Molecular studies in Duchenne/Becker muscular dystrophies of Argentine patients.

FLORENCIA GILIBERTO,; LEONELA LUCE ; IRENE SJIJAN

Duchenne Muscular Dystrophy:Symptoms, Managment and Prognosis.

NOVA publishers. NOVA SCIENSE PUBLISHERS, INC

Lugar: New York 11788-3619, USA. ; Año: 2015;

Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked diseases caused by mutations in the dystrophin gene. These dystrophinopathies affect males, being females asymptomatic carriers. However, some females manifest symptoms mainly due to inactivation of their normal X-chromosome. Mutations in the dystrophin gene include gross deletions (65%), duplications (5%) and small mutations (30%). Several dystrophin isoforms are expressed in different tissues and its absence in Central Nervous System (CNS) is related with cognitive impairment. There is a correlation between genotype and phenotype in which frameshift mutations result in DMD while in-frame mutations originate a milder phenotype, BMD. Thus, molecular characterization of mutations is crucial to predict the clinical course in patients. Owing to the lack of cure, it is important to prevent the birth of affected boys providing carrier-status assessment. The main approaches for molecular diagnosis of dystrophinopathies are: 1) Detection and characterization of mutations; 2) Genoptype/Phenotype correlation; 3) Carrier status assessment; 4) Prenatal diagnosis and; 5) Genetic analysis of symptomatic females. The methodology implemented to carry out these diagnostic approaches was: - Multiplex and simplex PCR of deletion-prone regions; - Multiplex ligation-dependent probe amplification (MLPA); - Segregation analysis of up to 11 short tandem repeat [STR-(CA)n] loci; - AR (human androgen receptor gene) assay for X-chromosome inactivation pattern. The methodology used allowed to detect all the deletions and duplication that account for 2/3 of the molecular alterations described in affected patients, and predict the frameshift correlation between genotype and phenotype. Deletions in the central and 3´ regions of the gene correlated with cognitive impairment. The study of a BMD family revealed several unexpected asymptomatic males with an in-frame deletion of exons 45-55. Segregation analysis allowed the identification of the at-risk haplotype and estimation of the probability of being carrier of the mutation in most of the analyzed individuals. STRs studies were able to detect germinal mosaicism, de novo mutations, gross deletions, meiotic recombination events, changes in allele length and chorionic villus maternal contamination. Most of the prenatal cases were diagnosed with certainties up to 95%. The genetic analysis of symptomatic female carriers with mild muscular weakness, increased serum creatin kinase and low levels of muscular dystrophin, showed a skewed X inactivation pattern. In conclusion, the results found in the BMD family with exons 45?55 deletion, supports previous predictions that are the optimal multiexon skipping target in antisense gene therapy to transform the severe Duchenne muscular dystrophy into the milder BMD, or even asymptomatic, phenotype. The skewed inactivation pattern observed in symptomatic females unravels the reason of disease manifestation. Finally, the molecular methodology used proved to be efficient for characterizing the disease-causing mutation in affected individuals and for assessing the carrier status in healthy relatives. These findings helped inform precise genetic counseling and contributed to characterization of the disease in the Argentine population.