buffered saline added with a MRI contrast agent (6.25 mM of Gd-DOTA; Guerbet Laboratories, Roissy, France). The contrast agent was used here to reduce MRI acquisition time. After removing surrounding skin and muscles, the skulls containing intact brains were immersed in the fixing solution for four days, and then transferred to a Fomblin (FenS chemicals, Goes, Netherlands) bath for at least seven days after brain fixation. This schedule provided homogeneous distribution of the Gd-DOTA throughout the whole brain [START_REF] Gimenez | Microscopic DTI accurately identifies early glioma cell migration: correlation with multimodal imaging in a new glioma stem cell model[END_REF].

MRI acquisitions

Ex vivo 3D MRI acquisitions with a high spatial resolution were performed at 9.

Quantitative analysis of brain volumes

Brain regions were defined by MRI segmentation on the 3D T1w MR images. Each cerebral structure was manually delimited using regions of interest (ROI) drawn every five slices on the coronal orientation of the 3D T1w MRI, using Fiji software [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. Using the Segmentation editor plug-in (http://fiji.sc/Segmentation_Editor) the whole cerebral structure was reconstructed by interpolation between ROIs. Then, all ROIs were manually corrected based on the Allen mouse brain atlas (http://atlas.brain-map.org/atlas). Measurements were obtained for the following structures: the anterior commissure (ac), the brainstem (BS), the caudate putamen (CPU), the cerebellum (CB), the colliculi (CO), the corpus callosum (cc) the cortex (CX), the fimbria (fi), the globus pallidus (GP), the hippocampal formation (HF), the hypothalamus (HP), the olfactory bulb (OB) and the thalamus (TH). Each structure was color-coded, and its 3D representation and volume were determined using the 3D viewer plug-in in the Fiji software (https://imagej.net/3D_Viewer). For each region, the volume was calculated as the number of voxels × the voxel volume. All segmentations were done blind to the genotype. All data passed the normality test (except the BS for WT), we thus used a student t-test for group comparison.

Behavioral tests

Open Field: Locomotor activity of mice was video-recorded via a camera mounted above an open field and connected to a computer. Horizontal activity was analyzed using Ethovision XT14 software (Noldus, Wageningen, Netherlands). In the open field condition, the activity of four mice was simultaneously monitored in a white arena (L50 x l50 x h45 cm) for 30 min.

Within each genotype, mice were randomly placed in the different arenas.

The Marble Burying Test was performed as described previously [START_REF] Delotterie | Chronic administration of atypical antipsychotics improves behavioral and synaptic defects of STOP null mice[END_REF]. More precisely, the mouse was placed in a clear plastic box (L42 × l26 × h15 cm), containing 24 glass marbles (1.6 cm in diameter) evenly spaced (six rows of four marbles) on 4 cm of litter. Thirty minutes later, the animals were removed from the cages, and the number of marbles buried more than two thirds in the litter was scored.

Resident-Intruder Test: home-cage social interaction was assessed, as described [START_REF] Andrieux | The suppression of brain cold-stable microtubules in mice induces synaptic defects associated with neuroleptic-sensitive behavioral disorders[END_REF].

The duration of sniffing investigation displayed by a resident male mouse in response to presentation of an intruder male mouse was measured. In this test, wild-type and Svbp KO mice were housed individually during 1 or 3 weeks prior to testing to allow establishment of a homecage territory. The intruder mice, housed in groups, were unfamiliar to the resident mouse. In each trial, an intruder was placed in a corner of the resident home-cage (L36 × l20 × h14 cm), and the exploration activity of the resident was video-recorded for 5 minutes. The time spent sniffing in close contact with the intruder was measured manually from video recordings.

The Hamlet test is a novel complex environment device that allows mice to be trained in groups in a more ecological space than the housing cage [START_REF] Crouzier | Topographical memory analyzed in mice using the Hamlet test, a novel complex maze[END_REF]. The maze (160 cm diameter)

is composed of a central place, streets expanding from it in a star shape and 5 houses that have been functionalized. The houses contained a food dispenser (physiological function encoded:

Eat), two water dispensers (Drink), a novomaze ® (Viewpoint, Lissieu, France) (Hide), a running wheel (Run) or a compartment for a stranger mouse (Interact). Animals were placed in the Hamlet in groups of 7 from the same housing cage, for 4 h per day during a 2-weeks training period. The videotracking system (Viewpoint) recorded activity in each house in terms of number of entries and duration of presence.

