Person:

Kunkel, Louis

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.

Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2). Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2cl501/cl501, exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8–15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.

Background: Desmuslin is an α-dystrobrevin-interacting protein expressed primarily in heart and skeletal muscle. The desmuslin protein interacts with and is closely related to desmin, a protein encoded by a locus mutated in some forms of hereditary distal myopathy. As a muscle-specific intermediate filament protein, desmuslin is also a candidate for myopathies of unknown etiology. Results: The desmuslin gene was localized to chromosome 15q26.3 by electronic screening of the human DNA sequence database. Primer pairs were designed to amplify the 5 exons of the desmuslin gene in 11 overlapping DNA segments. The desmuslin gene was screened for mutations in 71 patients with various forms of myopathy for which there was no known cause. In this analysis, 10 common and 2 rare amino acid altering single-nucleotide polymorphisms were identified, all of which were seen in a control population of individuals thus making these unlikely causes of the phenotype. Interestingly, one of the single-nucleotide polymorphisms found in a patient resulted in a premature stop codon in the first exon. The nonsense mutation was also detected in the patient's unaffected father and one unaffected control; it was detected in 0.44% (2/454) of unrelated chromosomes and is therefore predicted to have a homozygous frequency of 0.002%. Conclusion: No causative mutations were found in the desmuslin gene. However, the single-nucleotide polymorphisms mapped in this study represent a well-mapped group that can be used for disequilibrium studies of this region of chromosome 15q26.3.

Background: One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results: An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper. Conclusion: This automated process allows laboratories to discover DNA variations in a short time and at low cost.

Background: The development of large-scale gene expression profiling technologies is rapidly changing the norms of biological investigation. But the rapid pace of change itself presents challenges. Commercial microarrays are regularly modified to incorporate new genes and improved target sequences. Although the ability to compare datasets across generations is crucial for any long-term research project, to date no means to allow such comparisons have been developed. In this study the reproducibility of gene expression levels across two generations of Affymetrix GeneChips® (HuGeneFL and HG-U95A) was measured. Results: Correlation coefficients were computed for gene expression values across chip generations based on different measures of similarity. Comparing the absolute calls assigned to the individual probe sets across the generations found them to be largely unchanged. Conclusion: We show that experimental replicates are highly reproducible, but that reproducibility across generations depends on the degree of similarity of the probe sets and the expression level of the corresponding transcript.