Identification of a Dutch founder mutation in MUSK causing fetal akinesia deformation sequence

Abstract

Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement. FADS can result from mutations in CHRNG, CHRNA1, CHRND, DOK7 and RAPSN; however, these genes only account for a minority of cases. Here we identify MUSK as a novel cause of lethal FADS. Fourteen affected fetuses from a Dutch genetic isolate were traced back to common ancestors 11 generations ago. Homozygosity mapping in two fetuses revealed MUSK as a candidate gene. All tested cases carried an identical homozygous variant c.1724T>C; p.(Ile575Thr) in the intracellular domain of MUSK. The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers. Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings. A functional assay in myocytes derived from human fetuses confirmed that the variant blocks MUSK-dependent motor endplate formation. Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing.

Figure 1

Co-segregation of a variant in MUSK with lethal FADS in a Dutch genetic isolate. (a) Schematic illustrating the common ancestry of 14 affected fetuses from 11 families. (b) Sanger sequencing chromatograms of fetal (top) and parental (bottom) DNA samples identifying the variant NM_005592.3: c.1724T>C; p.(Ile575Thr) and multispecies alignment of the affected residue (right). (c) Domain structure of MUSK depicting the extracellular domain containing IgG-like domains, a Frizzled domain (FZ, also named C6 box), a transmembrane domain (TM) and a cytoplasmic domain that contains a juxtamembrane (JM)/PTB-domain and the tyrosine kinase domain. Ile575 is located at the boundary between the JM and kinase domains, very close to the Dok7-interacting region and the autophosphorylation site Tyr554. Other mutations previously reported to cause CMS are also indicated. Mutations found in compound heterozygotes are color-matched^{, , , , ,} . All amino-acid numbers refer to MUSK isoform 1 (GenPept accession NP_005583.1).

Figure 2

Macroscopic appearance of four fetuses homozygous for the c.1724T>C; p.(Ile575Thr) variant in MUSK. (a) Case 6, stillborn at a gestational age of 33 weeks; (b) case 8, termination of pregnancy at 23 weeks; (c) case 10, died immediately after birth at 32 weeks; (d) case 11, termination of pregnancy at 22 weeks. Note the multiple contractures, facial appearance of hypertelorism and micro-retrognathia, club feet (b–d), oligodactyly of the right foot (c), plantar flexion of the feet (a). For details see Table 1. Bars indicate 5 cm.

Figure 3

Histopathological findings indicative for defects in neuromuscular synaptogenesis. (a) Skeletal muscle biopsies showed small and rounded atrophic fibers and an increased number of intracellular nuclei (arrowheads). (b and c) Immunostaining of type I (b) and type II fibers (c) indicated a relative loss of type I fibers. (d–g) Combined staining for nerve terminals (synaptotagmin-1) and AChR clusters (α-bungarotoxin-rhodamine) in quadriceps muscle indicated fewer and smaller neuromuscular junctions in case 8 (arrowheads in d, e; 23 weeks of gestation) compared with a control fetus (f, g; 22 weeks). (h–k) Combined staining for nerve terminals, AChR clusters and anti-phosphotyrosine (pTyr) monoclonal 4G10 in diaphragm indicated fewer clusters (arrowheads) and a loss of tyrosine kinase activity in MUSK^I575T/I575T endplates (h, i) compared with control tissue (j, k). Note that residual tyrosine kinase activity was detected in some mutant endplates (arrow), which may also result from unrelated tyrosine kinases.

Figure 4

Wild-type but not mutant MUSK augments AChR clustering in MUSK^I575T myocytes. (a) Primary myocytes derived from an affected fetus homozygous for MUSK^I575T were plated on Matrigel-coated coverslips and transduced with lentiviral vectors encoding either MUSK^WT or MUSK^I575T. As a control, non-transduced MUSK^I575T cells were used. Myocytes were treated with neuronal Agrin(3,4,8) and stained for nuclei (DAPI, displayed in green) and AChR clusters (Rhodamine-bungarotoxin, red). Representative examples from each experimental group are shown. (b) Mean±SEM values for the AChR cluster density, quantitated from 35 images from three coverslips of each group. ***P<0.001 (Student's t-test). (c) Immunoblot analysis of Flag-tagged MUSK^WT and MUSK^I575T constructs expressed in HEK293 cell lysates, suggesting similar yield and molecular mass.