Nebulin Gene Research Articles

Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy.

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.

Characterization of nebulette and nebulin and emerging concepts of their roles for vertebrate Z-discs

Nebulin is an 800 kDa large actin-binding protein specific to skeletal muscle and thought to act as a molecular template that regulates the length of thin filaments. Recently, a 100 kDa nebulin-like protein has been described in the avian cardiac muscle and referred to as nebulette. We have determined the full-length (8 kb) cDNA sequence of the human nebulette. Its open reading frame (3044 bp) encodes a 109 kDa protein that shares extensive similarity with the C-terminal region of human nebulin. The C-terminal regions of nebulin and nebulette are identical in domain organization and share a family of highly related C-terminal repeats, a serine-rich domain with potential phosphorylation sites, and an SH3 domain. Immunoelectron-microscopy suggests that the C-terminal 30 kDa of nebulin and nebulette filaments integrate into the Z-disc lattice, whereas their N termini appear to project into the I-band. Gene mapping studies assign the human nebulette gene to chromosome 10p12, whereas the nebulin gene has been previously assigned to 2q21. Evolutionary constraints appear to have maintained identical modular arrangements in these two independent genes. Comparison of nebulin and nebulette cDNAs demonstrates that a subgroup of repeats within the C-terminal regions is regulated tissue-specifically and stage-dependently during development of both molecules. This leads to a substantial diversity of nebulin and nebulette isoforms. Their further study is likely to provide insights into how they contribute to the molecular diversity of Z-discs from different muscle tissues and fiber types.

Assignment of the human nebulin gene (NEB) to chromosome band 2q24.2 and the alpha 1 (III) collagen gene (COL3A1) to chromosome band 2q32.2 by in situ hybridization; the FRA2G common fragile site lies between the two genes in the 2q31 band.

Assignment of the human nebulin gene (NEB) to chromosome band 2q24.2 and the alpha 1 (III) collagen gene (COL3A1) to chromosome band 2q32.2 by in situ hybridization; the FRA2G common fragile site lies between the two genes in the 2q31 band.

Molecular diversity of myofibrillar proteins: gene regulation and functional significance.

Myofibrillar proteins exist as multiple isoforms that derive from multigene (isogene) families. Additional isoforms, including products of tropomyosin, myosin light chain 1 fast, troponin T, titin, and nebulin genes, can be generated from the same gene through alternative splicing or use of alternative promoters. Myofibrillar protein isogenes are differentially expressed in various muscle types and fiber types but can be coexpressed within the same fiber. Isogenes are regulated by transcriptional and posttranscriptional mechanisms; however, specific regulatory sequences and transcriptional factors have not yet been identified. The pattern of isogene expression varies during muscle development in relation to the different origin of myogenic cells and primary/secondary fiber generations and is affected by neural and hormonal influences. The variable expression of myofibrillar protein isoforms is a major determinant of the contractile properties of skeletal muscle fibers. The diversity among isomyosins is related to the differences in the parameters of chemomechanical transduction as ATP hydrolysis rate and shortening velocity. Troponin and tropomyosin isoforms determine the variable sensitivity to calcium, whereas titin isoforms dictate the elastic properties of muscle fibers at rest. Both myosin and troponin isoforms contribute to the differences in the resistance to fatigue of muscle fibers.

Chromosomal localization of the mouse titin gene and its relationto “muscular dystrophy with myositis” and nebulin genes on chromosome 2

In the mouse, the genes for the structural components of the myofibril titin and nebulin, Ttn and Neb, map to proximal Chr 2, as does the gene for a muscle disease, “muscular dystrophy with myositis,” mdm. To facilitate the evaluation of Ttn and Neb as possible candidates for mdm, we have determined their relative map positions, using a Mus spretus/Mus musculus interspecific backcross. The gene order (distances in cM) cen-Vim-16.9 ± 4.7-Neb-7.6 ± 3.0-Ttn, Acra-18.0 ± 4.9-Pax-6-17.7 ± 4.9-ahas been determined. Considering the standard deviations, Neb, Ttn, and Acra could colocalize with mdm. Using Ttn and Neb probes, DNAs from mdm/mdm and mdm/+ mice were tested for restriction fragment variants in comparison to the M. musculus wildtype. No variants have been found with 11 restriction nucleases. Our data corroborate a conserved synteny comprising genes NEB, TTN, CHRNA1 on human Chr 2q.

Cloning and expression of human nebulin cDNAs and assignment of the gene to chromosome 2q31-q32

We have isolated two nonoverlapping cNDAs encoding human nebulin, a muscle-specific protein. Northern hybridization analysis shows that nebulin is encoded by a huge message at least 25 kb in length. By hybridizing two nonoverlapping cDNAs to DNA isolated from rodent × human cell hybrids, we assign this presumably single-copy gene to human chromosome 2; sublocalization studies indicate that the nebulin gene is on the long arm of the chromosome, in the region 2q31-q32.