Truncating Mutations of MAGEL2, a Gene within the Prader-Willi Locus, Are Responsible for Severe Arthrogryposis UMR-1169, INSERM et Université Paris Saclay Hôpital Bicêtre, France Dan Mejlachowicz

Incidence: 1 in 3000 Non syndromic AMC: Neuromuscular junction : CHRNG, CHRNA1, CHRND, CHRNB1, DOK7, RAPSN, CHAT Skeletal muscle: TPM2, MYH2, MYH3, MYH8, TNNI2, TNNT3, MYBPC1, DMPK, ACTA1, NEB, RYR1, SYNE1, PIEZO2, TTN Survival of motor neurons or myelination of peripheral nerve: GLE1, PIP5K1C, ERBB3, SMN1, TRPV4, BICD2, ECEL1, ADCY6, CNTNAP1, GPR126 The difficulty in establishing a genetic diagnosis for AMC individuals is due to the high genetic heterogeneity and/or the existence of some not yet identified disease causing genes Arthrogryposis multiplex congenita (AMC)

II:1II:2 III:1 III:2 III:3 U.S.: 22.w.g. Reduced mobility polyhydramnios unilateral club foot. Fetal death: 24 w.g. Fetopathological examination: severe microretrognathia, Club foot, flexion of elbows and knees, camptodactyly of the fingers U.S.: 23 w.g. Bilateral club foot polyhydramnios bilateral camptodactyly Fetal death : 24 w.g. Pathological examination: identical phenotype Identical phenotype Fetal death: 27 w.g. ab Hanitra Ranjatoelina-Randrianaivo, CHU St-Pierre La Réunion

I:1I:2 II:1II:2 III:1III:2III:3 Genetic mapping combined with Whole exome sequencing: NM_ : c.1996delC p.Gln666Serfs*36 Inherited from the unaffected father and grandmother Heterozygous 1bp deletion in MAGEL2 : one of the paternally expressed genes within the Prader-Willi syndrome locus (15q11.2)

MAGEL2 RNA analysis Marked reduction of MAGEL2 expression in patient MAGEL2 β-actin MWCCPPCP +RT-RT C: Control P: Patient Sequence analysis of RT-PCR product from Patient: paternally mutated allele only DNA RNA

Sanger sequencing of MAGEL2 in additional affected individuals or fetuses with arthrogryposis and/or reduced fetal mobility (n=84) Phenotype: discovered at birth Microretrognathia, bilateral camptodactyly, Bilateral club foot, Severe hypotonia, Respiratory distress, Death: postnatal day 2 Genetic testing (normal): Array CGH Sanger sequencing: CHRNG, CHRNA1, CHRNB1, CHRND, RAPSN, DOK7 CHAT; DMPK locus Paternal deletion of 15q11-q13 and maternal uniparental disomy 15 excluded. Fabienne Giuliano, CHU Nice De novo heterozygous 1bp deletion in MAGEL2 NM_ : c.2118delT; p.Leu708Trpfs*7 De novo : on the paternal or maternal allele ?

DNA RNA: Mutation only DNARNA: paternal allele only DNA I:1I:2 II:1 I:1I:2 II:1 MAGEL2: c.2118delTMAGEL2: rs Deleterious de novo MAGEL2 mutation occurred on the paternal allele

Fetal phenotype: Increased nuchal translucency 16 w.g: Reduced mobility Unilateral club foot Fetopathology examination Retrognathia Unilateral club foot Cleft palate Alexandra Benachi et Jelena Martinovic, CHU Antoine-Béclère Heterozygous 1bp deletion in MAGEL2 Targeted exome sequencing (AMC): NM_ : c.1996delC; p.Gln666Serfs*36 Inherited from the unaffected father and grandmother

Cassidy et al. PWS phenotype: to varying extents reduced fetal mobility, polyhydramnios, distal joint contractures, neonatal hypotonia with respiratory distress, early-childhood onset obesity, intellectual disability, hypogonadotropic hypogonadism and short stature PWS “genotype” paternal deletion of 15q11-q13 (65–75% of individuals) maternal uniparental disomy 15 (20–30%) more rarely: imprinting defect or microdeletion of SNORD116 cluster PWS locus : five paternally expressed genes (MKRN3, MAGEL2, NDN, NPAP1 and SNURF-SNRPN) and a family of six paternally expressed snoRNA genes or clusters. CONTIGUOUS GENE SYNDROME ?

c.1652delT c.1802delC c.3124C>T c.3181_82delAT c.2118delTc.1996delC MAGEL2 5’3’ Schaaf et al Mejlachowicz et al Here: 5 affected individuals from 3 unrelated families, Fetal phenotype very similar to those previously reported in PWS Heterozygous truncating mutations on the paternal allele of MAGEL2. Mutations either inherited from or occurred de novo on the paternal allele Truncating mutations in MAGEL2 are responsible for this condition Recently, truncating mutations of MAGEL2 have been identified on the paternal allele of four affected individuals with features resembling PWS and called Schaaf-Yang syndrome (2013) Intragenic mutations of the paternal allele of MAGEL2 result in a large clinical spectrum ranging from severe fetal phenotype (our data), to syndromic intellectual disability or autism (Schaaf-Yang syndrome) MAGEL2 intragenic mutations recapitulate the PWS phenotype

MAGEL2 imprinting variance Matarazzo and Muscatelli (2013) detected Magel2 transcripts in brain of mice deleted for the paternal allele of Magel2 indicating an incomplete silencing of the maternal allele. Paternally Magel2-null mice have reduced embryonic viability (in 10 to 30% embryos) but otherwise normal embryonic growth in survivors, followed by post-natal growth retardation, excessive weight gain recapitulating aspects of the PWS phenotype Guo et al. reported an imprinting variance of Magel2 in pig during fetal development. The imprinting pattern of MAGEL2 (as well as other genes at the PWS locus) may be regulated during development by trans effect on the maternal allele. Clinical expression of PWS syndrome might depend on the temporal or spatial variation in expression of the maternal MAGEL2 allele during development ?

Altogether, these data strongly suggest that MAGEL2 is a PWS-determining gene In the absence of paternal deletion of 15q11-q13 or maternal uniparental disomy 15, search for intragenic mutations on the paternal allele of MAGEL2 should be proposed Fetuses with reduced movements, polyhydramnios and distal arthrogryposis, Newborns with severe undiagnosed central hypotonia Children when PWS is suspected clinically Mejlachowicz et al CONCLUSION AND PERSPECTIVE

UMR-1169 Inserm et Université Paris Saclay Dan Mejlachowicz Flora Nolent Jérome Maluenda Judith Melki Hanitra Ranjatoelina-Randrianaivo, CHU St- Pierre-La Réunion Fabienne Giuliano, CHU Nice Alexandra Benachi et Jelena Martinovic, CHU Antoine-Béclère Ivo Gut, CNAG, Barcelona, Spain Damien Sternberg, CHU Pitié-Salpétrière Annie Laquerrière, CHU Rouen ACKNOWLEDGEMENTS PHRC, AFM, Inserm COLLABORATION