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Mutations of DEPDC5 cause autosomal dominant focal epilepsies

Saeko Ishida1,2, Fabienne Picard3, Gabrielle Rudolf4,5, Eric Noé1,2, Guillaume Achaz2,6,7, Pierre Thomas8, Pierre Genton9,10, Emeline Mundwiller11, Markus Wolff12, Christian Marescaux4, Richard Miles1,2, Michel Baulac1,2,13, Edouard Hirsch4, Eric Leguern1,2,14 & Stéphanie Baulac1,2

The main familial focal epilepsies are autosomal dominant yet been identified for FFEVF, linkage to 22q12 has been reported in nocturnal frontal lobe epilepsy, familial temporal lobe several families4,8–11. epilepsy and familial focal epilepsy with variable foci. We have previously described the electroclinical characteristics of A frameshift mutation in the DEPDC5 (encoding DEP 19 families with autosomal dominant focal epilepsies, subdivided into domain–containing 5) was identified in a family with ADNFLE, FTLE and FFEVF12. Two large multiplex French families (N focal epilepsy with variable foci by linkage analysis and exome and S) with diagnosed FFEVF were selected from this cohort for link- sequencing. Subsequent pyrosequencing of DEPDC5 in a age analysis using a high-density genome-wide scan with 10,000 SNPs. All rights reserved. cohort of 15 additional families with focal epilepsies Both families mapped to the FFEVF-associated on identified 4 nonsense mutations and 1 missense mutation. 22q12, with maximum logarithm of odds (LOD) scores of 2.08 (fam- Our findings provided evidence of frequent (37%) loss-of- ily N) and 1.81 (family S). Haplotype reconstruction confirmed the function mutations in DEPDC5 associated with a broad segregation of a disease haplotype in both families (Supplementary spectrum of focal epilepsies. The implication of a DEP Figs. 1 and 2). America, Inc. (, Egl-10 and Pleckstrin) domain–containing We then sequenced the entire exome of subjects N-III:4 and protein that may be involved in membrane trafficking and/or N-III:6 and sought rare coding variants in the 22q12 candidate G protein signaling opens new avenues for research. region. In both individuals, we found the same single-base deletion, c.1122delA, in exon 16 of the DEPDC5 gene (also known as KIAA0645; Epilepsy is a frequent neurological disorder characterized by sponta- NM_001242896). This variant caused a frameshift, p.Leu374Phefs*30, © 2013 Nature neous, recurrent seizures. Focal epileptic seizures are thought to origi- introducing a premature stop codon 29 amino acids downstream. nate within networks limited to one hemisphere. Several autosomal The c.1122delA variant fully segregated with the phenotype within dominant, non-lesional focal epilepsies have been described as spe- the family (Fig. 1). npg cific age-related and electroclinical syndromes: autosomal dominant We next sought DEPDC5 mutations in 15 additional probands from nocturnal frontal lobe epilepsy (ADNFLE; MIM 600513)1, familial the families with autosomal dominant focal epilepsies (ADNFLE, temporal lobe epilepsy (FTLE; MIM 600512)2, including mesial and n = 7; FTLE, n = 4; FFEVF, n = 4)12, including family S in which lateral forms (also termed autosomal dominant epilepsy with auditory disease was linked to the 22q12 locus. Massively parallel pyrose- features (ADEAF; MIM 604619)3, and familial focal epilepsy with quencing screening of all 43 exons and splice regions of DEPDC5 variable foci (FFEVF; MIM 604364), characterized by focal seizures identified 4 further nonsense mutations (encoding p.Arg239* in that are initiated in distinct cortical regions in different family mem- family S, p.Arg328* in family L, p.Gln372* in family Q and p.Gln1523* bers4. The only gene mutations currently identified are those linked in family B) and 1 missense mutation (p.Arg485Gln in family O) in to ADNFLE (CHRNA4, CHRNA2, CHRNB2 and KCNT1, encoding, 5 families (Fig. 2a, Table 1 and Supplementary Fig. 3). All muta- respectively, the α4, α2 and β2 subunits of the neuronal nicotinic tions were shown to segregate with the phenotype within the families, acetylcholine receptor and a potassium channel subunit)5,6 and to except for the mutation encoding p.Gln1523* in family B, where addi- ADEAF (LGI1, encoding epitempin)7. Although no causal have tional family members were unavailable for analysis (Fig. 1). In family

