Apports des nouvelles technologies de la génomique dans les maladies rares Judith Melki Inserm UMR-1169 & University of Paris Sud CHSF, CHU Bicêtre [email protected] 7681 ccatcttctc tttgtgcatg ttggtctccg tgtcccaatt tcccctttct atagggactg cagtcctaat gaattagacc ccaacaaacg acctgatatt aacttgatta cctccataaa 7801 gatcctattt ctaaataagg ccacattttt ttgagatact aggaattagg acatcaatgt atcttttatg tgacagacat ttcaacccat tagagttacc taacctccct cctaacacca 7921 cttccccttt ataaaatgag gataaaagtg ctgacctcac agggctgtgg agaacctggg gctatgcatg tagaaggatt agcacagtgc ctggcacatg gctggaaggc atcaaatgtt 8041 agctagtatt attatgaaat ggggatatag agccttagag ctcaatttat tttgctttgc ttatacagaa gtccatatgg ataacatttt cctccaactc taaagggcat aatgattttt 8161 cataacagcg taagttgatt tttacatctt gtactttaca aaggaactat atatttgaat aaaatttact ttttatttga gtattgccat gtattcatac tatgatacaa ttgccttgaa 8281 taaatacctt actcccagta agtaaataaa ccctaaatgt taaaaatctg aacaatttaa acatggctag aaaatgcacc ttctatatta ttcctaaaat aaaagaaata aaggctctaa 8401 aatgcaatat tgaattcccc caaccatgct gatgtaggta aactgtattt cagatattgg gaaatagcct cataaactga gaagaacacg gcttttagat tcaagtacat atggattcaa 8521 cttccacctt tacccctaca gctctgtgac cagtgggaag ttatgtagct ttgttcagcc ttggtttctt catctgcgaa attaggaaaa taatactcct tcaaaagtga gagagcgtaa 8641 cctgcagtgg atgaatggat aaacaaaatg tgctatgtac atataatgga atattattca gccttaaaaa ggaaggaaat tctgacacac gctataacat ggattaacct tgagaacatt 8761 atgctaagtg aaataaacca gtcatcaata gacagatact gtattattcc acttatgtga ggtacctaaa gtcatcaaat tcatagaggt aaaaaacata aaggcttttg ccaaggtcca 8881 gggggagggt agaatgagga gctactgttc actgggtaga gagctttaat tttgcaagat gaaaagagtt ctggaaatgg ctgctcatga tggtttcaaa acgatgtgaa tatccctaat 9001 gccactaaac tgtacacttt aaaaatggtt acgatggcaa atcttatgtt atatatattt tactctaatt tttaaaataa atttaaaaaa taaatcctaa aacattgttt aaaatgtgaa 9121 atagtttaag gaaatcccct aatgtgacgc ctagaataga agcaggtagc tattagacac attccttctt tagttttctc cctcctcccc accataaata gttgcaaaaa taattggagt 9241 gtgcacatag ccaaagattt aatgccacat agccaaacaa acaccagata attcaggaac ccttgattct gaagtgaagc ttataagaag atgaaccaca ttggatcagg aaataagaaa 9361 Leaccagtccat génome atgttgcaat taacttgttc humain tgtgattggg agcaagtcac = 500 ttagcttctt 000 tggacctgtt pages ttctcatctg tcaaataagg comme agggttgaac celletaggtaatct -aaatgaaatcci 9481 caagtcctta gaggcttgta ttacataaat caagtcaaga catggtattt aagaatgaag ggtcatagtt tagcatgcta ataattcttc ttcatgcaaa aacataggag ggggaaataa 9601 atatctttta tcgtaatacc atgataaatt tgctgggtgg ggggaggaat agattataag acaggccgaa aggagcaatt aatagcgaaa tgtcacacta ttctatatca aatgttatgc 9721 atttaaaaga atatgtcagt tttgcaagat gaacaagttc tagaaatgtg ttgcacaatg atgtgtatgt agttaacaac tctgcactgt acatttaaaa tggtgaagat ggtaaatttt 9841 atattatggg ttttttgcca caacttaaaa aaagaatatg ggcaactatt ttctttcttt aacatcctca tttttcaaaa acaataccag tggttttcaa gcttttttac aaagagcaaa 9961 tccattcttc taacaaagtt tcataagaaa aataactgta aaaaaaaaat atggagttga aagtgaggca tgagatggag gtaacgaata ttcccagtat gagcctctct cccttcttgt 10081 tccttgggcc tgctcctgag ttctgcaaag actccctagt gctccaggag gctggtttaa aaatcaatgc tttatactct acaaagaaat gtaggccagg cacaatggct cacacctgta 10201 atcccagcac tttgggaggc caaggtgggg agatcacttg aggtcaggag ttcaagacca gcctggccaa catggcgaaa ctctatctgt