AN IMPROVED BIOCHEMICAL-GENETIC APPROACH IN EARLY DIAGNOSIS OF LGMD2A: NEW FUNCTIONAL PROTEIN TEST AND RAPID GENE MUTATION ANALYSIS

The strategies so far employed for the diagnosis of limb-girdle muscular dystrophy type 2A (LGMD2A) are the screening of calpain-3 protein deficiency or gene mutations in patients selected on their LGMD phenotype. Due to the phenotype variability and the incomplete sensitivity of methods so far used for protein and mutation analysis, many patients remain undiagnosed with serious clinical and genetic consequences. To obtain a higher ascertainment rate, we propose an improved diagnostic approach based on: wider inclusion criteria of patients' phenotype; new biochemical method to test calpain autocatalytic activity in cases with normal protein expression; gene mutation screening by D-HPLC, which is a rapid, fully-automated, low-cost and very high sensitive method. In 230 selected patients, previously excluded from known forms of muscular dystrophies, the conventional calpain-3 immunoblot have identified 74 protein-deficient patients. In 45 of 230 patients we identified gene mutations by a preliminary allele-specific mutation analysis. As LGMD2A is considered to affect about 30-50% of all LGMD patients, we plan to extend our screening to the remaining cases by the new test we developed to detect the abnormal calpain-3 activity despite its expression is normal. Our previous experience showed that mutant patients with normal protein expression but abnormal enzyme activity are 20% of all LGMD2A so far diagnosed, thus suggesting the diagnostic use of this test to increase detection rate. Moreover, we will search calpain-3 gene mutations by D-HPLC analysis in all patients, independently of their protein expression. This project offers the opportunity to many patients to receive a LGMD2A molecular diagnosis. This issue has important implications on clinical level (early diagnosis, prevention of clinical complications, future trials with drugs or gene therapy) and genetic counselling (possibility to offer heterozygote and prenatal diagnoses).