Dimitra Kiritsi

Johannes S Kern

Hauke Schumann

Jürgen Kohlhase

Cristina Has

Prof. Dr Leena Bruckner-Tuderman
email: bruckner-tuderman@uniklinik-freiburg.de

Molecular mechanisms of phenotypic variability in junctional epidermolysis bullosa

Keywords: Dermatology, genetic screening/counselling, gene therapy

Background Junctional epidermolysis bullosa (JEB), a group of hereditary skin fragility disorders, is associated with a wide variety of phenotypes, although all forms are characterized by trauma-induced skin blistering and tissue separation at the dermalepidermal junction zone. A subgroup, coined JEB-other, is associated with mutations in the COL17A1 gene encoding collagen XVII or, more rarely, with mutations in the laminin 332 genes LAMA3, LAMB3, or LAMC2. The objective of this study is comprehensive genotypephenotype analysis in JEB-other patients with COL17A1 mutations and elucidation of disease mechanisms underlying different skin phenotypes.

Methods and results COL17A1 mutations and their clinical and cellular consequences were systematically analyzed in 43 patients with JEB-other. Cell culture, RT-PCR and protein biochemistry were applied to assess the effects of splice-site mutations, i.e. the nature and amounts of transcripts and polypeptides synthesized and their association with the phenotypic outcome. Thirty-four distinct COL17A1 mutations were disclosed, 12 of them novel. mRNA and protein analyses demonstrated that patients with only about 12-14% of the physiological collagen XVII levels had mild cutaneous involvement and a long life span.

Conclusions

In contrast to complete null phenotypes, presence of minor amounts of collagen XVII protein in JEB skin is associated with mild phenotypic manifestations. The data have significant implications for design of molecular therapies for JEB, since they suggest that already a low extent of collagen XVII restoration will improve skin stability and alleviate symptoms.

INTRODUCTION

JEB is genetically and clinically heterogeneous, but in all forms trauma-induced tissue separation occurs at the level of the lamina lucida of the dermal-epidermal basement membrane [START_REF] Yancey | Non-herlitz junctional epidermolysis bullosa[END_REF] . The mostly lethal JEB-Herlitz is caused by null mutations in the LAMA3, LAMB3, or LAMC2 genes encoding laminin polypeptides and JEB with pyloric atresia by mutations in the integrin genes ITGB4 and ITGA6. A phenotypically variable group, coined JEB-other, is associated with missense mutations in the above laminin genes or with mutations in COL17A1 encoding collagen XVII [START_REF] Fine | The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB[END_REF] . The COL17A1 phenotypes exhibit a wide range of severity and, in addition to skin blistering, different degrees of mucosal involvement, enamel defects, dystrophy or loss of nails, and alopecia can occur. The causes for the phenotypic variability are not well understood. Although more than 60

COL17A1

mutations have been reported in the literature (http://www.hgmd.cf.ac.uk/ac/index.php), the only clear genotype-phenotype correlation is that patients with complete lack of collagen XVII in the skin exhibit severe, generalized blistering with mucosal and adnexal involvement, whereas patients with reduced amounts of normal or with mutated collagen XVII in the skin display more moderate symptoms [START_REF] Varki | Epidermolysis bullosa. I. Molecular genetics of the junctional and hemidesmosomal variants[END_REF][START_REF] Pasmooij | Localized and generalized forms of blistering in junctional epidermolysis bullosa due to COL17A1 mutations in the Netherlands[END_REF] .

The COL17A1 gene (NM_000494.3) on chromosome 10q24.3 comprises 56 exons [START_REF] Giudice | Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180[END_REF] . Its product, collagen XVII, is a type II transmembrane protein. Each collagen XVII is a trimer of three 180 kDa α1(XVII)-chains with an intracellular N-terminal domain of 466 amino acids, a transmembrane stretch of 23 amino acids and an extracellular C-terminus of 1008 amino acids. The ectodomain contains 15 collagenous (COL) subdomains, interrupted by 16 noncollagenous sequences (NC) and is proteolytically shed from the cell surface by ADAMs proteinases, yielding a shorter form of the protein which remains stable in the extracellular matrix [START_REF] Franzke | Collagenous transmembrane proteins: recent insights into biology and pathology[END_REF] . Thus, functionally, collagen XVII plays a dual role as a cell surface receptor and as a matrix component. During steady-state homeostasis, it maintains adhesion of basal keratinocytes to the basement membrane. Shedding of the ectodomain releases the cell from some of its binding partners and allows it to become motile during epidermal differentiation or during wound healing and tissue regeneration [START_REF] Nishie | Ectodomain shedding generates Neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid[END_REF] .

Recent pilot studies on causal therapies for genetic skin diseases have shown that relatively small biological changes, e.g. moderately increased levels of a missing protein in the skin, can have substantial clinical effects [START_REF] Uitto | Progress in epidermolysis bullosa research: toward treatment and cure[END_REF] . Specifically, fibroblast-based cell therapies of collagen VII-deficient dystrophic epidermolysis bullosa demonstrated that about 35 % of physiological levels of the collagen confer greatly improved skin integrity [START_REF] Wong | Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa[END_REF][START_REF] Kern | Mechanisms of fibroblast cell therapy for dystrophic epidermolysis bullosa: high stability of collagen VII favors long-term skin integrity[END_REF] and, in a human pilot trial, gene therapy for JEB partially restored laminin 332 in the skin and significantly ameliorated the phenotype [START_REF] Mavilio | Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells[END_REF] . This was corroborated by a recently described laminin gamma-2 hypomorphic mouse [START_REF] Bubier | A mouse model of generalized non-Herlitz junctional epidermolysis bullosa[END_REF] , which has low levels of laminin 332 in the skin and a moderate phenotype, in contrast to the perinatally lethal laminin 332 knockout mice [START_REF] Ryan | Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells[END_REF][START_REF] Kuster | IAP insertion in the murine LamB3 gene results in junctional epidermolysis bullosa[END_REF][START_REF] Meng | Targeted inactivation of murine laminin gamma2-chain gene recapitulates human junctional epidermolysis bullosa[END_REF] . These observations suggest that the multiprotein complexes at the dermalepidermal junction are highly stable with robust interactions of the binding partners, which can compensate a significant, but not complete, loss of one component. On this background, we addressed the role in skin integrity of a major component of the epidermal adhesion complex, collagen XVII, in a large cohort of patients with collagen XVII-deficient JEB.

PATIENTS AND METHODS

Patients and tissue samples

Forty-three patients of Caucasian ancestry and European origin were investigated. They were suspected to have EB and presented at the Freiburg EB-center for clinical evaluation and diagnosis, or DNA and skin samples were provided by collaborating centers. Following informed consent, EDTA-blood and skin samples were obtained from the patients and, if available, from family members. The study was approved by the ethics committee of the University of Freiburg.

