Mutations in the satellite cell gene MEGF10 cause a recessive congenital myopathy with minicores

Abstract

We ascertained a nuclear family in which three of four siblings were affected with an unclassified autosomal recessive myopathy characterized by severe weakness, respiratory impairment, scoliosis, joint contractures, and an unusual combination of dystrophic and myopathic features on muscle biopsy. Whole genome sequence from one affected subject was filtered using linkage data and variant databases. A single gene, MEGF10, contained nonsynonymous mutations that co-segregated with the phenotype. Affected subjects were compound heterozygous for missense mutations c.976T > C (p.C326R) and c.2320T > C (p.C774R). Screening the MEGF10 open reading frame in 190 patients with genetically unexplained myopathies revealed a heterozygous mutation, c.211C > T (p.R71W), in one additional subject with a similar clinical and histological presentation as the discovery family. All three mutations were absent from at least 645 genotyped unaffected control subjects. MEGF10 contains 17 atypical epidermal growth factor-like domains, each of which contains eight cysteine residues that likely form disulfide bonds. Both the p.C326R and p.C774R mutations alter one of these residues, which are completely conserved in vertebrates. Previous work showed that murine Megf10 is required for preserving the undifferentiated, proliferative potential of satellite cells, myogenic precursors that regenerate skeletal muscle in response to injury or disease. Here, knockdown of megf10 in zebrafish by four different morpholinos resulted in abnormal phenotypes including unhatched eggs, curved tails, impaired motility, and disorganized muscle tissue, corroborating the pathogenicity of the human mutations. Our data establish the importance of MEGF10 in human skeletal muscle and suggest satellite cell dysfunction as a novel myopathic mechanism.

Fig. 1

Clinical symptoms of an unclassified autosomal recessive congenital myopathy. a Three siblings from a non-consanguineous nuclear family (1030) presented with childhood weakness, respiratory problems, and scoliosis. b The arm in subject 1030-1 illustrated elbow contracture to approximately 20°. Biceps and triceps strength in subject 1030-1 were 3+/5 (Medical Research Council scale). c The necks of subjects 1030-1 and d 1030-6 showed neck atrophy and contractures, more prominent in subject 1030-6. Neck flexion strength was 2/5 in 1030-1 and 0/5 in 1030-6. Neck extension strength was normal in both subjects

Fig. 2

Dystrophic and myopathic pathology in family 1030 muscle biopsy. a Hematoxylin and eosin (H&E) staining of sectioned muscle biopsy tissue taken from the left biceps of female subject 1030-1 at age 8 years showed rounded fibers with markedly excessive variation in fiber diameter, fatty replacement, and increased internalized nuclei. Some of the large, hypertrophic fibers were 150 μm in diameter. Inflammation and necrosis were not observed, but scattered basophilic regenerating fibers were present. Magnification is ×10; scale bar is 200 μm. b Modified Gomori trichrome staining of 1030-1 biceps highlighted the increased endomysial connective tissue, as well as a single focus of myonecrosis (arrow). Magnification is ×20; scale bar is 100 μm. c NADH reductase histochemistry of 1030-1 biceps showed many fibers with scattered clearings or “moth-eaten” appearance, reflecting disruption of the myofibril by minicores (arrows), predominantly in the darker type I fibers. Magnification is ×40; scale bar is 50 μm. d H&E staining of muscle biopsy tissue taken from the right thigh of female subject 1030-4 at age 9 years showed clusters of polygonal myofibers with abundant fatty replacement. There was excessive variation in fiber diameter with frequent hypertrophic fibers, 75–95 μm in diameter. Degeneration, necrosis, or basophilic regenerating fibers were not seen. Magnification is ×20; scale bar is 100 μm

Fig. 3

Morphological and birefringence abnormalities in megf10 morphant zebrafish. a Zebrafish treated with anti-megf10 morpholinos were more likely to die or exhibit an abnormal phenotype than control fish. Abnormal phenotypes were defined as bent or severely curled tails, unhatched eggs, or impaired swimming, including unproductive quivering, rotating in circles, or reduced motility in response to touch. Total numbers of fish examined in each category are given in parentheses after the treatment group label. The remainder of fish in each category appeared unaffected or had non-specific phenotypes, which included four COMO-treated fish (one at each dose) with a slightly curled tail, inflated yolk sac, or bloated appearance, and six fish treated with 6 ng of MO3 that had inflated precardia. b Rows from top to bottom are wild-type, 6 ng COMO, 6 ng MO1, and 6 ng MO2. Columns from left to right are brightfield ×5 magnification, birefringence ×5 magnification, brightfield ×11.25 magnification, and birefringence ×11.25 magnification. Compared to control fish, MO1-treated fish showed decreased birefringence, and MO2-treated fish had marked disruption of somite boundaries, a thinner tail with less muscle tissue overall, and substantially decreased birefringence

Fig. 4

Myopathic features of megf10 morphant zebrafish muscle. Immunohistochemistry was performed with antibodies against β-dystroglycan (left) and myosin heavy chain (right) on wild-type fish and morphants treated with 6 ng COMO, 6 ng MO1, and 6 ng MO2 (top to bottom). Wild-type and COMO-treated fish showed myosepta with straight boundaries and tightly packed myofibers. Compared to control fish, MO1-treated fish showed subtle curving of myosepta and loosely packed myofibers with occasional prominent gaps (arrow), and MO2-treated fish showed less distinct somite boundaries and abundant gaps between myofibers (e.g., in somites indicated by arrows), creating an overall disorganization of the fiber pattern