Apports des nouvelles technologies de la génomique dans les maladies rares

Judith Melki Inserm UMR-1169 & University of 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 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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 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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.

Mutation of ADCY6, which encodes an adenylate cyclase that synthesizes cAMP, causes AMC suggesting that ADCY6 acts in the GPR126-cAMP pathway, which regulates myelination of Schwann cells.

Laquerrière et al. 2014 Loss-of-function mutations in LGI4, a secreted involved in Schwann cell myelination, are responsible for arthrogryposis multiplex congenita

Coll. Bruno Reversade Xue*, Maluenda*, Marguet* et al. 2017 LGI4 (leucine-rich glioma-inactivated 4) is a ligand secreted by Schwann cells that regulates peripheral nerve myelination via its receptor ADAM22 expressed by neurons.

As a secreted protein that is required for Schwann cell development and differentiation, LGI4 may be regarded as a novel therapeutic target to stimulate myelination in humans.

MAGEL2

Neuromuscular junction Lower motor neuron CHRNG, CHRNA1, CHRNE, Skeletal muscle GLE1, ERBB3 , BICD2 CHRND, RAPSN, DOK7, TNNI2, TNNT3, MYH3, PIP5K1C, TRPV4, SMN1, CHRNB1, COLQ, CHAT, MUSK, TPM2, MYH2, MYH8, ECEL1, UNC50 NALCN MYH13, RYR1, DMPK, FKTN, NEB, MYBPC1, SYNE1, COL6A2, TTN, ACTA1, MTM1, PIEZO2, CFL2, AMPD1, SCN4A, LMOD3

Axoglial : CNTNAP1 Laquerrière et al. 2014 Defective node of Ranvier Ravenscroft et al. 2015 GLDN formation or axoglial interaction Mejlachowicz et al 2015 ADCY6 represent novel disease Maluenda et al. 2016 GPR126 mechanisms in severe AMC Xue et al. 2017 LGI4 Abiusi et al. 2017 Vein of Galen aneurysmal malformation (VGAM)

VGAM is a congenital anomaly of the cerebral vasculature representing 30% of all pediatric vascular malformations. Incidence : ~1 in 50,000.

The outcome is particularly poor when anomalies such as cerebral defects or signs of cardiac dysfunction are associated.

Coll. A. Ozanne, G Saliou We conducted whole exome sequencing in 19 unrelated patients presenting this malformation and subsequently screened candidate gene in a cohort of 32 additional patients using either targeted exome or Sanger sequencing. In a cohort of 51 patients, we found 5 affected individuals with heterozygous mutations in EPHB4

AA5614 AA5615 AA5616 AA5717 AA5718 * * * * I:1 I:2 I:1 I:2 I:1 I:2 I:1 I:2 I:1 I:2

II:1 II:1 II:1 II:1 II:1

c.570dupG c.2484+1G>T c.2484+2insT c.2609T>A c.319T>C p.His191Alafs*32 p.Met814_Val829del p.Val870Glu p.Cys107Arg Knockdown of EPHB4 in embryos leads to specific anomalies of dorsal cranial vessels including dorsal longitudinal vein(3), the ortholog of the median prosencephalic vein, the embryonic precursor of the vein of Galen. This model allowed to demonstrate EPHB4 loss of function mutations in this disease by the ability to rescue the vascular defect in KD-zebrafish co-injected with WT but not truncated EPHB4 mimicking the patient mutation. The EPHB4 receptor plays a unique role in vascular development, where it regulates venous differentiation. EphB4 is highly enriched in veins, and is the only EphB receptor that specifically binds to ephrinB2.

The identification of EPHB4 pathogenic mutation in patients presenting capillary malformation or vein of Galen aneurysmal malformation should lead to careful follow up of pregnancy of carriers for early detection of anomaly of the cerebral vasculature in order to propose optimal neonatal care.

Endovascular embolization indeed greatly improved the prognosis of patients.

Vivanti et al. 2018 Added value of new genomics technologies in undiagnosed patients

• Genotype well correlated with clinical phenotype : Genetic heterogeneity Very rare disease: gene not tested or genetic testing not available

• Mutation in a known gene but atypical phenotype: Gene: non tested (added value of whole exome sequencing instead of targeted) Large phenotypic spectrum (atypical presentation)

• Mutation in a gene with known therapies

• New gene : Added value of clinical network for very rare diseases ++ Limits of WES

No candidate gene ?

« GENETIC CAUSES » Poor coverage of the mutation by WES Intronic mutations Microdeletion/duplication neither detected by aCGH nor WES Somatic mosaicism Uniparental disomy Epigenetic modifications

Repeat expansion ? Mutations in highly homologous genes ?

Oligogenic mechanism ? Added value of WES ++ New genetic disease mechanism ?

« CLINICAL CAUSES» Acuity of the clinical phenotype (including disease course) with update for variant interpretation Siblings suffering from distinct diseases ? Sampling error ? Phenocopy ? New genomics approaches: numerous applications

I- New diagnostic tools II- To end an expensive and potentially invasive diagnostic testing III- To provide accurate estimate of recurrence risk and genetic information of at-risk relatives IV- To enlarge the clinical spectrum of disease V-To identify new disease genes

To identify new disease gene mechanism Starting point to elucidate pathophysiology and start therapeutics strategies To improve care of patients: pharmacogenomics, gene-based therapy based on gene function, correction of gene mutation or replacing the defective gene or protein

Collaboration Inserm and Paris Sud University UMR-1169, CHU Bicêtre

J. Devaux, Aix-Marseille Université, P. Francesc, Institute of Health Carlos III, Spain Alexandre Vivanti Jérôme Maluenda I. Gut, CNAG, Barcelona, Spain Emanuela Abiusi Flora Nolent NG. Laing, University of Western Australia, Australia Loic Quevarec Adrien Camus A. Laquerrière, CHU Rouen, France Turczynski Sandrina Dan Mejlachowicz A. Ozanne, CHU-Bicetre, France Cynthia Grondin B. Reversade, Institute of Medical Biology, Judith Melki M. Tawk, U1195, CHU-Bicetre, France H. Topaloglu, Hacettepe University Ankara,

Collaboration Acknowledgments • AFM PHRC French National Network • PHRC, AP-HP CRMR-AnDDIRares • Agence de Biomedecine • Inserm • University Paris Sud