Immunofluorescence staining

Immunofluorescence staining of the skin was performed using a panel of antibodies to components of the epidermal basement membrane zone as described [START_REF] Herz | Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes[END_REF] . For diagnostic purposes, the following domain-specific collagen XVII antibodies were used: the polyclonal antibody NC16A, which binds to the NC16A domain of collagen XVII, and the monoclonal NC16A1 recognizing amino acids 513-525 [START_REF] Franzke | Collagenous transmembrane proteins: collagen XVII as a prototype[END_REF][START_REF] Schacke | Two forms of collagen XVII in keratinocytes. A full-length transmembrane protein and a soluble ectodomain[END_REF]19] . The sections were observed with an Axiophot fluorescence microscope (Carl Zeiss, Göttingen, Germany). To quantify the collagen XVII expression in situ, 3 skin sections of each patient were stained with the NC16A1 antibody and photographed with the confocal laser scanning microscope (LSM510, Carl Zeiss, Göttingen, Germany). The fluorescence signal was quantified using the image processing program Image J [START_REF] Collins | ImageJ for microscopy[END_REF] version 1.43 (http://rsbweb.nih.gov/ij/docs/index.html), after background subtraction.

Keratinocyte cultures

Primary epidermal keratinocytes were isolated from skin biopsies of patients 28, 38 and 15 and of a normal control, and cultivated in keratinocyte growth medium (Invitrogen, Karlsruhe, Germany), as described [START_REF] Konig | Transforming growth factor-beta stimulates collagen VII expression by cutaneous cells in vitro[END_REF] . They were used for RNA and protein isolation in passage 2 at 80% confluence.

Mutation detection and RNA analysis

Genomic DNA (gDNA) was extracted from EDTA-blood using the QiAmp DNA mini kit (Qiagen, Hilden, Germany). For amplification of all COL17A1 exons and exon/intron boundaries primers were used as described [START_REF] Giudice | Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180[END_REF][START_REF] Gatalica | Cloning of the human type XVII collagen gene (COL17A1), and detection of novel mutations in generalized atrophic benign epidermolysis bullosa[END_REF] , and sequencing was performed on an ABI 7330XL DNA analyzer. Mutations were confirmed by resequencing in both directions.

Total RNA was extracted from subconfluent cells with QiaAmp RNA blood mini kit (Qiagen Hilden, Germany). Reverse transcription (RT) was performed with Advantage RT-for-PCR Kit (BD Biosciences, Heidelberg, Germany) with 0.5 µg of total RNA. The primers for RT-PCR of COL17A1 were as follows: exon 18F 5'-gaggaggtgaggaagctgaa and exon 23R 5'tcccctttctctccagatcc, amplicon of 467bp; exon 50F 5'-attcagaggcatcgttggac and exon 52R 5'-ctgtgctcatggaagagctg, amplicon of 633bp; exon 51F 5'-gctgaaaacagcgacagctt and exon 54R 5'-gtcacgttgctgtaggcaga with a product of 532bp. The PCR products were separated on 1.5% agarose gels. The RT-PCR products were subcloned to TOPO TAcloning vector (Invitrogen, Karlsruhe, Germany), and for each reaction, 10 to 15 clones were sequenced using the M13-RV primer.

Immunoblotting

For immunoblotting, cultured keratinocytes were extracted with a buffer containing 0.1 M NaCl, 20 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, Pefabloc and EDTA [START_REF] Schacke | Two forms of collagen XVII in keratinocytes. A full-length transmembrane protein and a soluble ectodomain[END_REF][START_REF] Schumann | Three Novel Homozygous Point Mutations and a New Polymorphism in the COL17A1 Gene: Relation to Biological and Clinical Phenotypes of Junctional Epidermolysis Bullosa[END_REF] . Protein content was quantified with a micro Lowry assay (DC Protein Assay, Bio-Rad, Munich, Germany).

Normalized amounts of the proteins were subjected to SDS-PAGE, immunoblotted and incubated with the following anti-collagen XVII polyclonal antibodies: ecto-4, recognizing epitopes within the 50 most C-terminal amino acids and NC16A [START_REF] Schacke | Two forms of collagen XVII in keratinocytes. A full-length transmembrane protein and a soluble ectodomain[END_REF][START_REF] Schumann | The shed ectodomain of collagen XVII/BP180 is targeted by autoantibodies in different blistering skin diseases[END_REF][START_REF] Tasanen | Hemizygosity for a glycine substitution in collagen XVII: unfolding and degradation of the ectodomain[END_REF] . The blots were incubated with antibodies to β-tubulin (Abcam, Cambridge, UK) or GAPDH (clone 6C5, Millipore, Temecula, California) to control loading. Densitometric quantification of the bands was performed with the software Gel-Pro Analyzer software (MediaCybernetics, Inc., Bethesda, MD, USA).

RESULTS

Mutation detection strategy and novel COL17A1 mutations

Forty-three European patients with JEB-other were included in this study (Table 1). Most mutations were located in exons 51 and 52. Notably, splice-site mutations occurred preferentially in intron 51. Using this approach, the mutation detection rate reached 98.8 % (85/86 alleles). Thirty-four distinct mutations were disclosed, suggesting that most families have their own private mutation (Figure 1). The majority, 69%, were nonsense mutations, insertions and deletions predicted to lead to a premature termination codon (PTC).

Missense mutations comprised 19%, and splice-site mutations 12% of the survey. The mutations were distributed over the entire COL17A1 gene (Figure 1). Two mutations were found to be recurrent in European patients. The non-population-specific mutation p.Arg1226X [START_REF] Varki | Epidermolysis bullosa. I. Molecular genetics of the junctional and hemidesmosomal variants[END_REF][START_REF] Pasmooij | Localized and generalized forms of blistering in junctional epidermolysis bullosa due to COL17A1 mutations in the Netherlands[END_REF][START_REF] Nakano | Junctional epidermolysis bullosa in the Middle East: clinical and genetic studies in a series of consanguineous families[END_REF] was found in almost 10%, and p.Gly803X [START_REF] Darling | Premature termination codons are present on both alleles of the bullous pemphigoid antigen 2/type XVII collagen gene in five Austrian families with generalized atrophic benign epidermolysis bullosa[END_REF] in approximately 7% of the mutated alleles.

Phenotypic variability

To assess disease severity the following clinical parameters were recorded during multiple patient visits: extent of skin and mucosal involvement, nail dystrophy and alopecia. Twentyseven of the 43 patients had a disseminated blistering and a generalized disease phenotype [START_REF] Darling | Generalized atrophic benign epidermolysis bullosa[END_REF] , whereas 11 had a localized phenotype. No clinical data was available of five patients. All patients had dystrophic toe nails and the only patients with normal finger nails were 28, 35 and 36. For almost half of the patients, dental anomalies were reported, including enamel defects and subsequent susceptibility to caries and/or parodontitis [START_REF] Wright | Oral manifestations in the epidermolysis bullosa spectrum[END_REF] .

On molecular level, the individuals with severe clinical phenotypes were homozygous or compound heterozygous for PTC or splice-site mutations and exhibited the full spectrum of symptoms: generalized blisters, alopecia and nail anomalies [START_REF] Yancey | Non-herlitz junctional epidermolysis bullosa[END_REF][START_REF] Tosti | Nail involvement in epidermolysis bullosa[END_REF] (Figure 2A-C In order to explain the phenotypic variability, we focused on biological consequences of splice site mutations, which are delineated in the following.

Splice-site mutations associated with mild phenotypes in elderly individuals

The 80 year-old patient 28 presented with blisters mostly on the gluteal area and the thighs, as she was restricted to bed because of joint problems, and with toe nail dystrophy (Figure 2 E-F). Mucous membranes and hair were normal. Her two sisters, 79 and 82 years-old (patients 35, 36), were very mildly affected, with toe nail dystrophy as the only symptom (Figure 2D). These 3 patients were the only ones in our cohort, who did not have dystrophic finger nails.

