Analysis of Single Nucleotide Polymorphisms (SNPs) of the small-conductance calcium activated potassium channel (SK3) gene as genetic modifier of the cardiac phenotype in myotonic dystrophy type 1 patients.

Myotonic dystrophy type 1 (DM1) is the most frequently inherited neuromuscular disease in adults. It is a multisystemic disorder with major cardiac involvement most commonly represented by first-degree atrioventricular heart block (AVB), followed by different degrees of bundle-branch and intraventricular blocks In search for candidate genes, modifiers of the AVB phenotype in DM1, the expression of the small-conductance calcium activated potassium channel (SK3) gene was analysed in muscle biopsies from DM1 patients. The association between SK3 polymorphisms and the AVB phenotype was then studied analyzing 40 DM1 patients with AVB and 40 age-matched DM1 affected individuals with no ECG abnormalities. [CTG]n repeat length and cardiac clinical picture were also assessed for correlation. QRT-PCR experiments showed an over-expression of the SK3 transcript in DM1 muscle biopsies compared to healthy controls. However, no statistical association between the AVB phenotype and either the [CTG]n expansion length or the presence of specific SNPs in the SK3 gene were detected. These findings suggest that modifier genes, other than SK3, should be identified in order to explain the cardiac phenotypic variability among DM1 patients.

Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes.

BACKGROUND: The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. OBJECTIVE: To obtain unbiased information on the consequences of CAPN3 mutations. PATIENTS: 530 subjects with different grades of symptoms and 300 controls. METHODS: High throughput denaturing HPLC analysis of DNA pools. RESULTS: 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. CONCLUSIONS: A non-invasive and cost-effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103.

Molecular diagnosis in LGMD2A: mutation analysis or protein testing?

Limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) is caused by mutations in the CAPN3 gene encoding for calpain-3, a muscle specific protease. While a large number of CAPN3 gene mutations have already been described in calpainopathy patients, the diagnosis has recently shifted from molecular genetics towards biochemical assay of defective protein. However, an estimate of sensitivity and specificity of protein analysis remains to be established. Thus, we first correlated protein and molecular data in our large LGMD2A patient population. By a preliminary immunoblot screening for calpain-3 protein of 548 unclassified patients with various phenotypes (LGMD, myopathy, or elevated levels of serum creatine kinase [hyperCKemia]), we selected 208 cases for CAPN3 gene mutation analysis: 69 had protein deficiency and 139 had normal expression. Mutation search was conducted using SSCP, denaturing high performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS-PCR), and direct sequencing methods. We identified 58 LGMD2A mutant patients: 46 (80%) had a variable degree of protein deficiency and 12 (20%) had normal amount of calpain-3. We calculated that the probability of having LGMD2A is very high (84%) when patients show a complete calpain-3 deficiency and progressively decreases with the amount of protein; this new data offers an important tool for genetic counseling when only protein data are available. A total of 37 different CAPN3 gene mutations were detected, 10 of which are novel. In our population, 87% of mutant alleles were concentrated in seven exons (exons 1, 4, 5, 8, 10, 11, and 21) and 61% correspond to only eight mutations, indicating the regions where future molecular analysis could be restricted. This study reports the largest collection of LGMD2A patients so far in which both protein and gene mutations were obtained to draw genotype-protein-phenotype correlations and provide insights into a critical protein domain.