Using the DMN cDNA sequence (AF359284) as a query to blast the public human genome database, we found a genomic clone (AC018999) that included the entire desmuslin coding sequence. The alignment identified 5 exons which are schematically indicated in Figure . Eleven sets of primer pairs were developed to amplify the 5 exons and the flanking intron sequences (Table ). The primers were designed to amplify overlapping segments of the desmuslin coding sequence as indicated in Figure .
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 (more ...)
Eleven PCR primer sets that allow the amplification of the entire coding sequence and surrounding intronic sequence of the DMN gene.
Desmin, a desmuslin-interacting protein, has been implicated in hereditary distal myopathies implying that desmuslin itself may also be involved in myopathies of unknown etiology. DNA samples isolated from 71 dystrophic and myopathic patients (as described in Table ) were analyzed by direct sequencing to determine whether the phenotype was the result of a desmuslin mutation. All observed variants were subsequently tested in ≤ 156 control individuals. These analyses revealed 10 common and 2 rare SNPs [19
] that alter an amino acid codon; 7 result in non-conservative amino acids changes (Table ).
Summary of clinical diagnoses and pathological findings of patient samples studied for alterations in DMN.
Summary of SNPs in theDMN gene.
None of the SNPs seem associated with the patient phenotypes as each was also found in the control population. Nine silent SNPs, which did not alter an amino acid, were also identified in the patient population; several of these SNPs showed a high degree of heterozygosity. Most were tested for and detected in the control population, thus making desmuslin an unlikely candidate to be involved in these myopathies, but this first pass can be followed by others in a larger set of patient samples.
Interestingly, SNP 3, a C598T substitution resulting in a premature stop codon in exon 1, was detected in a single Nemaline myopathy patient. Neither the proband's mother nor any of the other patients was found to carry this mutation (Figure ). The C598T mutation removed a Pvu II site, thus permitting detection of the alteration by restriction digest analysis of a PCR product generated from genomic DNA. The assay is outlined schematically in Figure and documented in Figure . Using this Pvu II assay, the C598T change was detected in the unaffected father of the patient, making it unlikely that heterozygosity for the C598T change is the causative mutation in the myopathic proband.
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 (more ...)
The removal of the Pvu II site allowed rapid screening of additional patients and control samples by DNA amplification of the region and subsequent digestion of the PCR product with Pvu II. One of 312 control chromosomes was found to have lost the Pvu II site, and the heterozygous presence of the C598T mutation was confirmed by DNA sequencing of the PCR product. Thus it is likely that the C598T mutation is not responsible for the myopathic phenotype of the proband. This is a surprising result considering that a single copy of this stop codon is present in 0.44% (2/454) of the population and 0.002% of the population is predicted to carry this nonsense mutation in a homozygous state. The phenotype of the latter individuals is not known.
There is the possibility that either the heterozygous C598T DMN mutation combined with another mutation in a different gene or the C598T in a homozygous state causes muscular dystrophy or myopathy of unknown genetic pathogenesis. The C598T DMN mutation is a good candidate to be a modifier for muscular and/or cardiac phenotypes, considering: (i) the frequency of the mutation in the normal population in the heterozygous as well as homozygous state; and (ii) the key role of desmuslin in connecting the Z-discs to the extracellular matrix. The desmuslin SNPs should be examined in a larger set of myopathy and muscular dystrophy patients to test this hypothesis. More specifically, it should be determined whether there is a statistically significant difference in the frequency of individual or clustered SNP haplotypes between the patient and the control populations.
The desmuslin coding sequence is highly polymorphic with 9 SNPs that do not alter an amino acid and 12 that do. Ten of these variations have minor allele frequencies that are represented in over 10% of the chromosomes sampled. These 10 are spaced within a genomic interval of ≤ 28 kilobases, or approximately 1 per 2800 bases. These highly informative markers are thus very useful for genetic analysis. The presence of so many SNPs within the coding sequence of a protein is somewhat unexpected and is much greater than that documented for other genes. This degree of variability may reflect a redundancy of desmuslin at the Z-line where other IF proteins with potentially overlapping functions are located [21