1Institut National de la Santé et de la Recherche Médicale (INSERM) U975, Institut du Cerveau et de la Moelle Epinière (ICM), Hôpital Pitié-Salpêtrière, Paris, France. 2Université Pierre et Marie Curie–Paris 6 (UPMC), Paris, France. 3Department of Neurology, University Hospitals of Geneva (HUG), Geneva, Switzerland. 4Neurology Department, Strasbourg University Hospital, Strasbourg, France. 5Strasbourg University (UDS), Strasbourg, France. 6Unité Mixte de Recherche (UMR) 7138, Centre National de la Recherche Scientifique (CNRS), Paris, France. 7UMR 7241, Collège de France, Paris, France. 8Service de Neurologie, Hôpital Pasteur, Nice, France. 9Centre Saint Paul, Henri Gastaut, Marseille, France. 10Mediterranean Institute of Neurobiology (INMED), Marseille, France. 11Genotyping/Sequencing Platform of ICM, Hôpital Pitié-Salpêtrière, Paris, France. 12Department of Pediatric Neurology, University Children’s Hospital, Tuebingen, Germany. 13Epilepsy Unit, Assistance Publique–Hôpitaux de Paris (AP-HP) Groupe Hospitalier Pitié-Salpêtrière, Paris, France. 14Département de Génétique et de Cytogénétique, AP-HP Groupe Hospitalier Pitié-Salpêtrière, Paris, France. Correspondence should be addressed to S.B. ([email protected]).

Received 21 December 2012; accepted 7 March 2013; published online 31 March 2013; doi:10.1038/ng.2601

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Family N: c.1122delA Family L: c.982C>T Database of Genomic Variants. DEPDC5 I mutations occurred significantly more often I 1 2 1 2 in our group of affected individuals (5/32; +/+ m/+ P = 5 × 10−12, Fisher exact test). II II Four out of six mutations (encod- 2 3 7 1 6 5 4 8 2 1 m/+ +/+ +/+ +/+ m/+ +/+ m/+ ing p.Arg239*, p.Arg328*, p.Gln372* and p.Leu374Phefs*30 alterations) are predicted III III 1 2 3 4 9 5 6 10 7 8 1 2 to result in degradation of the mutated tran- +/+ +/+ m/+ m/+ m/+ m/+ +/+ m/+ +/+ m/+ m/+ script by the nonsense-mediated mRNA IV decay (NMD) system, whereas the nonsense Family Q: c.1114C>T 1 2 3 4 5 6 7 mutation encoding p.Gln1523*, located in +/+ m/+ m/+ +/+ m/+ I the last exon of the gene, is not likely to be 1 2 targeted by the NMD system. To confirm Family S: c.715C>T +/+ m/+ that the mutation encoding the p.Arg239* II I ?? alteration leads to NMD, we treated cultured 1 4 2 3 5 1 2 m/+ lymphoblasts from three mutation carriers III II (S-III:10, S-IV:7 and S-IV:9) and one spouse 1 1 2 3 4 5 6 (S-III:11) with emetine, an NMD inhibi- Family B: c.4567C>T tor. With sequencing of DEPDC5 cDNA III 2 2 I the mutation encoding p.Arg239* was only 4 5 3 7 6 10 11 12 13 14 15 18 16 17 1 2 m/+ +/+ m/+ +/+ +/+ m/+ m/+ detected when NMD was inhibited (Fig. 2c), II showing that this nonsense mutation speci­ IV 3 2 1 4 fically leads to transcript degradation. 12 3 4 5 6 10 78 9 12 11 +/+ m/+ m/+ m/+ m/+ DEPDC5 haploinsufficiency is likely to be III 1 2 3 4 the cause of disease in individuals carrying V this mutation. 6 1 2 3 4 5 All rights reserved. m/+ m/+ m/+ m/+ IV We identified mutations in one-third of 1 2 3 the families in our cohort (6/16, 37%), and, m/+ overall, 20 subjects with epilepsy carried a Family O: c.1454G>A Focal epilepsy DEPDC5 mutation. The age of disease onset

