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).