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JMG Online First, published on January 22, 2015 as 10.1136/jmedgenet-2014-102730 New loci


MuSK: a new target for lethal fetal akinesia deformation sequence (FADS) Maria Wilbe,1 Sara Ekvall,1 Karin Eurenius,2 Katharina Ericson,1,3 Olivera Casar-Borota,1,3 Joakim Klar,1 Niklas Dahl,1 Adam Ameur,1 Göran Annerén,1 Marie-Louise Bondeson1 ▸ Additional material is published online only. To view please visit the journal online ( jmedgenet-2014-102730). 1

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden 2 Department of Women’s and Children’s Health, Uppsala University, Uppsala, Sweden 3 Department of Pathology and Cytology, Uppsala University Hospital, Uppsala, Sweden Correspondence to Dr Maria Wilbe, Science for Life Laboratory, Department of Immunology, Genetics and Pathology, BMC, Uppsala University, Husargatan 3, Box 815, Uppsala SE-751 08, Sweden; [email protected] Marie-Louise Bondeson, Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala SE-751 85, Sweden; [email protected] Received 22 August 2014 Revised 14 December 2014 Accepted 15 December 2014

ABSTRACT Background Fetal akinesia deformation sequence syndrome (FADS, OMIM 208150) is characterised by decreased fetal movement (fetal akinesia) as well as intrauterine growth restriction, arthrogryposis, and developmental anomalies (eg, cystic hygroma, pulmonary hypoplasia, cleft palate, and cryptorchidism). Mutations in components of the acetylcholine receptor (AChR) pathway have previously been associated with FADS. Methods and results We report on a family with recurrent fetal loss, where the parents had five affected fetuses/children with FADS and one healthy child. The fetuses displayed no fetal movements from the gestational age of 17 weeks, extended knee joints, flexed hips and elbows, and clenched hands. Whole exome sequencing of one affected fetus and the parents was performed. A novel homozygous frameshift mutation was identified in muscle, skeletal receptor tyrosine kinase (MuSK), c.40dupA, which segregated with FADS in the family. Haplotype analysis revealed a conserved haplotype block suggesting a founder mutation. MuSK (muscle-specific tyrosine kinase receptor), a component of the AChR pathway, is a main regulator of neuromuscular junction formation and maintenance. Missense mutations in MuSK have previously been reported to cause congenital myasthenic syndrome (CMS) associated with AChR deficiency. Conclusions To our knowledge, this is the first report showing that a mutation in MuSK is associated with FADS. The results support previous findings that CMS and/or FADS are caused by complete or severe functional disruption of components located in the AChR pathway. We propose that whereas milder mutations of MuSK will cause a CMS phenotype, a complete loss is lethal and will cause FADS.


To cite: Wilbe M, Ekvall S, Eurenius K, et al. J Med Genet Published Online First: [please include Day Month Year] doi:10.1136/ jmedgenet-2014-102730

Fetal akinesia deformation sequence (FADS) (OMIM 208150), also known as Pena-Shokeir syndrome I or arthrogryposis multiplex congenita (AMC) with pulmonary hypoplasia, is a rare condition associated with malformations caused by reduced fetal movement (fetal akinesia). About 30% of affected individuals are stillborn and many live born infants survive only a short time due to complications of pulmonary hypoplasia. FADS is characterised by arthrogryposis, fetal akinesia, intrauterine growth restriction, developmental abnormalities such as cystic hygroma, pulmonary hypoplasia, cleft palate, cryptorchidism, cardiac defects, intestinal malrotation and

sometimes pterygia of the limbs. It shows phenotypic overlap with the lethal type of multiple pterygium syndrome (LMPS, OMIM 253290).1 FADS is most often inherited as an autosomal recessive trait,2 but X-linked or dominant inheritance have also been suggested.3 4 Several mutations have been described in FADS and/or LMPS, explaining around 24% of the cases, and involving genes in the motor neuron development and survival, genes encoding components of the neuromuscular junction (NMJ), adult skeletal muscle proteins, and fetal myostructural proteins.5 The mutations located in genes associated with the NMJ include the subunits of the acetylcholine receptor (AChR): CHRNA1 (OMIM 100690),6 CHRND (OMIM 100720)6 and CHRNG (OMIM 100730);7 and genes signalling with muscle, skeletal receptor tyrosine kinase (MuSK) in the prepatterning process: RAPSN (OMIM 601592)8 and DOK7 (OMIM 610285).9 Mutations in SYNE1 (OMIM 608441), also involved in the prepatterning process, leads to AMC,10 and mutations in CNTN1 (OMIM 600016), a neural adhesion and NMJ protein, have been described to cause a congenital lethal myopathy11 (figure 1). Other non-lethal diseases involved in the NMJ pathway include congenital myasthenic syndrome (CMS, OMIM 608931) and Escobar syndrome (OMIM 265000). CMS can be classified as presynaptic, synaptic or postsynaptic. Approximately 75% of CMS cases are postsynaptic, the majority of which are caused by AChR deficiency.12 The emergence of high throughput DNA sequencing technologies has made it possible to sequence more rapidly and less expensively the coding parts of a patient’s genome, to enable unbiased searching for novel disease genes and identify potentially damaging mutations. We performed whole exome sequencing (WES) of a family trio, where the parents had five affected fetuses/children with a severe lethal form of FADS and one healthy child. Diagnosis of the fetuses was unknown until identification of the diseasecausing gene was completed. This study reports a novel lethal homozygous insertion in the gene encoding MuSK. The frameshift mutation leads to a premature stop codon and likely a non-functional protein, causing the FADS phenotype.