acaaaaaaca ccaaaattag ccaggcatga 10321 tggcgcatgc ctgtagtccc agctactcat gaggctgagg tgggaggaac gcttgagccc aggaagttga ggcggcattg agctgagata gcaccactgc actccagcct gggtgacaga 10441 gcaaggccct atctcaaaaa aaaaaaaaaa aaaagaaaag aaatgtaaat ggccctcacc atgcattcga ctgggaatta atggtggtag agctttgttc aactgagccc cacaatccat 10561 ctaaccattt actgcaatac tgtaaaagtg gaccttagag ggggtatttc tatctgcttt caggaagaga tattatggat atactagagg taaaaaaccc ccaagtttag cacattctca 10681 ataaaaatgt gtataaatga ctgaattcat gaagggaccc aagctccagg aaaagcatag caggagaagt 9attttattaa tctatccatt cattcattta tttattcgaa agcatttctt 10801 taacatcaat tatattccag attattgttaGénome agcacaggga nucléaire ataaaaacat gaatcagata: 3,2 cagtccctac 10 cctcaaggaa bp (3 ctcactctct 200 ggtacaagagMb) acatgtttca aaaggtcatt 10921 gtaaagaaat gtggaaaagg caagaattat gcacagaggg ctatgggagt accaagccag ctgtgaggga gccagggaag acttcttgaa ggaaggatta agagctagct gctcaatgac 11041 aggagaaggt catggaaggt gtagacaaaa taatgagcat gtgtccatgt gtgggaagtg gcttggtgtg ttgggaagaa cacaacacag agaaggaatg ggagacatga agccacaggc 11161 agggttgtgg agagttttgc atgtgagtag ggcctgtctg catttttctc ~25 ccttggcttt 000 gcctggctcc gènes tttccttcca tcccagggga cctccccctg acactccaca gagctttgag 11281 ttctataatc tgtagcttat tcccactcca tggagaaaga ggaaagaagg ctaagaggaa gaaggaaagg gcatttcacc tcctcagtgg aagctaccat agaaagtcag atcaggccgg 11401 gcacggtggc tcatgcctgt aatcccagcg ctttgagagg ctgcggctgg cggatcacct gaggtcagga gtttgagacc agcctgggca acatggtgag accccgtctc tactaaaaat 11521 acaaaaatta gccaggcatg gtgctgtgtg cccgtagtcc cagctactgt ggaggctgag gcaggagaat tgcttgaacc caggaggcag aggttgcagt gagccaagat agtgccaccg 11641 cactccagcc tgggcaacag agcgaaaact ctatctcaaa aaaaaaaaaa aaaaaaaaaa aaaagatcac aacgtttacc cataaaagaa aacaacaatg ttgcttcatg agtccttgat 11761 gggtttctga gaggcagaag catttgacct gaaggtgctg tgtggaaagg gcccgctgag aactcctcct ccaccaactc cccaggacca agctatctat aggtgctggg tgttcacggc 11881 tgacttgccc agcatcaggg aggcctggtc cctcaacctc agttcaaggc cctcaggtac ttggagctca agacttcccc tccttaggac actctacttt ccagctttgt ttaatgaaaa 12001 cattcacttc tgattcaata gcattagaaa acccagattt catttccctt tacaacagca tgaacacctg aatcgttccc ctactgggag tctctttgga gtttgaatca aaatgctgac 12121 cataatggag ggacttctct ctcttatatg aacatccact agataaacat caaacaaacg tagatatgaa aatgctgaca tggggcagac aggaatgaaa atctgttagg aaagacctca 12241 gaattcccta tactctttct aagtagatag agatacagat aaacatccca aattttaagt ttttaaatct tttagttgat aaaaatcaaa gcagtgactg atcccatgtg gtccagacca 12361 ctgttctcat ctaaggcaac ctcagaaacc caacagcccc attgagtaga tttagaccac aggttactgg gagaagatgg atttcaggaa cagcaaaaat caaaagcaaa tattcttttg 12481 aagcttgcaa aatcactgtt ttagaatctg aattatattg gcaaccagag agaatcaagc attcttcttc atttcccaaa atactaacat ttcccttgtg ggaactgatt ctcgatttct 12601 atttattaca aaaggaagaa aaacatgttg aggtcttaca ttctcctatt ttttttcact tttccattgt cctgaatagc aaagtaccat attactcaga atatgatttt gcatatgcta 12721 tcagataaat ctaaaacatt tttgtggcta aatttagatg tttgtataac atactggggg aaaatcactt aagtgatttt ggcactaata ctcaaaagac gtgtttctac actttcagta 12841 aaactacaac aaacgtgtta aatatttggt aaaaacaggg aggcatggtc aaaatttgaa aatcaaaaag tcttaaatga agaaagataa gatgatggct ctacattccc atgaactagt 12961 acaacaatta ttttttttaa aacatacata tactgtttct agagcagttt tgggttcact acgaaattaa