In this family, the novel splice-site mutation c.1744-2A>C at the acceptor splice site of exon 20 was combined with the nonsense mutation p.Arg1169X. RT-PCR with primers spanning exon 18-23 revealed three abnormal transcripts (Figure 3A). The PCR products were subcloned, the clones sequenced and the transcripts identified. Transcript 1 (12.5% of the clones) contained in-frame skipping of exons 21 and 22 (90 nucleotides) and resulted in the deletion of 30 amino acids, spanning part of the COL15 domain. In transcript 2 (25% of the clones), three nucleotides in exon 21 were skipped leading to the deletion of Gly582

within the COL15 domain. The T2 band presumably also contains the transcripts that do not bear the splice-site mutation, but correspond to the allele carrying the p.Arg1169X mutation. Transcript 3 (12.5% of the clones) led to activation of a cryptic acceptor splicesite in intron 20 and to partial out-of-frame retention of 95 nucleotides of this intron (Figure 3A). 50% of the clones contained the normal sequence, which probably resulted from the allele with the p.Arg1169X mutation. It is likely that mutated collagen XVII is translated from transcripts 1 and 2. The first would contain a partial deletion of the COL15 domain and the latter a single amino acid deletion of Gly582 (Figure 3B, lower panel). Immunoblotting of keratinocyte lysates of patient 28 showed that only about 3-4% of collagen XVII was present, as compared to control cells (Figure 3B, upper left panel). Since mutated polypeptides are often sensitive to proteolytic degradation, we subjected a skin biopsy of patient 28 to immunofluorescence staining using collagen XVII antibodies. This revealed a reduced signal, corresponding to approximately 14% of collagen XVII levels in control skin (Figure 3B, upper right panel). Taken together, the data indicate that a fraction of the translated polypeptides was degraded, but also that about 14% of normal collagen XVII levels confer reasonable skin stability and a mild blistering phenotype in vivo.

The 67 year-old male patient 38 presented with blisters mostly on thighs and hands (Figure 2G). He had nail dystrophy (Figure 2H), but no mucosal involvement. The frontal alopecia was of androgenetic type and not associated with JEB. Mutation screening disclosed two novel mutations, the splice-site mutation c.4156+1G>A at the donor splice site of exon 52 and the nonsense mutation p.Arg169X. RT-PCR with primers spanning exons 51-54 generated four aberrant transcripts (Figure 4A). Transcript 1 (25% of the clones) led to partial, out-of-frame skipping of exon 52 and to a PTC after 155 codons. In transcript 2 (25% of the clones), 210 nucleotides of exon 52 were skipped resulting in the deletion of amino acids 1316 -1385 of the COL3 and NC3 subdomains. Transcript 3 (50% of the clones) comprised in-frame skipping of exon 52 (390 nucleotides) and led to the deletion of 130 amino acids and absence of the COL4 and COL3 subdomains (Figure 4A). The fourth, minor one could not be identified. Immunoblotting of patient keratinocyte lysates revealed small amounts of a shorter, truncated collagen XVII polypeptide of about 165 kDa, likely a product of transcript 3 (Figure 4B, upper left panel). Semiquantiative analysis of band intensity showed that this corresponded to about 4 % of controls. Immunofluorescence staining of a skin biopsy revealed reduced collagen XVII staining, corresponding to about 12% of controls (Figure 4B, upper right panel).

The novel splice-site mutation c.3766+5G>A associated with complete loss of collagen XVII and a generalized phenotype

In contrast to the mutations described above, the outcome of the novel splice-site mutation c.3766+5G>A, at the donor splice site of exon 51 was associated with a severe phenotype in the 1 year-old patient 15. He suffered from congenital and persisting generalized blistering, mucosal involvement and nail dystrophy. In accordance, three of the four transcripts representing more than 80% of the clones, resulted in frame-shift and PTC. One minor transcript could not be identified in the analyzed clones. Only transcript 4 (18% of the clones) led to in-frame skipping of exon 51. This deletion is predicted to encompass amino acids 1207-1255 and to eliminate the NC5, COL5 and NC6 domains of the protein (Figure 5). However, the truncated collagen XVII was obviously unstable and susceptible to degradation, since neither immunofluorescence staining of the skin (not shown) nor immunoblotting of keratinocyte extracts revealed presence of collagen XVII protein (Figure 5).

DISCUSSION

Here we describe genotype-phenotype correlations in a cohort of 43 European JEB patients with collagen XVII mutations. The group exhibited a wide spectrum of clinical variability ranging from mild, predominantly acral blistering to severe phenotypes with generalized blistering, mucosal involvement, extensive nail dystrophy and universal alopecia. In general, the milder pathological manifestations were associated with missense or splice-site mutations and the presence of at least some collagen XVII in the skin. The hallmark of the severe phenotypes was total lack of collagen XVII in the skin, but different mutation mechanisms were found to underlie the deficiency. Delineation of such mechanisms is a pivotal for understanding the molecular pathology and a prerequisite for development of novel, biologically valid therapies.

Particularly interesting was the observation that splice-site mutations allowing low expression of collagen XVII generate mild disease manifestations, as has been suggested in individual case reports [START_REF] Ruzzi | A homozygous nonsense mutation in type XVII collagen gene (COL17A1) uncovers an alternatively spliced mRNA accounting for an unusually mild form of non-Herlitz junctional epidermolysis bullosa[END_REF][START_REF] Franzke | C-terminal truncation impairs glycosylation of transmembrane collagen XVII and leads to intracellular accumulation[END_REF] . Illustrative examples of this were the elderly patients 28, 35, 36 and 38, who had had very few symptoms during past several decades. Occasional skin fragility had been suspected to represent an acquired condition. However, with advancing age, diseases such as diabetes or increasing physical immobility aggravated the blistering, and the patients were finally diagnosed with JEB in their 7 th or 9 th decade of life. These observations suggest that comorbidities are modifying factors of JEB and should alert medical professionals to critically re-evaluate individuals suspected to have an acquired skin blistering disorder, which does not respond to therapy.