I Generalized epilepsy ranged from 0 to 39 years (mean of 12.9 ± America, Inc. 1 2 3 4 5 10.9 (s.d.)). DEPDC5 mutations were not lim- Lesional focal epilepsy ited to families with the FFEVF phenotype II Electrical seizure but were also found in a family with two indi- 12 3 5 4 6 7 8 9 10 11 12 +/+ Undefined epilepsy viduals exhibiting a typical nocturnal fron- 13 m Mutated tal lobe epilepsy (family B) and two other III © 2013 Nature 12 3 4 5 6 7 8 9 10 11 12 + Not mutated families (L and O) with individuals with tem- m/+ m/+ poral lobe seizures. A phenotype of FFEVF Figure 1 Pedigrees of families with segregation of DEPDC5 mutations. Identified DEPDC5 was attributed to family N because frontal

npg mutations included c.1122delA (p.Leu374Phefs*30) in family N, c.715C>T (p.Arg239*) seizures have been diagnosed in one fam- in family S, c.982C>T (p.Arg328*) in family L, c.1114C>T (p.Gln372*) in family Q, c.1454G>A ily member (N-IV:5), and frontal electrical (p.Arg485Gln) in family O and c.4567C>T (p.Gln1523*) in family B. Individual N-IV:4 showed seizures were identified in another (N-IV:4), frontal spikes in electroencephalography (EEG) but no clinical seizure. A question mark whereas seizures in other family members indicates individuals born in the 1870s with doubtful epilepsy histories. Diagonal lines indicate deceased individuals. originate in the temporal lobe. Penetrance was incomplete in families N, S and O, in agreement with reports of low penetrance of O, the father (O-II:8) may have transmitted the mutation encod- 62% (ref. 14) or 50% (ref. 4) in families with FFEVF. Seven asympto- ing p.Arg485Gln, as it was not inherited from the mother (O-II:7). matic obligate carriers (N-II:2, S-II:5, S-III:4, S-III:7, S-III:10, S-IV:5 The arginine at position 485 is a highly conserved (Fig. 2b). and O-II:8) and four asymptomatic at-risk individuals aged 8–42 years Different prediction software tools—MutationTaster, SIFT and (N-III:5, N-IV:7, S-IV:7 and S-V:2) carried the mutations. Our data PolyPhen-2—predicted that the arginine-to-glutamine alteration strongly support the causal role of DEPDC5 in a subset of our families at position 485 is disease causing, tolerated and possibly damaging, with focal epilepsies. However, the observed low penetrance and respectively. This p.Arg485Gln substitution was not detected in a variable expressivity (in age at onset and localization of seizure focus cohort of 450 controls of matched ancestry. None of the six DEPDC5 in various brain areas) suggest that other gene(s) or epigenetic and variants was present in dbSNP135 or the 1000 Genomes Project data- environmental factors modulate the phenotype. base or in the 6,503 exomes for which variants are currently listed in Because five out of the six mutations introduce a premature stop the National Heart, Lung, and Blood Institute (NHLBI) exome vari- mutation, loss of function of DEPDC5 presumably causes the epi- ant server (EVS) database. One nonsense mutation (1/11,813) and leptic phenotype. It will now be crucial to determine the function 1 frameshift mutation (1/11,775) have been identified in the 6,503 of the DEPDC5 gene product and examine how its loss causes epi- exomes listed in the EVS database, and no copy number variations lepsy. DEPDC5 transcript was first identified in brain cDNA disrupting the DEPDC5 coding sequence have been reported in the libraries15. This gene is strongly expressed at rather constant levels

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a DEPDC5 gene c.715C>T c.982C>Tc.1114C>Tc.1122delAc.1454G>A c.4567C>T

