MLPA identification of dystrophin mutations and in silico evaluation of the predicted protein in dystrophinopathy cases from India

In this study, 415 clinically diagnosed patients were tested for mutations by Multiplex ligation dependent probe amplification (MLPA). Muscle biopsy was performed in 34 patients with negative MLPA. Phenotype-genotype correlation was done using PROVEAN, hydrophobicity and eDystrophin analysis. We have utilized bioinformatics tools in order to evaluate the observed mutations both at the level of primary as well as secondary structure.

Mutations were identified in 75.42% cases, of which there were deletions in 91.6% and duplications in 8.30%. As per the reading frame rule, 84.6% out-of frame and 15.3% in-frame mutations were noted. Exon 50 was the most frequently deleted exon and the exon 45–52 region was the hot-spot for deletions in this cohort. There was no correlation noted between age of onset or creatine kinase (CK) values with extent of gene mutation. The PROVEAN analysis showed a deleterious effect in 94.5% cases and a neutral effect in 5.09% cases. Mutations in exon 45–54 (out of frame) and exon 46–54 (in-frame) regions in the central rod domain of dystrophin showed more negative scores compared to other domains in the present study. Hydrophobicity profile analysis showed that the hydrophobic regions I & III were equally affected. Analysis of deletions in hinge III hydrophobic region by the eDystrophin programme also predicted a hybrid repeat seen to be associated with a BMD like disease progression, thus making the hinge III region relatively tolerant to mutations.