Based on the data in the present study, we predict that both the percentage of transcripts not causing PTCs and the location of the splice-site mutation play a role in the outcome on protein level. All splice-site mutations described here targeted collagenous subdomains of the collagen XVII molecule. Some, but not all of them had devastating effects on the stability of collagen XVII and led to degradation of unstable molecules by tissue proteinases. Mutations within collagenous domains are known to perturb the G-X-Y repeat motif and affect the stability of the ectodomain, leading to unfolding and degradation of the collagen XVII molecule [START_REF] Tasanen | Hemizygosity for a glycine substitution in collagen XVII: unfolding and degradation of the ectodomain[END_REF][START_REF] Huilaja | Glycine substitution mutations cause intracellular accumulation of collagen XVII and affect its post-translational modifications[END_REF][START_REF] Vaisanen | Molecular mechanisms of junctional epidermolysis bullosa: Col 15 domain mutations decrease the thermal stability of collagen XVII[END_REF] . Biochemical analyses of transcripts and mutant polypeptides, as performed here, may become useful prognostic markers for determining the course of the disease at an early stage and be helpful for counselling the patients relating to genetic and environmental contributions to their disease and quality of life. As reported for other EB forms, modifying factors might influence the phenotype [START_REF] Titeux | A frequent functional SNP in the MMP1 promoter is associated with higher disease severity in recessive dystrophic epidermolysis bullosa[END_REF][START_REF] Dang | Differential expression of pyloric atresia in junctional epidermolysis bullosa with ITGB4 mutations suggests that pyloric atresia is due to factors other than the mutations and not predictive of a poor outcome: three novel mutations and a review of the literature[END_REF] . Our results suggest that environmental conditions related to physical activity and trauma, as well as comorbidities are aggravating circumstances. Modifier genes and epigenetic factors are certainly involved in modulation of phenotypic variations. In the case of missense and truncating mutations, misfolded proteins are subjected to intracellular degradation, processes which are likely to be subject of inter-individual variations [START_REF] Bateman | Genetic diseases of connective tissues: cellular and extracellular effects of ECM mutations[END_REF] .

In terms of therapeutic perspectives, an important finding in this study is that already a low percentage of functional collagen XVII translated from the transcripts of splice-site mutations can confer a mild skin phenotype, in contrast to the null variants. The natural history of JEB in four aged individuals with 2-4% of collagen XVII in vitro and about 12 -14 % of normal collagen XVII levels in vivo demonstrated that for successful biological therapies it will not be necessary to achieve 100 % restoration of collagen XVII in the skin, but already smaller increases will improve dermal-epidermal stability and alleviate symptoms in JEB.

  ). They were significantly younger than the group of patients with milder phenotypes, namely patients 28-38 (mean age 14.4 versus 48.5 years) (Figure2D-J).Particular emphasis was put on various localized phenotypes, which may be difficult to recognize. Missense mutations were typically associated with modest-mild phenotypes, as seen in patients 29, 30, 32-34, and 37. Patients 29, 30, 33 and 34, who harboured the same mutation Arg1303Gln, exhibited a somewhat unusual phenotype with mild,

predominantly acral blistering and mucosal involvement, which was associated with light fibrotic changes such as lacrimal duct obstructions, microstomia, esophageal stenoses or finger contractures (Figure

2I-J

).

Intriguing was the constellation observed in patients 28, 35, 36 and 38 with minimal, localized blistering and toe nail dystrophy (Figure

2D-H

). They had had mild traumainduced blistering since childhood, but only sought medical attention when age-related health conditions aggravated the blistering. This was first misinterpreted as an acquired bullous disease, but recalcitrance to therapies finally led to the diagnosis of JEB at the advanced age of 67 -80 years.

Acknowledgements and funding

We thank all patients who participated in this study and the physicians who sent us samples. The excellent technical support by Gabriele Grüninger, Vera Morand, Margit Schubert and Kaethe Thoma is gratefully acknowledged. This work was supported by the Network Epidermolysis bullosa grant from the Federal Ministry for Education and Research (BMBF), the CRC850 / B6 grant from the German Research Foundation DFG, the Excellence Initiative of the German Federal and State Governments and Freiburg Institute for Advanced Studies, School of Life Sciences, and by the K. Kriezis scholarship from the National and Kapodistrian University of Athens to D.K.

Competing interests: None

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MD, PhD Gregor Kuhlenbäumer
email: g.kuhlenbaeumer@neurologie.uni-kiel.de

PhD Julia Hullmann

PhD Silke Appenzeller

Prof G Kuhlenbäumer

Novel genomic techniques in the analysis of monogenic disorders

Keywords: monogenic, disease gene identification, exome sequencing, SNP array monogenic, exome sequencing, disease gene identification

HAL is

Introduction

During the last years enormous technical progress has been achieved in the areas of DNAsequencing and genotyping-array technology. Resequencing of the whole human genome can now be achieved within weeks instead of months to years and genotyping-array technology allows the detection of genomic duplications and deletions on a whole genome scale within days and with unprecedented accuracy. These technologies are leading to new strategies in the analysis of monogenic diseases allowing the identification of causative mutations in disorders which are not amenable to linkage-based positional cloning [START_REF] Biesecker | Exome sequencing makes medical genomics a reality[END_REF][START_REF] Hayden | Genomics shifts focus to rare diseases[END_REF]. In this review we will focus on two technologies: Gentoyping arrays and exome sequencing, and the strategies which can be applied to filter the many detected, previously unknown SNPs for the causative mutation. Exome sequencing is defined as the selective resequencing of all exons in a genome. Since most mutations causing monogenic disorders are exonic this approach is especially promising for the analysis of monogenic disorders. A number of studies has already proven that exome sequencing alone is able to indentify genes involved in monogenic disorders [START_REF] Lalonde | Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing[END_REF]Ng, et al., 2010a;Ng, et al., 2010b;Ng, et al., 2009].

Why do we still need to study monogenic disorders?

Why do we need to study rare monogenic disorders in the age of complex genetics elucidating the genetic basis of more important common complex disorders [START_REF] Peltonen | Lessons from studying monogenic disease for common disease[END_REF]]? 1. Compared to infrequent complex disorders many monogenic disorders are not this rare. Monogenic variants of common complex disorders can be very frequent, especially in F o r P e e r R e v i e w Kuhlenbäumer,4 selected ethnic groups. The Leucine-Rich-Repeat-Kinase-2 (LRRK2, MIM609007) G2019S mutation is for example responsible for a large proportion of all Parkinson's disease (PD) cases in North African Arabic-and Ashkenazi Jewish patients [START_REF] Lesage | LRRK2 G2019S as a cause of Parkinson's disease in North African Arabs[END_REF][START_REF] Ozelius | LRRK2 G2019S as a cause of Parkinson's disease in Ashkenazi Jews[END_REF].

2. Rare monogenic variants of common diseases contribute eminently to our understanding of complex disorders, e.g. in Alzheimer's disease (AD). The current view of AD pathogenesis, the so called amyloid hypothesis, is largely based on the discovery of mutations in three genes (APP, MIM104760; PS1, MIM104311 and PS2, MIM600759) causing monogenic AD [START_REF] Ertekin-Taner | Genetics of Alzheimer's disease: a centennial review[END_REF].

3. Monogenic disorders can be modeled in animal-and cell-systems mainly because they have a strong phenotypic effect. Animal models may be used as pathogenetic as well as therapeutic disease models. In contrast most genetic variants known to be associated with complex disease to date are difficult to analyze in model systems because 1.) they have only marginal phenotypic effects and 2.) the really causative variant cannot be discerned from variants in close linkage disequilibrium.

4. Mutations causing monogenic diseases provide information about the function of the affected gene.

Please note, that for simplicity all phrases and words in the following paragraphs are meant to refer to monogenic disorders only and not to their complex counterparts unless stated otherwise. [START_REF] Botstein | Discovering genotypes underlying human phenotypes: past successes for mendelian disease, future approaches for complex disease[END_REF].

Positional cloning starts with the ascertainment of large families inheriting the disease under investigation in a Mendelian fashion. The disease gene is mapped by linkage analysis [START_REF] Ott | Analysis of human genetic Linkage[END_REF]. Linkage analysis allows the delineation of a genomic candidate region containing the disease gene. All genes in the candidate region are analyzed for mutations. Causality of genetic variants in a gene is assumed to be shown if the variants meet the following requirements.

