Materials and Methods
Genetic testing was performed on an amniocentesis sample from M1. Aneuploidy FISH analysis was performed on 100 nuclei from uncultured amniocytes and tested chromosomes X, Y, 13, 18, and 21. CMA was performed on cultured amniocytes using the Affymetrix CytoScan HD platform according to manufacturer’ instructions. MPseq was performed on DNA extracted from amniocytes using AutoPure LS (Qiagen, Hilden, Germany). MPseq libraries were prepared using Nextera Mate Pair Library Preparation Kit (Illumina, San Diego, CA), which were subsequently purified and used for short-read library preparation using TruSeq DNA Library Prep kit (Illumina, San Diego, CA). Purified libraries were sequenced in an Illumina HiSeq 2500 using RapidRun mode to obtain 101bp reads. MPseq data was processed with BIMAv3[10] and analyzed with SVAtools version 0.24.9 [8]. Polymerase chain reaction (PCR) experiments were performed to amplify breakpoint junctions identified by MPseq. Gene expression experiments were performed on total mRNA purified from cultured amniocytes of the proband and a normal control male with normal SMAD2 copy number and no phenotypic evidence of dextrocardia. Relative mRNA levels were determined by real-time RT-PCR analysis using Applied Biosystems TaqMan Assays (Thermo Fisher Scientific, Waltham, MA) targeting the SMAD2 exon junctions 2/3, 4/5, 6/7, and 9/10. Three technical replicates were used to calculate standard deviation for both the proband and control RNA expression (primers used are listed in Supplementary Table 1).