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).