1.) The variants segregate with the disease phenotype in the families.

2.)

The variants are unknown, meaning that they are neither found in databases (e.g. dbSNP, ) nor in controls.

3.)

The variants lead to a (non-conservative) amino acid exchange, a protein truncation or a splicing aberration. 4.) Different variants in the same gene segregating with the disease are found in different affected families.

In the best case these data are backed by: 5.) Functional data supporting causality [START_REF] Collins | Positional cloning moves from perditional to traditional[END_REF].

6.) Finding de-novo mutations in some families.

Experience has shown that this approach is highly reliable. The main advantage of linkage analysis compared to genome or exome sequencing is the identification of a small candidate region increasing the a priori likelihood that an identified variant in this region is causal. In most cases only one variant fulfilling the above mentioned criteria is found. The main F o r P e e r R e v i e w Kuhlenbäumer,6 drawback of linkage analysis is the requirement for large families. To achieve statistical significance a multigenerational pedigree with 6-12 affected individuals is required for autosomal dominant disorders. Autosomal recessive disorders in consanguineous families can often be solved in small families using homozygosity mapping but non-consanguineous families with suspected causative compound-heterozygote mutations are not amenable to homozygosity mapping because the affected children do not inherit identical copies of the candidate region from both parents. In addition phenocopies and significantly reduced disease penetrance strongly impede linkage-based disease gene identification.

Novel genomic techniques: Overview of Next-Generation-DNA-sequencing and

Genotyping-array technology

Next-Generation-Sequencing (NGS) bears some fundamental differences compared to conventional Sanger-sequencing. Sanger-sequencing of candidate genes is directed to a specific target, mostly by sequencing PCR-amplified-DNA. Sanger-sequencing generates long "reads", meaning that one reaction deciphers 400-800 bases allowing easy sequence assembly. In principle, one sequence suffices to identify homozygous and heterozygous sequence variants because every reaction contains and determines both possible alleles.

Sanger-sequencing always follows the same technical principle but NGS uses a number of different technical approaches. The following characteristics of NGS are somewhat generalized and not all of them apply to all methods. Technical details can be found in other reviews [START_REF] Dufva | Introduction to microarray technology[END_REF][START_REF] Mardis | The impact of next-generation sequencing technology on genetics[END_REF][START_REF] Tucker | Massively parallel sequencing: the next big thing in genetic medicine[END_REF]. NGS as such is based on sequencing clonally amplified single molecules of DNA. NGS needs between 10-50 reads of the same base to reliably identify a heterozygous sequence variant, because every read shows only one of the possible two alleles in a stochastic distribution. NGS generates comparatively short reads making assembly more difficult. The main advantage of NGS is the massive amount of generated sequence data. Depending on the method used between ~400 and ~3000 megabases can be generated in one "run" of the instrument [START_REF] Mardis | The impact of next-generation sequencing technology on genetics[END_REF]. For comparison, the human genome has a size of ~3000 megabases. While NGS is much cheaper than Sangersequencing on a per base basis, it is still very expensive on a genome scale. Because the overwhelming majority of all single base mutations causing monogenic disorders are located in exons or in splice sites in the immediate vicinity of exons, it should be sufficient to sequence all exons, the so called "exome" to detect most mutations causing monogenic diseases. The exome has a size of only ~30 megabases. Exome sequencing is therefore currently much cheaper than whole genome sequencing. The starting material used for mutation analysis is usually whole genomic DNA. Therefore exonic DNA has to be enriched from genomic DNA to perform exome sequencing. A number of enrichment-methods is currently available. Most of them use hybridization of fragmented genomic DNA to immobilized exonic target DNA [START_REF] Mamanova | Target-enrichment strategies for next-generation sequencing[END_REF][START_REF] Summerer | Enabling technologies of genomic-scale sequence enrichment for targeted high-throughput sequencing[END_REF]Turner, et al., 2009]. None of these methods is perfect and able to extract pure exonic DNA which homogenously covers the whole target. Therefore not all sequences produced by exome sequencing will be "on-target", meaning within the targeted exons.

Genotyping-arrays also called SNP-chips are devices allowing the simultaneous determination of the genotypes of 10 4 to 2x10 6 SNPs [START_REF] Bier | DNA microarrays[END_REF]. Genotyping-array technology has been available for years and is technically mature. Genotyping-arrays are now the method of choice for linkage analysis because they are faster and allow to construct a much denser linkage map than conventional Short-Tandem-Repeat marker based linkage analysis. Most current genotyping-arrays also contain probes of non-polymorphic DNA allowing the detection of small genomic duplications and deletions in the range of some Kuhlenbäumer,8 kilobases (=Copy Number Variants,CNV). Analysis of the signal intensities is used to determine CNVs [START_REF] Komura | Genome-wide detection of human copy number variations using high-density DNA oligonucleotide arrays[END_REF][START_REF] Peiffer | High-resolution genomic profiling of chromosomal aberrations using Infinium whole-genome genotyping[END_REF]. Normal diploid DNA contains two copies of each DNA fragment while genomic duplications contain three copies and deletions only one copy. Hence, probes within heterozygous duplications have a signal intensity of about 1.5 and probes within heterozygous deletions of 0.5, while homozygous duplications show a signal intensity of 2.0 and homozygous deletions of 0 compared to wildtype segments with two copies..

New strategies in the analysis of monogenic disorders

In the following paragraphs we will describe the novel strategies in the analysis of monogenic disorders made possible by the technologies introduced in the previous paragraphs, starting with the genetic characteristics of suitable monogenic disorders which were previously intractable by classing positional cloning. Sections on CNV-analysis and NGS describe the application of these techniques to monogenic disorders in more detail and give examples from the most recent literature. Three further paragraphs are devoted to the question how the causative mutant can be filtered out from the background of thousands of inocuous variants present in every exome. The last sections deal with possible problems, diagnostic applications and finally give a summary and an outlook.

Genetic characteristics of Mendelian disorders suited for analysis by novel genomic techniques

Positional linkage based disease gene identification is a proven reliable strategy and will remain the gold-standard if suitable families are available. In this context, NGS may be used Formatted: Font: Not Bold Deleted: . Deleted: Hybridization is a quantitative process and therefore these probes can be used to detect genomic duplications and deletions (= Copy Number Variants, CNV).

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The high density of probes allows the reliable detection of small duplications and deletions in the range of some kilobases. Kuhlenbäumer,9 to sequence whole candidate regions determined by linkage analysis. However, in many cases large families are not available or one might even find single affected individuals only. These situations could be simply due to ascertainment problems, but the more common issue is that the disease under investigation confers a severe reproductive disadvantage. Either statistical power is too small for linkage analysis in the families or, in the case of single patients only, linkage analysis is principally not applicable. A number of scenarios is conceivable (Fig. 1).