Exons 12 15 16 21 43 c

p.Arg239* Emetine

*

+ DEPDC5 p.Arg239*p.Arg328*p.Gln372*p.Leu374Phefs*30p.Arg485Gln p.Gln1523 S-IV:9 protein c.715C>T/+ DUF3608 DEP – Amino acids 1 100 381 1,179 1,260 1,603 b Arg485 + S-III:11 Homo sapiens RA QC LT TC RS V R ER ES HS RK S ASS CD +/+ Pan troglodytes RA QC LA TC RS V R ER ES HS RK S ASS CD – Bos taurus RA QR LS TC RS V R ER ES HS RK S ASS CD Canis lupus RA QR LA TC RS V R ER DS HN RK S ASS CD Rattus norvegicus RA QR LA TC RS V R EQ EN HN RK S ASS CD Mus musculus RA QR LA TC RS V R EQ EN HS RK S ASS CD Gallus gallus RA QR CT TF RS V R ER ES RT RK S SSS YD Xenopus tropicalis RA QR SI TF RA V R - -E RQ VR KS SC SY D Danio rerio RA QR ST NF RS S R ER E SVS RK SW GS AD melanogaster AQ ST CS LQ RV V R AKK TS VP SL ET YA Y Anopheles gambiae VH GT SS LQ RI A R TK KT SVPS LD GF GT

Figure 2 DEPDC5 mutations. (a) Schematics of the exon-intron structure of the DEPDC5 gene with the position of mutations shown based on the human genomic sequence (NM_001242896) (top) and of the DEPDC5 protein with the positions of alterations indicated (bottom). DEPDC5 is a multidomain protein carrying an N-terminal domain of unknown function termed DUF3608 and a DEP domain at its C terminus. The p.Arg239*, p.Arg328*, p.Gln372* and p.Leu374Phefs*30 alterations are clustered in the DUF3608 domain. (b) Multiple-protein alignment showing conservation of the arginine residue at position 485 in the encoded by the orthologs of DEPDC5 across species. (c) Sequence chromatograms of one affected (S-IV:9) and one unaffected (S-III:11) member of family S, showing the presence of a mutation encoding p.Arg239* in the DEPDC5 cDNA of lymphoblasts from S-IV:9 pretreated with emetine but not in untreated cells. All rights reserved.

in both the developing and adult human brain (Human Brain that DEPDC5 may be a G protein substrate. The DEPC5 ortholog Transcriptome), consistent with a role in epilepsy. We attempted to gene product in , Yjr138p (also known as SEA1), is part of an obtain clues from sequence analysis and database mining. The RefSeq evolutionary conserved multiprotein SEA complex involved in mem- America, Inc. collection reports five putative isoforms of DEPDC5 of 1,572, 559, brane trafficking16. DEPDC5 may also be involved in oncological 1,594, 1,603 and 1,503 amino acids in length for isoforms 1–5, respec- processes, as (i) it was reported to be deleted in two glioblastomas17 tively. One of the mutations (encoding p.Gln1523*) lies within an exon and (ii) an intronic SNP (rs1012068) in the DEPDC5 locus has been absent from isoform 2, suggesting that this isoform is not involved in associated with hepatocellular carcinoma risk18. pathology. Alignment with orthologs, using reciprocal best-BLASTP In conclusion, this work provides clear evidence that loss-of-function © 2013 Nature hits, and phylogeny showed that the DEPDC5 protein is highly con- mutations in DEPDC5 are a major cause in a broad spectrum of auto- served in metazoa and fungi (but absent from plants) (Supplementary somal dominant focal epilepsies. It shows that a single gene, DEPDC5, Fig. 4), indicating a core role for this ancient eukaryotic factor. is a common genetic actor in epileptic syndromes with different brain

