Genomic organization and single-nucleotide polymorphism map of desmuslin, a novel intermediate filament protein on chromosome 15q26.3

Abstract

Background: Desmuslin is an alpha-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.

Figure 1

Schematic representation of the desmuslin gene and protein. Desmuslin protein structure, genomic organization, approximate location of SNPs identified in this study and primer pairs used to amplify the entire coding sequence are indicated. A comparison of the DMN cDNA sequence with its related genomic DNA led to the identification of 5 exons ranging from 71 bp (exon 3) to 2447 bp (exon 4). The exact start of transcription in exon 1 and the termination of transcription at the end of exon 5 are unknown. Unlike the brain DMN cDNA, muscle DMN cDNA does not contain the sequence between exons 4 and 5 (indicated by an open box). The locations of the 21 SNPs are indicated by arrows; the 12 SNPs that alter an amino acid codon are shown in red and the 9 silent SNPs are shown in black. The exact position of each of the SNPs is described in Table 3. The positions of the 11 primer pairs are also shown below the respective exons.

Figure 2

Analysis of SNP3 (C598T) in patient and control samples. (A) Sequence results of the C598T patient and his mother. At nucleotide number 598, the patient has a C and T, indicated by N. The patient's unaffected mother is homozygous for C. (B) Pvu II site amplified with 3F and 3R. The size of PCR product amplified with 3F and 3R is 790 bp and contains three Pvu II restriction sites. When the PCR product from a control subject is digested with Pvu II, four products of 321 bp, two of 138 bp and two of 10 bp are generated. Since the C598T removes the first Pvu II restriction site, digestion of the amplification product will produce a fragment of 459 bp instead of 321 bp and 138 bp. (C) Electrophoresis of PCR product digested with Pvu II shows that the patient and his unaffected father both have a band of 459 bp. The 138 bp fragment in the mother and control is twice as strong as those of the patient and the father. Similarly the 321 bp band in the mother and control is stronger than in the patient and his father.