Supplemental data

Supplemental data includes additional methods, six figures and two tables.

Figures titles and legends

  contrast, we have WGS data for both families harboring the p.Q28* disease haplotype and the uniformity of coverage means we are better able to exclude such scenarios. The genotypephenotype correlation is further corroborated by the results for Family 2 where a different truncating SVBP variant was detected in patients exhibiting the same phenotype. Detailed phenotypic descriptions of the 8 affected individuals described here allows us to start to delineate the range of phenotypic features associated with defective detyrosination in humans. In combination with the 4 patients reported by Iqbal et al (29), it is clear that the disorder comprises several common features as well as a number of variable characteristics. All patients presented with intellectual disability (moderate to profound), delayed speech and delayed gross motor development. Microcephaly was another common feature in human patients and this mirrors what was observed in Svbp KO mice where there was a 7% loss in global brain volume. Neuroradiological assessment of human MRI data (Figure 1 d,e)

  4 T (Bruker Biospec Avance III; IRMaGe facility) using a volume coil for transmission and a head surface cryocoil for reception. A 3D T1W gradient-echo MRI sequence was used for brain segmentation and volumetric analysis (repetition time: 35.2 ms, echo time: 8.5 ms, flip angle: 20°, field of view: 12 • 9 • 16 mm 3 , isotropic spatial resolution: 50 µm, 4 signal accumulations, total acquisition time per brain: 2 h 32 min).

Figure 1 .

 1 Figure 1. Pedigrees and brain imaging results for the three families described in this study.

Figure 2 .

 2 Figure 2. Impact of SVBP deficiency on detyrosination parameters

Figure 3 .

 3 Figure 3. Effect of SVBP deficiency on neurite outgrowth and axonal differentiation. Hippocampal

Figure 4 .

 4 Figure 4. Svbp deletion induce brain morphological defects

Figure 5 .

 5 Figure 5. Svbp KO mice exhibit behavioural defects

Table S1 :

 S1 Phenotypes of affected individuals in families described in this study. -, not known or missing data. AED, Anti epilepsy drugs. Reported centiles are calculated based on growth charts available at www.rcpch.ac.uk/resources/uk-who-growth-charts-2-18-years and Freeman et al 1995 (PMID: 7639543).