npg We note also that DEPDC5 and its orthologs occur as single gene localization and electroclinical expression, including ADNFLE, FTLE copies, even within genomes known to be ancient polyploids, such and FFEVF. Thus, seizure initiation sites may be dissociated from as Dario rerio and . This evidence, together underlying genetic mechanisms, in striking contrast to previous with the dominance of epilepsy-causing mutations, suggests that gene data, which link LGI1 mutations with a seizure focus localized in the dosage may be critical for normal brain function. Transmembrane lateral temporal lobe and mutations in nicotinic acetylcholine subunit prediction using hidden Markov model (TMHMM) analysis also genes with a seizure focus localized in the frontal lobe. Understanding showed that the DEPDC5 protein has no transmembrane domain the relationship between loss of DEPDC5 function and the genesis of and no with known ion channels. The presence of a DEP pathological neuronal synchronies in different epileptic networks and domain, a globular protein motif of about 80 amino acids found in syndromes will provide new, clinically relevant insights and knowledge many proteins involved in G protein signaling pathways, suggests that may be useful in genetic counseling. With no known homology between the DEPDC5 protein and other pro- teins, new pathogenic mechanisms seem likely. Table 1 Mutations identified in the DEPDC5 gene DEPDC5 is the second gene after LGI1 found Allele to be mutated in familial focal epilepsies not Coding mutation frequency encoding ion channel or transmitter receptor Family Phenotype Genomic position (hg19) (NM_001242896) Exon Protein alteration (EVS) subunits. In addition to channelopathies, we N FFEVF Chr. 22: g.32200188delA c.1122delA 16 p.Leu374Phefs*30 0% must consider alternative genetic pathways S FFEVF Chr. 22: g.32188751C>T c.715C>T 12 p.Arg239* 0% leading to epileptogenesis. L FTLE Chr. 22: g.32198725C>T c.982C>T 15 p.Arg328* 0% Q FFEVF Chr. 22: g.32200180C>T c.1114C>T 16 p.Gln372* 0% URLs. 1000 Genomes Project, http:// O FTLE Chr. 22: g.32210986G>A c.1454G>A 21 p.Arg485Gln 0% www.1000genomes.org/; BLASTP, http://blast. B ADNFLE Chr. 22: g.32302238C>T c.4567C>T 43 p.Gln1523* 0% ncbi.nlm.nih.gov/; Database of Genomic

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Variants, http://projects.tcag.ca/variation/; dbSNP, http:/www.ncbi. 1. Scheffer, I.E. et al. Autosomal dominant nocturnal frontal lobe epilepsy. A distinctive nlm.nih.gov/projects/SNP/; Human Brain Transcriptome, http:// clinical disorder. Brain 118, 61–73 (1995). 2. Berkovic, S.F. et al. Familial temporal lobe epilepsy: a common disorder identified hbatlas.org/; MERLIN, http://www.sph.umich.edu/csg/abecasis/ in twins. Ann. Neurol. 40, 227–235 (1996). merlin/; MutationTaster, http://www.mutationtaster.org/; NHLBI 3. Ottman, R. et al. Localization of a gene for partial epilepsy to chromosome 10q. Nat. Genet. 10, 56–60 (1995). Exome Variant Server, http://evs.gs.washington.edu/EVS/; Online 4. Klein, K.M. et al. Familial focal epilepsy with variable foci mapped to chromosome Mendelian Inheritance in Man (OMIM), http://www.omim.org/; 22q12: expansion of the phenotypic spectrum. Epilepsia 53, e151–e155 PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/; RefSeq, http:// (2012). 5. Baulac, S. & Baulac, M. Advances on the genetics of mendelian idiopathic www.ncbi.nlm.nih.gov/refseq/rsg/; SIFT, http://sift.jcvi.org/; trans- epilepsies. Clin. Lab. Med. 30, 911–929 (2010). membrane prediction using hidden Markov model (TMHMM), 6. Heron, S.E. et al. Missense mutations in the sodium-gated potassium channel http://www.cbs.dtu.dk/services/TMHMM/. gene KCNT1 cause severe autosomal dominant nocturnal frontal lobe epilepsy. Nat. Genet. 44, 1188–1190 (2012). 7. Kalachikov, S. et al. Mutations in LGI1 cause autosomal-dominant partial epilepsy Methods with auditory features. Nat. Genet. 30, 335–341 (2002). 8. Berkovic, S.F. et al. Familial partial epilepsy with variable foci: clinical features Methods and any associated references are available in the online and linkage to chromosome 22q12. Epilepsia 45, 1054–1060 (2004). version of the paper. 9. Callenbach, P.M. et al. Familial partial epilepsy with variable foci in a Dutch family: clinical characteristics and confirmation of linkage to chromosome 22q. Epilepsia Note: Supplementary information is available in the online version of the paper. 44, 1298–1305 (2003). 10. Morales-Corraliza, J. et al. Familial partial epilepsy with variable foci: a new family Acknowledgments with suggestion of linkage to chromosome 22q12. Epilepsia 51, 1910–1914 (2010). We thank the genotyping and sequencing platform of ICM for technical assistance, 11. Xiong, L. et al. Mapping of a gene determining familial partial epilepsy with the DNA and cell bank of ICM for DNA extraction and cell culture, P. Couarch for variable foci to chromosome 22q11-q12. Am. J. Hum. Genet. 65, 1698–1710 technical assistance, M. Gaussen for linkage analysis and C. Depienne for helpful (1999). discussions. S.I. received a grant from the French government. This study was 12. Picard, F. et al. Dominant partial epilepsies. A clinical, electrophysiological and supported by the program Investissements d’Avenir ANR-10-IAIHU-06. genetic study of 19 European families. Brain 123, 1247–1262 (2000). 13. Thomas, P., Picard, F., Hirsch, E., Chatel, M. & Marescaux, C. Autosomal dominant AUTHOR CONTRIBUTIONS nocturnal frontal lobe epilepsy. Rev. Neurol. (Paris) 154, 228–235 (1998). S.I. performed experiments and analyzed data. F.P. performed phenotyping and 14. Scheffer, I.E. et al. Familial partial epilepsy with variable foci: a new partial epilepsy collected clinical data. G.R. collected samples and extracted DNA. E.N. and E.M. syndrome with suggestion of linkage to chromosome 2. Ann. Neurol. 44, 890–899 (1998). participated in genetic experiments. G.A. carried out the bioinformatics analysis 15. Ishikawa, K. et al. Prediction of the coding sequences of unidentified human genes. on DEPDC5 and statistical analysis. P.T., P.G., M.W., C.M. and E.H. performed X. The complete sequences of 100 new cDNA clones from brain which can code