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In all scenarios it is assumed that all patients share the same clinical phenotype but not necessarily the same genetic cause of the disease:

1.) Only a number of small families and some single individuals are available (Fig. 1.1). The disease might be autosomal dominant or autosomal recessive. A number of examples demonstrating the feasibility of disease gene identification in this setting are already available and described in more detail in the following paragraphs. Theoretically linkage-analysis could be achieved using several hundred small families, e.g. affected sibling pairs. This approach has been used for complex disorders. However, such large numbers of families with a monogenic disorder are hardly ever available and in most disorders high locus heterogeneity would severely hamper this approach. Recessively inherited diseases with unrelated parents due to compound heterozygote mutations are not amenable to homozygosity mapping and will be excellent targets for disease gene identification by exome sequencing.

2.) The disease is seemingly sporadic because it is found in multiple single individuals with unaffected parents (Fig. 1.2). This may be the case for diseases leading to death or severe handicap in childhood as well as disorders directly impairing fertility. That these diseases might in many instances be in fact monogenic and caused by de-novo mutations has been shown for some disorders by candidate gene sequencing and for others by exome sequencing [Hoischen, et al., 2010;Ng, et al., 2010a;[START_REF] Saitsu | De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy[END_REF]. Alternatively, the disease Deleted: . Kuhlenbäumer, 10 might be autosomal recessive with only one affected child per family. This could e.g. be the case if the disease leads in to stillbirths most cases.

3.) Multiple small families with presumed X-linked inheritance are available (Fig. 1.3). In this scenario, targeted NGS of all genes on the X-chromosome will be a solution [START_REF] Tarpey | A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation[END_REF].

4.) It can also be envisaged to apply exome sequencing in distantly related individuals of large families who share a common founder many generations ago. These individuals share only a small proportion of their genome allowing to reduce the number of putatively disease causing mutations. Linkage based cloning will still be the method of choice for large families with monogenic disorders. However, sometimes significant linkage can not be established despite sufficient family size. This might be due to misclassification of patients, problems with low disease penetrance, clinically identical phenocopies and further to date unknown causes. In addition a number of rather common disorders exists for which many families with presumed monogenic inheritance have been collected and for some of theses diseases even linkage has been established in a few families but no causative mutations could be identified.

Two prototypical examples are Restless-legs syndrome and Essential tremor [START_REF] Deng | Genetics of essential tremor[END_REF][START_REF] Winkelmann | Genetics of restless legs syndrome: a burning urge to move[END_REF]]. It will be very interesting to see whether exome sequencing can contribute to the elucidation of the genes underlying these disorders.

Identification of genomic duplications and deletions using genotyping-arrays

Genomic rearrangements, especially larger genomic duplications or deletions, are the second most common cause of monogenic diseases. Very small genomic duplications or deletions can be detected by NGS or Sanger-sequencing. Genotyping-array analysis for CNVs will often be performed before exome sequencing because it costs only between 300$ and 700$ Deleted: The strategy and the results of the latter will be described in more detail in the following paragraphs. sets are suspicious. One study for example screened 3699 controls negative for the candidate CNV, others even more [START_REF] Helbig | 15q13.3 microdeletions increase risk of idiopathic generalized epilepsy[END_REF][START_REF] Shinawi | A small recurrent deletion within 15q13.3 is associated with a range of neurodevelopmental phenotypes[END_REF]. Duplications and deletions detected in genotyping-arrays can be verified using methods like quantitative PCR, Fluorescence In Situ Hybridization (FISH) and long range PCR [START_REF] Helbig | 15q13.3 microdeletions increase risk of idiopathic generalized epilepsy[END_REF][START_REF] Shinawi | A small recurrent deletion within 15q13.3 is associated with a range of neurodevelopmental phenotypes[END_REF]. Unknown rearrangements segregating with the disease in families and present in multiple genetically independent affected individuals are excellent, most likely causal candidate mutations. The same is true for de-novo duplications or deletions in patients since de-novo mutations are extremely rare. In many cases, a monogenic disease is caused by CNVs in a subset of patients while others bear a point mutation in a gene contained within the duplication or deletion. Two good examples are Charcot-Marie-Tooth disease type 1A (CMT1A), caused in most patients by a duplication on chromosome 17p11.2 harboring the PMP22 gene (MIM601097) but in some cases also by point mutations in the same gene [START_REF] Kuhlenbäumer | Clinical features and molecular genetics of hereditary peripheral neuropathies[END_REF] and early infantile epileptic encephalopathy caused by CNVs as well as point mutations involving the STXBP1 gene (MIM602926) [START_REF] Saitsu | De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy[END_REF].

Next-Generation-DNA sequencing: Identification of point mutations

If whole exome sequencing is applied to monogenic disorders, it is not trivial to identify the causative variant among the many identified variants present in the exome. The a priori likelihood that a variant is causal is much lower than in the classic linkage based approach, because the whole exome and not only the genes in a small candidate region identified by linkage analysis are sequenced. Disease causing mutations are in most cases either missense mutations which change an amino acid, nonsense mutations which introduce a premature stop codon or small duplications/deletions causing a frameshift altering the amino acid sequence C-terminal of the mutation which often leads to a premature stop codon and protein truncation. Currently available exome sequencing data suggest that ~5000-10000 of these three types of mutations are found in the average Caucasian exome [START_REF] Ng | Genetic variation in an individual human exome[END_REF]Ng, et al., 2009]. The overwhelming majority of these variants are most likely benign.

Approximately 400 to 700 of these variants are absent in SNP-databases, in the eight fully sequenced exomes available in the HapMap database and the currently available data of the 1000 Genomes project [START_REF] Lalonde | Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing[END_REF][START_REF] Ng | Genetic variation in an individual human exome[END_REF]Ng, et al., 2010b;Ng, et al., 2009]. In a small family, many of these variants will segregate with the disease by chance and many identified variants will not be present in a reasonable number of controls (100-1000), because only rare variants have been selected for examination beforehand. In addition, it is costly and time consuming to check all variants for segregation and absence in controls by Sanger-sequencing or other genotyping-methods. Completion of the thousand genomes project establishing the complete genome sequence of more than 1000 humans will enable us to eliminate a large number of variants which are present at low frequencies in the general population but not found in current SNP-databases (http://www.1000genomes.org).

Nonetheless, data will have to be painstakingly filtered to allow reliable disease gene identification.

Approaches for filtering the identified variants (Fig. 2)

