Novel missense mutations and unexpected multiple changes of RYR1 gene in 75 malignant hyperthermia families.

Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder of skeletal muscle characterized by disturbance of intracellular calcium homeostasis in the sarcoplasmic reticulum. Mutations of the ryanodine receptor 1 (RYR1) gene account for most cases, with some studies claiming up to 86% of mutations in this locus. However, RYR1 gene is large and variants are common even in the normal population. We examined 54 families with MH susceptibility and 21 diagnosed with equivocal MH. Thirty-five were selected for an anesthetic reaction, whereas the remainder for hyperCKemia. In these, we studied all 106 exons of the RYR1 gene. When no mutation was found, we also screened: sodium channel voltage-gated, type IV alpha subunit (SCN4A), calcium channel voltage-dependent, L type, alpha 1S subunit (CACNA1S), and L-type voltage-gated calcium channel alpha 2/delta-subunit (CACNL2A). Twenty-nine different RYR1 mutations were discovered in 40 families. Three other MH genes were tested in negative cases. Fourteen RYR1 amino acid changes were novel, of which 12 were located outside the mutational 'hot spots'. In two families, the known mutation p.R3903Q was also observed in malignant hyperthermia-nonsusceptible (MHN) individuals. Unexpectedly, four changes were also found in the same family and two in another. Our study confirms that MH is genetically heterogeneous and that a consistent number of cases are not due to RYR1 mutations. The discordance between in vitro contracture test status and the presence of a proven causative RYR1 mutation suggests that the penetrance may vary due to as yet unknown factors.

Frequency of LGMD gene mutations in Italian patients with distinct clinical phenotypes.

BACKGROUND: The frequency of various limb-girdle muscular dystrophy (LGMD) molecular diagnoses has previously been investigated only in cohorts of patients presenting LGMD phenotype. METHODS: A total of 550 muscle biopsies underwent multiple protein screening (including calpain-3 functional assay) and extensive gene mutation analysis to examine the frequency of LGMD subtypes in patients with distinct clinical phenotypes (severe childhood-onset LGMD, adult-onset LGMD, distoproximal myopathy, and asymptomatic hyperCKemia). RESULTS: The percentage of molecularly ascertained cases directly relates with the degree of clinical involvement: 60% of total LGMD (77% of childhood-onset, 46% of adult-onset, 66% of distoproximal myopathy) and 14% of hyperCKemia. The higher number of molecular diagnoses in severe phenotypes might suggest that genes selected for our screening are those more frequently associated with severe LGMD, and that the hyperCKemia group includes heterogeneous diagnoses. The probability of obtaining a molecular diagnosis increases when a protein defect is found in a muscle biopsy: in such cases, we diagnosed 87% of LGMD and 76% of hyperCKemia. CONCLUSIONS: Diagnosing 77% of childhood-onset limb-girdle muscular dystrophy (LGMD) and 60% of total LGMD is an important result. The missing identification of gene mutations in about 40% of patients with typical LGMD phenotype suggests that unknown genetic or nongenetic etiologies are still to be recognized. Dysferlin, caveolin-3, and emerin protein defects invariably corresponded to primary disorders (100%), whereas a lower correlation was found for sarcoglycans (77%) and calpain-3 (84%). The different efficiency of genetic diagnosis after the identification of a protein defect in the various disorders is possibly due to different pathogenetic effects of mutations.

Early onset calpainopathy with normal non-functional calpain 3 level.

Limb girdle muscular dystrophy 2A (LGMD2A), caused by calpain 3 deficiency, is currently diagnosed through the immunodetection of muscle protein by Western blot (WB) analysis . However, WB may provide normal results in patients with LGMD2A. The case of a female (3y 6mo of age) is described. She was found to be affected by asymptomatic hypercreatine-kinaesaemia during routine biochemical analysis at 10 months of age and had developed myopathic signs at the last neurological assessment. The WB of muscle biopsy performed at 28 months of age showed a normal quantity and pattern of bands for calpain 3. Despite this finding, on molecular analysis she was found to be a compound heterozygote for two mutations of the calpain 3 (CAPN3) gene (R110X and G222R). Autocatalytic activity assay showed a loss of function of calpain 3. This is the first genetically confirmed case of very early onset calpainopathy with a normal amount of protein at WB. Molecular analysis is also suggested in very young patients with normal WB.