All rights reserved. phenotyping of families. R.M. and M.B. contributed to the writing of the manuscript. for large proteins in vitro. DNA Res. 5, 169–176 (1998). E.L. supervised the project and contributed to the writing of the manuscript. 16. Dokudovskaya, S. et al. A conserved coatomer-related complex containing Sec13 S.B. designed the study, supervised data analysis and wrote the manuscript. and Seh1 dynamically associates with the vacuole in Saccharomyces cerevisiae. Mol. Cell Proteomics 10, M110 006478 (2011). COMPETING FINANCIAL INTERESTS 17. Seng, T.J. et al. Complex rearrangements in astrocytic tumors The authors declare no competing financial interests. identified using microsatellite and chromosome 22 tile path array analysis. Genes Chromosom. 43, 181–193 (2005). America, Inc. 18. Miki, D. et al. Variation in the DEPDC5 locus is associated with progression to Reprints and permissions information is available online at http://www.nature.com/ hepatocellular carcinoma in chronic hepatitis C virus carriers. Nat. Genet. 43, reprints/index.html. 797–800 (2011). © 2013 Nature npg

Nature Genetics VOLUME 45 | NUMBER 5 | MAY 2013 555 ONLINE METHODS read depth, and detects variants (SNPs and indels). Only positions included in Families and subjects. Sixteen unrelated families from western Europe with the bait coordinates were conserved. Genetic variation was annotated with the non-lesional focal epilepsies were studied12. Mutations in CHRNA4, CHRNB2 IntegraGen in-house pipeline, consisting of gene annotation (using RefSeq), and LGI1 have been excluded in all families. Local ethics committee (CCPPRB detection of known polymorphisms (using dbSNP135 and the 1000 Genomes of Pitié-Salpêtrière Hospital, Paris, 69-03, 25/9/2003) approved this study. Project database) and characterization of mutations as exonic, intronic, Informed consent was obtained from all participants or their parents. silent or nonsense.