Exploiting the characteristics of Mendelian inheritance

The first step will be to exploit the characteristics of Mendelian inheritance (Fig. 2, "Genetic filter"). If a number of unrelated families and single individuals with the same clinical phenotype are available the first and most powerful step is to look for different mutations affecting the same gene in the affected individuals from unrelated families. That this approach is feasible has recently been shown e.g. for the autosomal-recessive Millersyndrome (MIM263750) [Ng, et al., 2010b] and the Fowler-syndrome (MIM610865) [START_REF] Lalonde | Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing[END_REF]. In Miller-syndrome exome sequencing was performed in four affected individuals belonging to three independent non-consanguineous families. It was unknown if Miller-syndrome is autosomal recessive or autosomal dominant. Therefore both models were taken into account. About 400 to 500 candidate variants not present in any SNP-database were identified in each patient. Thirty-one and 33 of these variants were compatible with recessive inheritance in each of the two sequenced siblings from family one but both probands of this family taken together showed only 9 SNPs compatible with recessive inheritance. Adding the single probands from families two and three allowed to reduce the number of candidate genes to only one in the recessive and eight in the dominant model. The authors calculated the Bonferroni corrected p-value that two different variants of the ~500 candidate variants are found in the same gene in each of three independent affected individuals is p~0.000015 arguing strongly against appearance by chance. Sanger-sequencing of the implicated DHODH gene (MIM126064) in three additional singleton cases revealed compound heterozygote mutations in every affected individual. In Fowler-syndrome two patients with this recessively inherited disorder sufficed to pinpoint the FLVCR2 gene (MIM610865) [START_REF] Lalonde | Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing[END_REF]. A number of other very recent reports suggests that even single patients may suffice to identify recessive mutations causing monogenic disorders, especially if enough is known about the pathomechanism to filter candidate mutations [START_REF] Byun | Whole-exome sequencing-based discovery of STIM1 deficiency in a child with fatal classic Kaposi sarcoma[END_REF]Gilissen, et al., 2010;[START_REF] Rios | Identification by whole-genome resequencing of gene defect responsible for severe hypercholesterolemia[END_REF]. Naturally, it is easier to reduce the number of candidate mutations for an autosomal recessive disease than for an autosomal dominant disease because only genes with both alleles bearing an unknown or a very rare variant have to be considered. Nevertheless exome sequencing can also be applied Kuhlenbäumer, 14 successfully to autosomal dominant disorders. Ten unrelated probands with autosomal dominant Kabuki-syndrome (MIM147920) were analyzed but exome sequencing did not identify a candidate gene in which all affected individuals showed an unknown variant [Ng, et al., 2010a]. This problem could be due to locus heterogeneity, to mutations outside the targeted exome or to the fact that exome sequencing failed to detect all mutations. However, further analyses allowed to find mutations in the MLL gene (MIM159555) in 7 out of the 10 sequenced exomes. Sanger sequencing revealed small frameshift indels not detected by NGS in 2 of the remaining 3 patients and sequencing of the MLL gene in 43 additional, seemingly sporadic patients detected mutations in 26 of these. This investigation proves that exome sequencing is able to identify genes causing monogenic disorders even in the presence of moderate locus heterogeneity. The degree of locus heterogeneity still permitting the identification of disease genes will depend to a large degree on the number of families available for exome sequencing. Interestingly, all mutations in the MLL gene had arisen denovo in the 12 patients for whom parental DNA was available underpinning that seemingly sporadic disorders might in fact be due to dominant de-novo mutations. It seems not unlikely that severe, often syndromal disorders conferring a large reproductive disadvantage and always affecting only one sibling might often be caused by de-novo mutations. It has been estimated that on average only two de-novo mutations are present per exome. [START_REF] Nachman | Estimate of the mutation rate per nucleotide in humans[END_REF]] Exome sequencing of one healthy triplet consisting of the parents and one offspring did not identify any de-novo mutation in the exome of the offspring [START_REF] Hedges | Exome sequencing of a multigenerational human pedigree[END_REF]. Therefore exome sequencing of triplets consisting of an affected child and the healthy parents will be an ideal way to drastically reduce the number of potentially disease causing candidate variants. In the best case, only one de-novo variant will be found per-patient.

Sanger-sequencing of the identified gene in further individuals affected by the same disease might reveal additional de-novo mutations and make causality highly likely. The demonstrates the feasibility of this approach [Hoischen, et al., 2010]. If one considers to identify the gene causing a monogenic disorder in one large family, some degree of filtering could be achieved by sequencing two or more distantly related individuals which share only a fraction of their genome. If the two related individuals have a common distant known or unknown founder the shared part of the genome will be reduced by 2 n per generation. The variants identified in different genes have to be tested for segregation in the families as an additional filter. As previously mentioned, the causative variants of rare disorders should usually be absent in ethnically matched unaffected controls with most published reports using 200-1000 controls. However, using modern genotyping techniques one could easily analyze 5000-10000 controls to differentiate between rare non-causal variants present also in the unaffected population and causative mutations. If a large number of controls is used for a disorder with a moderate disease frequency, it becomes mandatory that all controls individuals are screened negative for the investigated phenotype or that individuals positive for a variant under question can be re-examined clinically. In addition, recessive disorder might be caused by mutations found in the unaffected population at a low frequency in the heterozygous state. A recent study describes that severe brain malformations can be caused by recessive mutations in the WDR62 gene (MIM 613583) [START_REF] Bilguvar | Whole-exome sequencing identifies recessive WDR62 mutations in severe brain malformations[END_REF]. One particular mutation was present in a homozygous or compound heterozygous state in some of the patients but also in 0.2% of the ethnically matched controls in the heterozygous state.

Formatted: Font: Italic Formatted: Font: Italic Deleted: In principal, the same approach can be applied to Xchromosomal disorders using enrichment methods targeting only the Xchromosomal DNA. Filtering genetic variants to identify the potentially causative one will be facilitated by the much smaller size of the X-chromosomal exome compared to the whole exome resulting in a much smaller number of novel SNPs among which the disease causing one has to be identified. ¶ Deleted: . Most The incorporation of expression and functional data in the filtering process Functional, expression and evolutionary data as well as the computational prediction of functional effects of mutations can be incorporated in the filtering process (Fig. 2, "Functional filter"). Candidate genes can be ranked according to their function and expression. An easily understandable and successful example is the selection of ion channels as candidate genes for epilepsies. However, there are also numerous examples in which genes with an entirely unknown relation to the pathogenesis of the investigated disease were identified as disease genes. Expression data tell us which genes containing a mutation are expressed in the tissue affected by the disease. Genes which are not expressed in the target tissue are bad candidate genes. Disease causing mutations are more common in evolutionary conserved genes and in well conserved amino acid residues than in non-conserved ones.

There are numerous methods to include the assessment of evolutionary conservation in the filtering process [Mooney, 2005;[START_REF] Ng | Predicting the effects of amino acid substitutions on protein function[END_REF][START_REF] Ng | Genetic variation in an individual human exome[END_REF][START_REF] Tarpey | A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation[END_REF]. Computational prediction of functional SNP effects often employs evolutionary conservation as well as physicochemical properties of the affected amino acid in the protein [Mooney, 2005;[START_REF] Ng | Predicting the effects of amino acid substitutions on protein function[END_REF]. Details regarding these methods and algorithms can be found in a number of reviews [Mooney, 2005;[START_REF] Ng | Predicting the effects of amino acid substitutions on protein function[END_REF]. Well known examples for programs which can be used to identify possibly deleterious SNPs are the programs SIFT (Sorting Intolerant From Tolerant, http://sift.jcvi.org/), PolyPhen and the recently developed PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and SNAP (Screening for Non-acceptable Polymorphisms, http://cubic.bioc.columbia.edu/services/SNAP/) [START_REF] Adzhubei | A method and server for predicting damaging missense mutations[END_REF][START_REF] Kumar | Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm[END_REF][START_REF] Ramensky | Human non-synonymous SNPs: server and survey[END_REF]. Programs such as NNSPLICE can be used to assess mutations possibly affecting splicing [START_REF] Reese | Improved splice site detection in Genie[END_REF]. All programs are only partly reliable in predicting functional SNP effects and can therefore not be used to prove the deleterious effect of a SNP. However, they are good tools to supplement the other strategies and methods mentioned above and help to prioritize novel variants detected by exome sequencing for further analysis.