		V-6 (proband)	V-7	IV-3	IV-4	II-2	II-3 (proband)	II-1	II-3 (proband)
	SVBP variant									All have c.82C>T; p.Q28*
	(NM_199342.3) and ClinVar
	accession ID
	Ethnicity (degree of parental									Syrian (2nd-cousin) and Pakistani (1st-
	consanguinity)									cousin)
	Gender	Female	Female	Female	Male	Male	Female	Male	Female	3 Female, 1 Male
	Intellectual disability	Yes (moderate to severe)	Yes (severe)	Yes (moderate to severe)	Yes (moderate to severe)	Yes	Yes	Yes	Yes (severe)	4/4 (moderate to severe)
	Speech	Little expressive language, better	Little expressive language (few single words)	Few words	Little expressive language (few words -3	Few words	Only a few words aged 6 Only 10 words	Delayed speech and language	3 with delayed speech (1 too young for
		receptive language (few single words)			word sentences)			aged 21 years	development	assessment)
	Behavioural problem	Quiet	Loses temper, bangs head, cries, screams	-	-	-	-	-	Autistic behaviour	2 reported to be aggressive
	Height	106.9 cm aged 4 years 10 months (50th	90.7 cm aged 3 years 4 months (2nd-9th centile),	141.3 cm aged 45 years (<0.4th	158.4 cm aged 39 years (0.4th centile)	-	79 cm aged 2 years (2nd	Unable to stand-	96 cm age 3 years 6 months	<2 SD and -3.62 to -3.90 SD
		centile), 122.1 cm aged 7 years 7	131.2 cm aged 9 years and 10 months (9th-25th	centile)			centile)	flexion deformity	(25th centile)
		months (25th centile)	centile)					at hips and knees
	Head circumference	48.5 cm aged 5 years 4 months (0.4th	45.2 cm aged 3 years and 11 months (<0.4th	56.0 cm aged 45 years (50th-75th	55.5 cm aged 39 years (9th-25th centile) 49.7 cm aged 7	44.5 cm aged 2 years	46 cm aged 3	45 cm (<0.4th centile)	-2.63 to -6.43 SD
		centile)	centile)	centile)		years and 10	(<0.4th centile); 47.5 cm at	years (<0.4th
						months (0.4th	6 years 6 months (<0.4th	centile)
						centile)	centile)
	Spasticity	Yes (lower limb spasticity with brisk	Yes (increased tone in the lower limbs, with	No (walks only with the use of a	Yes (spastic paraparesis with brisk lower	Yes (generalized	Yes (generalized	Yes (spastic	Yes (progressive spasticity	No (4/4 with muscular hypotonia)
		tendon reflexes)	pathologically brisk reflexes and extensor plantar	walking frame, but lower limb tone	limb reflexes and extensor plantar	hypertonia	hypertonia predominantly	diplegia and non-	with delayed gross and fine
			responses)	and reflexes are normal)	responses; several operations to release	predominantly in	in both lower limbs, ankle	ambulant)	motor development)
					contractures at his hips and ankles)	both lower limbs,	clonus, easily elicited
						easily elicited	reflexes, unsteady spastic
						reflexes, unsteady	gait and toe walking)
						spastic gait)
	Gross motor development Delayed (walked at > 24 months)	Delayed (walked at 24 months)	Delayed (walked at 7 years)	Delayed (walked at 5 years)	-	-	'Walks on knees' Moderately delayed (walked	Delayed (walked at 2-3 years)
									at 14 months). Needs splints
	Brain imaging results	MRI showed irregular ventricular	MRI showed dysgenic corpus callosum with	MRI not available	MRI not available	-	MRI showed a thin corpus	-	MRI detected a thin corpus	CT scan performed for 2/4 and normal brain
		margins and a thin corpus callosum	dysmorphic ventricles; slight prominence of CSF				callosum, dilated ventricles		callosum and reduced	structures reported (data not shown)
		(data not shown)	spaces; normal cisterna magna, white matter,				with poor volume of white		periventricular white matter
			deep grey structures and calcification (data not				matter (Fig. 1d)		bulk with mildly enlarged
			shown)						ventricles (Fig. 1e)

Acknowledgments

We thank the in vivo experimental platform, the IRMaGE platform and the zootechnicians of the Grenoble Institute Neuroscience (GIN). We also thank Cyrielle Kint (www.diploid.com) and Christian Beetz (www.centogene.com) for reviewing exome data and helping facilitate international collaboration efforts and Gerardine Quaghebeur for reviewing MRI images for family 1.