gcagaaaata tagaatttcc atataaccat cccacaccat gcgtagcctc 13081 cctgatcaag atccctcacc agatggcacg tttgttacaa ctgattaacc tacattgata catccttagc acccacaggc catagttgaa ttagggttca ctacagtcta tgggtcttga 13201 caaatgtgta atgccatgta gccaccatta taacatcaca cggaatagtg tcattgtcct aaaaatcccc tgtgctcaac ttgttagtcc ttccttctcc ccagttctgg acaaccactg 13321 atctttttcc tgtctccata attttgactt ttccagaatg tcataaagtt ggaatcacgc agtatatagc cttttgagat gggcttcttt cacttaataa tatgcattta atgttcttcc 13441 ataatttttc actgtctaga tataccacag tttatccatt cacctactga agaagagctt ggttgcctcc aagttcaaca atttgctttt aaacttactt atttaaagag tggctagctc 13561 agaataattg ctgtctttat tttcttattt aatttcaaag gtgaccaaga caactcttaa ggaacgcatg aattcccctt agcaacaact ccaacagact gtctgacttt gcttgcttct Whole exome sequencing (WES) • NGS of all exons and intron-exon junctions • ~64 Mb (~25 000 genes) compared to whole genome (3200 Mb): ~ 1% • The majority of known mutations are located in these regions (90%, Stenson et al. 2009) Arthrogryposis multiplex congenita most often results from fetal hypo- or akinesia sequence Depending on the age of onset, fetal hypo or akinesia leads to: • Muscle atrophy • AMC • Pterygia • micrognathia • Pulmonary hypoplasia • Diaphragmatic defect Incidence 1: 3000 live births Non syndromic AMC: 75% of cases are of neuromuscular origin Genetic heterogeneity > 70 genes Patients inclusion: 286 unrelated affected individuals with AMC were included through collaboration with Geneticists, Fetopathologists and Neuropediatricians Methods: Whole exome sequencing (WES): 65 Targeted exome sequencing (TES): 153 TES negative WES: 54 250K microarray and Sanger sequencing : 14 Mutations in CNTNAP1 encoding CASPR, an essential component of Node of Ranvier domains, are responsible for AMC Phe1003fs; Phe1003fs; Cys968Phefs*11; p.Cys715Arg; homozygous homozygous homozygous Homozygous Ile999Trpfs*5; Ala143Glu; Cys974* Arg635*, homozygous homozygous Laquerrière et al. 2014 Clinical and morphological characteristics of patients carrying CNTNAP1 mutations The node of Ranvier, the flanking paranodal junctions and the juxtaparanodes underlie saltatory conduction of action potentials along myelinated axons, an essential process for neuronal function. The paranodal junctions consist of a complex containing the axonal proteins Caspr and contactin and the glial isoform of neurofascin. Laquerrière et al. 2014 Mutations in GLDN encoding gliomedin, an essential component of Node of Ranvier, are responsible for AMC 1 2 3 4 I:1 I:2 I:1 I:2 I:1 I:2 I:1 I:2 II:1 II:2 II:1 II:1 II:2 II:1 c.758delC c.1423G>C c.95C>A c.1240C>T c.541+1G>A c.1435C>T p.Pro253Leufs*51 p.Ala475Pro p.Ala32Glu p.Arg414* p.Arg479* Maluenda et al. 2016 These data indicate that mutations of GLDN or CNTNAP1 encoding essential components of the nodes of Ranvier and paranodes, respectively, lead to inherited nodopathies, a distinct disease entity among peripheral neuropathies. GLDN CASPR Mutations in ADCY6 are responsible for severe AMC with axoglial defects ADCY6: R1116C; homozygous; Loss of mbp expression in the PNS while the CNS mbp expression was comparable to controls. No defects in Schwann cell migration and axonal growth in the morphants.