METHODS Patients The parents were both in their 30s at the time of their first visit to the fetal medicine unit, Uppsala

Wilbe M, et al. J Med Genet 2015;0:1–8. doi:10.1136/jmedgenet-2014-102730

Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.


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New loci Figure 1 Fetal akinesia deformation sequence (FADS) and/or multiple pterygium syndrome (MPS) are associated with genes involved in the neuromuscular junction (NMJ). Several genes (CHRNA1, CHRND, CHRNG, RAPSN, DOK7, CNTN1 and SYNE1) involved in the NMJ have previously been associated with FADS. One additional gene, muscle, skeletal receptor tyrosine kinase (MuSK) (OMIM 601296) was identified that can be used in prenatal testing or genetic analysis for NMJ disorders.

University Hospital, Sweden, in 2003. There was no known consanguinity, but a common ancestor cannot be excluded due to the parents deriving from the same geographical area. At the time, they had one previous pregnancy, which ended in early miscarriage. The pedigree of the affected family in 2014 comprised two miscarriages, five affected children/fetuses, and one healthy child (figure 2A). The pregnancy in 2003 had a normal second trimester scan at their home clinic. In gestational week 23, the woman presented with high symphysis fundal measures of uterus at a standard check up with her midwife. Ultrasound examination revealed polyhydramnion and fetal hydrops and no fetal movements (figure 2B). The following examinations were conducted at the fetal medicine unit, Uppsala University Hospital. Table 1 shows the outcome of the pregnancy in 2003 and four additional pregnancies (2005, 2005, 2007, 2010), with fetuses/children presenting with findings consistent with FADS (figure 2C). All fetuses/children showed akinesia and joint contractions at ultrasound examination, and autopsy confirmed muscle atrophy and multiple joint contractions. In 2008 a healthy boy was born. Genomic DNA was extracted from EDTA blood ( parents), saliva (healthy sibling), and lung tissue (affected fetuses) using standard methods.

Whole-exome sequencing and data analysis DNA from a trio (mother, father and one affected fetus) was used as starting material for exome sequencing. For each sample, 100 ng of DNA was distributed to 12 separate primer pools and amplified according to the Ion AmpliSeq Exome Library Preparation protocol (Revision A.0, Life Technologies). The separate PCR products were pooled and primer sequences were partially digested. Adaptors were ligated and resulting amplicons were purified using Agencourt AMPure XP Reagent (Beckman Coulter) and eluted in amplification mix (Platinum PCR SuperMix High Fidelity and Library Amplification Primer Mix, Life Technologies). Size-selection and purification was conducted using Agencourt AMPure XP Reagent. The amplicons 2

were quantified using the Agilent Bioanalyzer instrument with Agilent High Sensitivity DNA kit. Emulsion PCR was performed on the Ion OneTouch 2 system using Ion PI Template OT2 200 Kit v3 chemistry, followed by enrichment using Ion OneTouch ES. Samples were loaded on an Ion PI chip Kit v2 and sequenced on the Ion Proton System using Ion PI Sequencing 200 Kit v3 (200 bp read length, Life Technologies) chemistry. DNA samples for the mother and the father were barcoded and multiplexed on one PI chip, while the fetal DNA was run on a separate PI chip. The sequencing generated over 30 million reads/sample of average length >150 bp. Alignment of reads to the human hg19 assembly and variant detection was performed using V.4.0 of the Torrent Suite Software. Single nucleotide polymorphism (SNPs) and indel data were stored in a local installation of the CanvasDB in-house database system13 ( CanvasDB) together with variant annotation information obtained from ANNOVAR14 and dbSNP137. R functions available from the CanvasDB system were used to identify potentially damaging variants that followed a recessive inheritance pattern (heterozygous in the parents and homozygous in the fetus or compound heterozygous in the fetus) and not present (

MuSK: a new target for lethal fetal akinesia deformation sequence (FADS).

Fetal akinesia deformation sequence syndrome (FADS, OMIM 208150) is characterised by decreased fetal movement (fetal akinesia) as well as intrauterine...
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