Funding

This work was supported by the Wellcome Trust (203141/Z/16/Z) and the NIHR Biomedical Research Centre Oxford. Additional funding for the WGS500 project was from Illumina. This research was also made possible through access to the data and findings generated by the Steering Committee: Peter Donnelly (Chair) 1 , John Bell 2 , David Bentley 3 , Gil McVean 1 , Peter Ratcliffe 1 , Jenny C. Taylor 1,4 , Andrew Wilkie 4,5 Operations Committee: Peter Donnelly (Chair) 1 , John Broxholme 1 , David Buck 1 , Jean-Baptiste Cazier 1 , Richard Cornall 1 , Lorna Gregory 1 , Julian Knight 1 , Gerton Lunter 1 , Gil McVean 1 , Jenny C. Taylor 1,4 , Ian Tomlinson 1,4 , Andrew Wilkie 4,5 Sequencing & Experimental Follow up: David Buck (Lead) 1 , Christopher Allan 1 , Moustafa Attar 1 , Angie Green 1 , Lorna Gregory 1 , Sean Humphray 3 , Zoya Kingsbury 3 , Sarah Lamble 1 , Lorne Lonie 1 , Alistair T. Pagnamenta 1 , Paolo Piazza 1 , Guadelupe Polanco 1 , Amy Trebes 1 Data Analysis: Gil McVean 1 (Lead), Peter Donnelly 1 , Jean-Baptiste Cazier 1 , John Broxholme 1 , Richard Copley 1 , Simon Fiddy 1 , Russell Grocock 3 , Edouard Hatton 1 , Chris Holmes 1 , Linda Hughes 1 , Peter Humburg 1 , Alexander Kanapin 1 , Stefano Lise 1 , Gerton Lunter 1 , Hilary C. Martin 1 , Lisa Murray 3 , Davis McCarthy 1 , Andy Rimmer 1 , Natasha Sahgal 1 , Ben Wright 1 , Chris Yau 6 1 The Wellcome Centre for Human Genetics, Roosevelt Drive, Oxford, OX3 7BN, UK 2 Office of the Regius Professor of Medicine, Richard Doll Building, Roosevelt Drive, Oxford, OX3 7LF, UK 3 Illumina Cambridge Ltd., Chesterford Research Park, Little Chesterford, Essex, CB10 1XL, UK 4 NIHR Oxford Biomedical Research Centre, Oxford, UK 5 Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK 6 Imperial College London, South Kensington Campus, London, SW7 2AZ, UK Genomics England Research Consortium: names and affiliations of authors John C. Ambrose 1 , Emma L. Baple 1 , Marta Bleda 1 , Freya Boardman-Pretty 1,2 , Jeanne M. Boissiere 1 , Christopher R. Boustred 1 , Mark J. Caulfield 1,2 , Georgia C. Chan 1 , Clare E. H. Craig 1 , Louise C. Daugherty 1 , Anna de Burca 1 , Andrew Devereau 1 , Greg Elgar 1,2 , Rebecca E. Foulger 1 , Tom Fowler 1 , Pedro Furió-Tarí 1 , Joanne M. Hackett 1 , Dina Halai 1 , James E. Holman 1 , Tim J. P. Hubbard 1 , Rob Jackson 1 , Dalia Kasperaviciute 1,2 , Melis Kayikci 1 , Lea Lahnstein 1 , Kay Lawson 1 , Sarah E. A. Leigh 1 , Ivonne U. S.Leong 1 , Javier F. Lopez 1 , Fiona Maleady-Crowe 1 , Joanne Mason 1 , Ellen M. McDonagh 1,2 , Loukas Moutsianas 1,2 , Michael Mueller 1,2 , Nirupa Murugaesu 1 , Anna C. Need 1,2 , Chris A. Odhams 1 , Christine Patch 1,2 , Daniel Perez-Gil 1 , Dimitris Polychronopoulos 1 , John Pullinger 1 , Tahrima Rahim 1 , Augusto Rendon 1 , Pablo Riesgo-Ferreiro 1 , Tim Rogers 1 , Mina Ryten 1 , Kevin Savage 1 , Kushmita Sawant 1 , Richard H. Scott 1 , Afshan Siddiq 1 , Alexander Sieghart 1 , Damian Smedley 1,2 , Katherine R. Smith 1,2 , Alona Sosinsky 1,2 , William Spooner 1 , Helen E. Stevens 1 , Alexander Stuckey 1 , Razvan Sultana 1 , Ellen R. A. Thomas 1,2 , Simon R. Thompson 1 , Carolyn Tregidgo 1 , Arianna Tucci 1,2 , Emma Walsh 1 , Sarah A. Watters 1 , Matthew J. Welland 1 , Eleanor Williams 1 , Katarzyna Witkowska 1,2 , Suzanne M. Wood 1,2 , Magdalena Zarowiecki 1 1 Genomics England, London, UK 2 William Harvey Research Institute, Queen Mary University of London, London, EC1M 6BQ, UK.

Search for other candidate pathogenic variants in WGS data from Family 1

In addition to the simple recessive model, we also considered a compound heterozygous model in the three regions that were IBD2 between all affected individuals but not homozygous (totalling 10 Mb), but no good candidates were found.

