Importance of muscle biopsy to establish pathogenicity of DMD missense and splice variants.

A precise genetic diagnosis of a dystrophinopathy has far-reaching implications for affected boys and their families. We present three boys with DMD single nucleotide variants associated with Becker muscular dystrophy presenting with myalgia, reduced exercise capacity, neurodevelopmental symptoms and elevated creatine kinase. The DMD variants were difficult to classify: AIII:1 a synonymous variant in exon 13 c.1602G>A, p.Lys534Lys; BIII:1 an essential splice-site variant in intron 33 c.4674+1G>A, and CII:1 a missense mutation within the cysteine-rich domain, exon 66 c.9619T>C, p.Cys3207Arg. Complementary DNA (cDNA) analysis using muscle-derived mRNA established splice-altering effects of variants for AIII:1 and BIII:1, and normal splicing in CII:1. Western blot analysis demonstrated mildly to moderately reduced dystrophin levels (17.6 - 36.1% the levels of controls), supporting dystrophinopathy as a probable diagnosis. These three cases highlight the diagnostic utility of muscle biopsy for mRNA studies and western blot to investigate DMD variants of uncertain pathogenicity, by exploring effects on splicing and dystrophin protein levels.