Genotyping and linkage analysis. A genome-wide screen was performed Screening of a cohort by pyrosequencing. All 43 exons and intron-exon on families N and S with Illumina 6K panel microarrays (Linkage-24 DNA junctions of DEPDC5 (except exon 2, which was screened by Sanger sequenc- Analysis BeadChip). LOD scores were calculated with MERLIN software ing) were analyzed by universal tailed amplicon sequencing (454 Sequencing assuming an autosomal dominant trait with 70% penetrance, a disease fre- Technology, Roche). This approach used the 454 GS Junior system and involved quency of 0.0001 and 0% phenocopy. Haplotypes were reconstructed according an initial PCR amplification with primer sets designed to amplify exons cor- to hg19 annotations. responding to the sequence under accession NM_001242896 (Supplementary Table 1), a second PCR aiming to incorporate multiplex identifier and 454 Exome sequencing. Exome sequencing was carried out in subjects III-4 and adaptors and, finally, an emulsion PCR step carried out according to the III-6 of family N. Targeted exome sequencing, library preparation, capture emPCR Amplification Method Manual (Roche). and sequencing, and variant detection and annotation were performed by IntegraGen (Evry, France). Exons of genomic DNA samples were captured Validation of exome and pyrosequencing variants by Sanger sequencing. using Agilent in-solution enrichment methodology with a biotinylated oligo- Mutations found through exome sequencing and pyrosequencing were vali- nucleotide probe library, and paired-end 75-base massively parallel sequencing dated by Sanger sequencing using the BigDye Terminator kit on an ABI Prism was carried out on an Illumina HiSeq2000. Sequence capture was performed 3730 DNA Analyzer (Applied Biosystems). Segregation analysis of within- according to the manufacturer’s instructions (Human All Exon kit V4+UTRs, family variants was carried out using the same primers as for pyrosequencing 70 Mb, Agilent). Briefly, 3 µg of each genomic DNA sample was fragmented (Supplementary Table 1). Mutation interpretation was assessed with Alamut by sonication and purified to yield fragments of 150–200 bp in length. Paired- version 2.2 (Interactive Biosoftware). The effects of amino-acid substitutions on end adaptor oligonucleotides from Illumina were ligated on repaired A-tailed protein function were predicted using MutationTaster, SIFT and PolyPhen-2. fragments, and fragments were purified and enriched by six PCR cycles. Purified libraries (500 ng) were hybridized to the SureSelect oligonucleotide Cell culture and mRNA experiments. Lymphoblastic cells from three muta- probe capture library for 24 h. After hybridization, washing and elution, the tion carriers (S-III:10, S-IV:7 and S-IV:9) and one spouse (S-III:11) from family eluted fraction was PCR amplified with 10 to 12 cycles, purified and quanti- S were treated (or not) overnight with 10 mg/ml emetine to inhibit NMD. Total All rights reserved. fied by qPCR to obtain sufficient DNA template for downstream applica- RNA was extracted with the Qiagen RNeasy Mini kit and reverse transcribed tions. Each eluted enriched DNA sample was then sequenced on an Illumina with the ThermoScript RT-PCR System (Invitrogen). DEPDC5 cDNA was HiSeq2000 as paired-end 75-base reads. Image analysis and base calling were amplified and sequenced using specific primers located in exons 12 and 15. performed using the Illumina Real-Time Analysis Pipeline version 1.14 with default parameters. Phylogenetic tree reconstruction. All amino-acid sequences encoded by America, Inc. orthologous genes were retrieved from NCBI and aligned using MUSCLE. Bioinformatics analysis. Bioinformatics analysis of sequencing data was based Visual inspection of the alignment with SEAVIEW showed that homology was on the Illumina pipeline (CASAVA 1.8). CASAVA aligns reads to the human present across several regions of the protein. Protein alignment was trimmed reference genome (hg19) with the alignment algorithm ELANDv2 (it performs to 454 homologous sites using GBLOCK. Tree reconstruction was performed multiseed and gapped alignments), calls SNPs on the basis of allele calls and by maximum likelihood using PhyML software with an LG matrix. © 2013 Nature npg

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