Additionally, several methods were used to search for copy number variants (CNVs), as described. 1 These included a) generating count profiles in 10 kb bins along the genome and correcting for noise due to biases in sequence composition, b) OncoSNP-SEQ 2 and c) ExomeDepth. 3 In addition to these, GenomeSTRiP 4 was used to search for intermediate-sized deletions (50-50,000 bp) that would have been missed by arrayCGH and Platypus. 5 We filtered the calls based on their frequency in other WGS500 samples (MAF < 0.5%). We considered the closest gene to each deletion as the affected gene, and, for the compound recessive model, treated large deletions and SNVs/small indels together. We found no rare structural variants within the linkage region.

We also considered noncoding variants with MAF < 0.5%. To narrow the search, we focused on variants that were within conserved blocks of DNA identified using phastCons 6 , and that fell in regulatory regions annotated by the Ensembl Variant Effect Predictor 7 , which were identified using information about various chromatin marks and transcription factor binding sites. This did not reveal any variants that seemed more likely to be causal than the p.(Q28*) variant in SVBP.

To allow for the possibility of a pedigree error, we analyzed the WGS data from individual V-6 alone to check that there were no more likely causal variants. We retained only high confidence variants with at least 5x coverage, a call quality of at least 20 and which passed upstream pipeline filtering. We also required for variants to be seen in both +ve and -ve strand reads. In a previous study, analysis of data from the WGS500 project identified regions of the genome where variant genotype calls are typically unreliable and varied based on which analytical pipeline had been used. 8 We therefore excluded variants situated in these regions as well.

We then applied a number of population allele frequency filters, removing variants that were seen at > 0.1% in at least one of the following datasets: the 1000 genomes project (phase3v5b), NHLBI ESP exomes (ESP6500SI-V2), ExAC (v0.3.1; using subpopulation with highest allele frequency), gnomAD (v2.0.1; using subpopulation with highest allele frequency).

We then restricted to coding variants (missense, in-frame indels, frameshifting indels, or stop gain, +/-2 bp splice site variants and variants predicted to disrupt splicing by MaxEntScan).

Lastly, we filtered for variants that were called homozygous and were seen in at least 80% of reads.

Sanger sequencing for variant validation and confirmation of segregation

For Sanger validation of the c.82C>T; p.(Q28*) variant we PCR amplified a 269 bp DNA fragment with primers 5'-GCTCGCTGTCAGTTCTCCTT-3' and 5'-AAGATCTGCCTCTTTCTGTACCA-3' in 25 µl reaction volumes using the FastStart Taq DNA Polymerase PCR kit (Roche).

Amplicons were run on a 1.8% agarose gel for size confirmation and then purified using exonuclease I and shrimp alkaline phosphatase. Sanger sequencing was performed using BigDye chemistry (v3.1).

RNA analysis of clinical samples

2.5 ml of whole blood was collected from all available family members (IV-2, IV-3, IV-4, V-6 and V-7) into PAXgene blood RNA tubes (PreAnalytiX). RNA was extracted using PaxGene blood RNA extraction kit (Qiagen) according to the manufacturer's guidelines. RNA was quantified using the nanodrop ND-1000 and RIN values were obtained using the 2100 Bioanalyser using RNA 6000 Pico chips (Agilent Technologies). RIN values were all 7.7-8.8, indicating good quality RNA. Reverse transcriptase (RT) reactions were performed using 500 ng of heat-denatured RNA as template with the QuantiTect kit (Qiagen) which uses a mix of oligo-dT and random primers. RT-negative reactions were set up in parallel to ensure there was no amplification from any residual genomic DNA.

A 265 bp segment spanning exons 2 and 3 of SVBP cDNA was amplified with primers SVBP-2F 5'-AGAAATATCCAGAAGTCAAGCCA-3' and SVBP-3R 5'-TCTCAAAGCTCTCAGGATCCC-3'. Amplicons were size-checked and then purified as described above. Sanger sequencing was performed using BigDye chemistry (v3.1) and run on an ABI 3730XL machine.

