Genetic testing
Diagnostic MLPA testing was performed on proband, targeting allDMD coding exons as well as its 5’UTR, 3’UTR, and DP427c regions. We identified a duplication spanning from the 5’UTR to exon 7 (g.(?_33229574)_(32827735_32717219)) (hg19). Subsequent CMA analysis of proband revealed the duplication to be 1.18Mbp in size, overlapping the 5’UTR and exons 1-7 of DMD (NM_004006) (arr[hg19] Xp21.1(32,741,375- 33,926,846)x3 mat) (Supplemental Figure 1). To further characterize the structural configuration of the duplication, PCR primers were designed bordering the identified duplication breakpoints, such that it could distinguish between direct tandem orientation, inverted tandem orientation, and insertion elsewhere in the genome (Supplemental Figure 2 and Supplemental Data). A long-range PCR protocol using these primers yielded a product ~1.8Kb in size (Supplemental Figure 3). Sanger sequencing of the amplified PCR product showed the duplication breakpoints to fall at Xp21.1:32,741,022 and Xp21.1:33,928,069 (hg19, Supplemental Figure 4 and Figure 2). Duplication breakpoint analysis revealed the presence of short palindromic sequences at the junction as well as microhomologies in neighboring regions (Supplemental Data).