Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy

Abstract

The congenital nemaline myopathies are rare hereditary muscle disorders characterized by the presence in the muscle fibers of nemaline bodies consisting of proteins derived from the Z disc and thin filament. In a single large Australian family with an autosomal dominant form of nemaline myopathy, the disease is caused by a mutation in the alpha-tropomyosin gene TPM3. The typical form of nemaline myopathy is inherited as an autosomal recessive trait, the locus of which we previously assigned to chromosome 2q21.2-q22. We show here that mutations in the nebulin gene located within this region are associated with the disease. The nebulin protein is a giant protein found in the thin filaments of striated muscle. A variety of nebulin isoforms are thought to contribute to the molecular diversity of Z discs. We have studied the 3' end of the 20. 8-kb cDNA encoding the Z disc part of the 800-kDa protein and describe six disease-associated mutations in patients from five families of different ethnic origins. In two families with consanguineous parents, the patients were homozygous for point mutations. In one family with nonconsanguineous parents, the affected siblings were compound heterozygotes for two different mutations, and in two further families with one detected mutation each, haplotypes are compatible with compound heterozygosity. Immunofluorescence studies with antibodies specific to the C-terminal region of nebulin indicate that the mutations may cause protein truncation possibly associated with loss of fiber-type diversity, which may be relevant to disease pathogenesis.

Figure 1

Overview of the layout of actin, tropomyosin, titin, and nebulin filaments in Z lines from vertebrate striated muscles. Titin: The N-terminal region spans the Z line, and, therefore, titin filaments from opposite sarcomeres fully overlap within the Z lines (12). Within the Z line, titin filaments connect to α-actinin Z filaments at multiple sites, which then cross-link the titin and thin filaments (–15). Nebulin:Nebulin filaments appear to insert about 30 nm away from the center of the Z line (10). Triangles on the left indicate the location of the six identified mutations (Figs. 2 and 3). On the right, arrows indicate the location of the epitopes to which antibodies were raised for immunofluorescence studies (Fig. 4).

Figure 2

Genomic organization of the 3′ end of NEB, corresponding to the repeat domains M163–M185, the serine-rich domain with potential RS-phosphorylation motifs, and the SH3-domain of the protein. The exons are shown as boxes numbered from 153a to 187, and the introns are shown as lines. Exons 177b, 177c, 177d, and exon 178b are exons that are not present in the published nebulin sequence (accession no. X83957) but identified in cDNA from one patient with nemaline myopathy. The C-terminal protein structure from the end of the super-repeat region (S21R1–S22R7) to the SH3 domain is shown below the exon/intron organization. The mutation sites are indicated by arrows. The alternatively spliced region of nebulin is shown as gray exons and protein domains.

Figure 3

Haplotypes of families 1 and 2 in the 4-centimorgan region on chromosome 2q21.2-q22 harboring the nebulin gene (7). Black symbols indicate affected persons and white symbols indicate unaffected persons.

Figure 4

RT-PCR analysis of the region spanning nebulin exons 162, 163, and 164 in the patient from family 4. The center lane shows that mRNA from the patient gave two abnormally spliced products, about 190 and 400 bp, respectively. Sequencing of the fragments revealed absence of exon 163 in the smaller fragment, and the larger fragment contained exon 163, as well as 100 bp of intron 163. Right lane, the expected 293-bp product from control mRNA; left lane, size marker φX174/HaeIII.

Figure 5

Cryostat sections from a control (a and b), patient 4 (c and d), and patient 3 (e and f) immunolabeled with antibodies to the SH3 (a, c, and e) and M176–M181 (b, d, and f) domains of nebulin. Note the striation pattern with both antibodies in the control (a and b), and the fiber-typing effect (enhanced in slow fibers) with the M176–M181 antibody in the control and patient 4 (b and d). The difference in fiber types with the SH3 antibody in patient 3 (e) does not relate to a specific fiber type, but note that some fibers appear to be negative. (Bars = 50 μm.)

Figure 6

Immunolabeling of muscle from patient 5 showing absence of labeling with the SH3 antibody in longitudinal and transverse orientation (a) but positive labeling with the M176–M181 antibody (b). Labeling is enhanced in the small fibers expressing slow myosin (c), but the larger fibers are not completely negative and show uneven labeling (arrow). (Bar = 50 μm.)