Autosomal-dominant distal myopathy associated with a recurrent missense mutation in the gene encoding the nuclear matrix protein, matrin 3

Abstract

Distal myopathies represent a heterogeneous group of inherited skeletal muscle disorders. One type of adult-onset, progressive autosomal-dominant distal myopathy, frequently associated with dysphagia and dysphonia (vocal cord and pharyngeal weakness with distal myopathy [VCPDM]), has been mapped to chromosome 5q31 in a North American pedigree. Here, we report the identification of a second large VCPDM family of Bulgarian descent and fine mapping of the critical interval. Sequencing of positional candidate genes revealed precisely the same nonconservative S85C missense mutation affecting an interspecies conserved residue in the MATR3 gene in both families. MATR3 is expressed in skeletal muscle and encodes matrin 3, a component of the nuclear matrix, which is a proteinaceous network that extends throughout the nucleus. Different disease related haplotype signatures in the two families provided evidence that two independent mutational events at the same position in MATR3 cause VCPDM. Our data establish proof of principle that the nuclear matrix is crucial for normal skeletal muscle structure and function and put VCPDM on the growing list of monogenic disorders associated with the nuclear proteome.

Figure 1

Refinement of the VCPDM Interval in the North American VCPDM Family A subset of markers used in this study is given in centromeric to telomeric orientation. The physical map positions are according to the UCSC March 2006 freeze. Patient V.34 carries the full-blown disease haplotype. Recombination events in patient V.37 and patient DM73.3 (newly identified patient from the North American family) excluded markers sara2AC and AC008667C (printed in red), respectively, delimiting a 5.37 Mb region.

Figure 2

Pedigree of the Bulgarian VCPDM Family Individuals of whom DNA samples were available are marked by asterisks.

Figure 3

Identification of the VCPDM Mutation (A) DNA sequence traces illustrating the c.254C→G, S85C mutation in the MATR3 gene (upper panel) and the corresponding wild-type sequence (lower panel). The position of the single nucleotide exchange is marked with an arrowhead. (B) Comparison of disease-associated haplotypes. The disease-associated haplotype in the North American family is shown under the column heading “NA”; the disease-associated haplotype in the Bulgarian pedigree is shown under the heading “BG.” The phases of alleles have been determined for all markers. Intragenic markers and the mutation in exon 2 of the MATR3 gene are boxed. The closest flanking and intragenic markers that show different alleles for the North American and Bulgarian samples are printed in red. (C) Multiple sequence alignment of human matrin 3 and related sequences. The mutant serine 85 residue is marked by an arrowhead.

Figure 4

Muscle Biopsy Findings in VCPDM The muscle biopsy specimen was taken from the left M. gastrocnemius of a 54-year-old male patient of the Bulgarian family. (A) The muscle parenchyma is largely replaced by fat and connective tissue. The remaining muscle fibers show a high degree of caliber variability. Hypertrophic muscle fibers containing nonsubsarcolemmal nuclei are marked by arrows. A hematoxylin stain was used on the cryostat section. The scale bar represents 250 μm. (B and C) Matrin 3 immunostaining (rabbit anti-matrin 3 antibody BL2526; Bethyl Labs, Montgomery, TX, USA) of cryosectioned muscle fibers showing variable degrees of immunoreactivity of myonuclei (black arrows, immunoreactive nuclei labeled by brown reaction product; white arrows, faintly stained or almost unlabelled myonuclei). Because of the vast proliferation of fat tissue, the cryostat sections had to be cut much thicker than normal, 15 μm, resulting in a blurry appearance of the nuclei. A hematoxylin counterstain was used. The scale bar represents 40 μm in (B) and 25 μm in (C). The inset in (C) shows a muscle fiber with intact sarcolemmal dystrophin immunoreactivity (Dys 1 antibody, Novocastra, Newcastle, UK) indicating preserved overall immunoreactivity of the biopsied tissue. The scale bar represents 50 μm. (D) Normal control muscle immunostained with the matrin 3 antibody: Strong immunoreactivity of all myonuclei. A hematoxylin counterstain was used on the cryostat section. The scale bar represents 50 μm. (E) Semithin section of biopsy tissue that was cut from the block of frozen tissue, thawed, fixed in 6% buffered glutaraldehyde, and embedded in epoxy resin. Two muscle fibers are surrounded by fibrotic connective tissue and fat cells. Dark, osmiophilic material has accumulated in both fibers. The arrow indicates a strongly osmiophilic deposit, most likely autophagic material. A paraphenylene diamine stain was used. The scale bar represents 20 μm. (F and G) Electron microscopy of ultrathin sections of the resin-embedded tissue. In (F), this muscle fiber shows an irregularly shaped nucleus with loose chromatin and numerous small perinuclear autophagic vacuoles containing osmiophilic, often myelin-like material. The scale bar represents 3 μm. In (G), two irregular, preapoptotic myonuclei are shown. One of the nuclei embraces an autophagic vacuole (indicated by an arrow), probably as a result of sarcoplasmic invagination. The scale bar represents 2 μm.