Materials and Methods
Clinical DMD MLPA analysis was performed on proband to confirm the presence of the duplication, assess its size, and confirm a DMD diagnosis. MLPA experiments included probes for each of the 79DMD coding exons, 3’UTR, 5’UTR, one probe for the alternative promoter/exon 1 DP427c, as well as control probes (MRC Holland). Capillary electrophoresis of PCR amplified ligated probe pairs was performed on ABI 3730xl (Applied Biosystems), and data analyzed with GeneMarker Software (SoftGenetics LLC, State College, PA, USA). CMA analysis was performed on proband using CytoScan HD Suite (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. CMA minimal and maximal probe positions for the duplication were used to design sequencing primers using Primer3Plus. Forward primer sequence: ctgtgttttgggccatttct and reverse primer sequence: tgggtttagccctaggacac produced a ~1.8Kbp product visualized in 2% agarose gel in a UV light box. The 1.8Kbp product was cleaned up using Exo-SAP-IT PCR Product Cleanup Reagent (ThermoFisher Scientific, Waltham, MA, USA) and Sanger sequenced on the ABI 3730xl (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Chromatogram files were analyzed in FinchTV (Geospiza, Inc.) and FASTA files exported and mapped to hg19 using BLAT in the UCSC genome browser [12].