Next-Generation-Sequencing of large candidate gene collections and combination of mapping and sequencing approaches

These "filtering" approaches rely on selecting only the functionally or positionally relevant parts of the exome for NGS. The approaches are conceptually similar to the traditional "candidate gene" or "linkage" approaches but NGS has the major advantage that massive amounts of candidate genes can be sequenced at once. The strategy requires selective enrichment of the DNA regions of interest, e.g. by using custom enrichment arrays. Due to the reduction of the amount of DNA which needs to be sequenced, this strategy does not only reduce the number of candidate variants but also allows to achieve higher sequencing coverage, i.e. more reads of every sequenced base. Such candidate gene approaches have already been successfully applied to identify causative mutations in human complex 1deficiency and autosomal recessive retinal-renal ciliopathy [START_REF] Calvo | High-throughput, pooled sequencing[END_REF][START_REF] Otto | Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy[END_REF]. In the latter very high locus heterogeneity necessitated the ability to identify disease genes in single individuals or families. The selection of ~13.000 ciliopathy "candidate exons" allowed to reduce the number of exons which needed to be sequenced by ~15-fold compared to the whole exome [START_REF] Otto | Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy[END_REF]. In addition, variants were filtered for those which mapped to homozygous stretches in the individual genome identified by the use genotyping arrays which facilitates the detection of truly homozygous but not of compound heterozygous mutations. Further successful examples of combined mapping/sequencing approaches are the Kuhlenbäumer, 18 selection of X-chromosomal genes in forms of X-linked mental retardation and identity by descent filtering in autosomal recessive hyperphosphatasia mental retardation syndrome (MIM 239300) caused by compound heterozygous mutations in the PIGV gene (MIM 610274) [START_REF] Krawitz | Identity-by-descent filtering of exome sequence data identifies PIGV mutations in hyperphosphatasia mental retardation syndrome[END_REF].

Problems of novel genomic methods in monogenic disease gene identification

One has to discern between technical and genetical/biological problems. Technical problems are e.g. inhomogeneous coverage of the target region. This problem will lead to a widely varying number of reads covering different regions of the exome which causes -in conjunction with unequal sequencing quality -the detection of false positive and false negative variants. False positive variants can be eliminated by Sanger-resequencing. In contrast, false negative variants are potentially a big problem because if the filtering strategy relies on the detection of mutations in the same gene in different affected individuals these might be missed. However, these problems will be alleviated by technical progress within the next years. The main genetical problem is locus heterogeneity. Locus heterogeneity is the presence of multiple distinct disease genes/disease loci for one clinical phenotype. Locus heterogeneity might complicate the application of the "genetic filters" already described. Some neurological disorders e.g. CMT (>40 loci), hereditary ataxia and hereditary spastic paraplegia show a high degree of locus heterogeneity. The main biological problem is proving causality of a mutation. Ultimately, causality has to be shown using cell-and animal model systems. There are two main arguments against using NGS in genetic diagnostics. The first one is that NGS of the whole exome or genome is still expensive compared to Sanger-sequencing of a few known disease causing genes for every condition. This problem will solved within the near future and one can predict that NGS will be cheaper than Sanger-sequencing of a number of possibly mutated genes. The second problem is an ethical one. NGS of whole exomes/genomes will not only discover the causative mutation for the investigated disease but also mutations causing other monogenic disorders and genetic risk factors for complex disorders. The first example for the former has been the identification of mutations causing primary ciliary dyskinesia in some patients who were primarily investigated because of Miller-syndrome [Ng, et al., 2010b]. Genetic risk factor determination for complex disorders e.g. APOE-genotype, the most important risk factor for complex AD, is a byproduct of exome/genome sequencing. A solution to this problem might be to discard all sequence-data outside the genes required for diagnosis before the analysis of the NGS-data. That genetic diagnostics using NGS are feasible has been shown very recently by the identification of a mutation in the SH3TC2 gene (MIM608206) in a family with CMT [START_REF] Lupski | Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy[END_REF].

Summary and outlook

Undoubtedly, novel genomic techniques will have a large impact on monogenic disease gene identification. Most importantly, they will enable us to tackle monogenic disorders not Kuhlenbäumer, 20 using genotyping arrays is already used in routine genetic diagnostics and NGS will most likely become the method of choice for diagnostic point mutation identification within in the next years. The next big step in the investigation of genetic disorders will presumably be the integration of data obtained by the analysis of noncoding RNAs and epigenetic marks.

Noncoding RNAs are transcripts that do not code for proteins and are involved in gene regulation. Epigenetic marks like DNA-methylation and Histone-modifications also regulate gene activity and are to a certain degree passed on from generation to generation. However, epigenetic modifications are to a much higher degree influenced by the environment than DNA itself is. Therefore environmental data will have to be taken into account as well. In addition, a recent publication demonstrates that NGS can also be applied to identify the mutations underlying linkage peaks for complex traits, at least if the linkage signal is mainly derived from a few large families in the cohort [START_REF] Bowden | Molecular basis of a linkage peak: Exome sequencing and family-based analysis identifies a rare genetic variant in the ADIPOQ gene in the IRAS Family Study[END_REF]. One can easily predict that the rapidly declining prices of NGS will lead to a surge in it's application to monogenic and complex disorders. Human Mutation

  The classic strategy of disease gene identification: positional cloning based on linkage analysisTo understand the new opportunities created by novel genomic techniques one has to comprehend the limitations of classical positional cloning

  directed to a target sequence. All DNA in the reaction mixture is sequenced. NGS generates comparatively short reads making assembly more difficult.

  Determination of the SNPgenotypes is usually based on allelespecific hybridization of genomic DNA followed by a detection step involving fluorophores.

  Duplications or deletions which are absent in databases and large control data

Formatted

  de-novo mutations in the SETBP1 gene (MIM611060) causing autosomal dominant Schninzel-Giedion syndrome (MIM 269150) by exome sequencing

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  amenable to classic linkage-based positional cloning due to insufficient family size and reveal how many seemingly sporadic disorders are in fact monogenic. Duplication/deletion analysis

  Figure legends:

Figure 1 :

 1 Figure 1: Example pedigrees of Mendelian disorders suited for analysis by novel genomic techniques. Circle -Female, Square -Male, Filled symbol -Affected.

Figure 2 :

 2 Figure 2: Analysis strategy for the analysis of monogenic disorders using novel genomic techniques. Ovals -Techniques and strategies, Rectangles -Results. The figure does not encompass filtering by a priori selection of large numbers of candidate genes or by the combination of mapping and sequencing approaches because these strategies act prior to the application of genotyping arrays and exome sequencing.

Figure 1 :Figure 2 :

 12 Figure 1: Example pedigrees of Mendelian disorders suited for analysis by novel genomic techniques. Circle -Female, Square -Male, Filled symbol -Affected.

John Wiley & Sons, Inc.Human Mutation

Acknowledgements

GK is a member of the Cluster of Excellence "Inflammation at Interfaces" of the Christian-Albrechts University Kiel. Work elucidating the genetic causes of monogenic disorders has been supported by grants to GK of the Deutsche Forschungsgemeinschaft (DFG), the "Innovative Medizinische Forschungsförderung" of the University of Münster, the "Intramurale Forschungsförderung" of the University of Kiel, the Heinrich-Hertz-Foundation and the "Association Francaise contre les Myopathies".

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