Quantitative PCR was also performed on the same cDNA samples using the SVBP-2F/3R primers and the iQ5 SYBR Green Supermix, using the iQ5 realtime-PCR machine (BIO-RAD). Ct values derived from a 234 bp ACTB amplicon were used to normalise expression. Expression was then compared to two control cDNA samples (prepared using the same method) using the 2 -ΔΔCt formula. 9 qPCRs were performed in triplicate and the experiment was performed three times.

Genotyping and backcrosses

PCR amplifications were performed directly on alkaline lysates of toe clips. Briefly, each toe was incubated 30 min at 95°C in 100 μL of alkaline solution (NaOH 25 mM, EDTA 0.2 mM, pH 12.0). Neutralization was performed by adding 100 μl Tris 40 mM, pH 5.0. Two microliters of lysates were analyzed by PCR (25 μL) with appropriate primers (Table S1) and GreenTaq Mix (Euromedex), according the following protocol: 95°C for 10 min, 33 cycles of [95°C for 30 sec / 58°C for 30 sec / 72°C for 30 sec], 72°C for 10 min. Indels in F0 mice were identified by Sanger sequencing of PCR products (Eurofins). The sequencing Sanger electropherograms obtained from mosaic F0 mice were analyzed using the TIDE software which decomposes sequencing traces and estimates the respective contribution of each PCR fragment. 10 We thus selected a mosaic F0 mouse carrying a 11 nt deletion introducing a frameshift and only four additional chimeric residues. This F0 mouse was further backcrossed with C57BL/6 mice to select the Δ11 deletion among the F0 mosaic indels. Tail fragments of the F1 mice and their successive backcrosses were lysed as above. Two microliters of lysates were then analyzed by PCR (25 μL), using forward primers targeting either the wild-type allele or the Δ11 deletion junction, a common reverse primer (Table S1) and GreenTaq Mix, according the following protocol: 95°C for 5 min, 33 cycles of [95°C for 30 sec / 50°C for 30 sec / 72°C for 30 sec], 72°C for 2 min. Amplicons were run on a 1.8% agarose gel for presence and size confirmation. PCR with the 5' WT-for primer generates a fragment only with the DNAs of wild-type and heterozygous mice. PCR with the 5' Δ11-for primer generates a fragment only with the DNAs of Svbp KO and heterozygous mice.

Y-Maze test

The Y-Maze test was performed as described previously. 11 Spontaneous alternation in a Ymaze is based on the innate tendency of rodents to explore new environments, and the shortterm storage and retrieval of relevant information are related to working memory. The apparatus consists of three walled arms (L20 x l8 x h16 cm) radiating at an angle of 120° from each other. Without prior habituation to the apparatus, each mouse was placed into the starting arm and allowed to move freely through the maze during 5 min. A mouse was considered to have entered an arm when all four paws were positioned in the arm runway.

An alternation was defined as entries into all 3 arms on consecutive choices. The alternation score (%) for each mouse was calculated as the ratio of the actual number of alternations to the possible number (defined as the total number of arm entries minus 2), as shown by the following equation: % alternation = [(Number of alternations) / (Total arm entries-2)] × 100.

Novelty Suppressed Feeding (NSF) test

The NSF test was performed as described previously. 12 It is an anxiety-based conflict test where the motivation to eat competes with fear of a brightly lit area. The NSF test was performed for 15 min. Briefly, animals had no access to food in their home cage for 20 h prior to performing the test. For the test, a single food pellet was placed on a brightly-lit white paper platform placed in the center of a plastic box (L37 x l57 x h20 cm) where the floor was covered with approximately 2 cm of bedding. The animal was placed in a corner of the box and the latency to eat was recorded.

Elevated Plus Maze

This labyrinth consists of two intersecting tracks, placed at 60 cm from the ground. One track had no walls (two open arms), while the other had dark walls 15 cm high (two arms closed).

Each arms measure L30 cm x l7 cm. The mice were placed in the center of the labyrinth, facing an open arm, and allowed to explore for 5 min. A camera placed above the labyrinth recorded the movements of the animal and the tests were analyzed using the Ethovision XT14 software (Noldus, Wageningen, Netherlands). Total distance travelled, number of entries and time spent in each arm were monitored.

Supplementary Figures and Tables Supplemental data include six figures and two tables