Search

Rationale: Congenital heart disease (CHD) is among the most common birth defects. Most cases are of unknown pathogenesis., Objective: To determine the contribution of de novo copy number variants (CNVs) in the pathogenesis of sporadic CHD., Methods and Results: We studied 538 CHD trios using genome-wide dense single nucleotide polymorphism arrays and whole exome sequencing. Results were experimentally validated using digital droplet polymerase chain reaction. We compared validated CNVs in CHD cases with CNVs in 1301 healthy control trios. The 2 complementary high-resolution technologies identified 63 validated de novo CNVs in 51 CHD cases. A significant increase in CNV burden was observed when comparing CHD trios with healthy trios, using either single nucleotide polymorphism array (P=7×10(-5); odds ratio, 4.6) or whole exome sequencing data (P=6×10(-4); odds ratio, 3.5) and remained after removing 16% of de novo CNV loci previously reported as pathogenic (P=0.02; odds ratio, 2.7). We observed recurrent de novo CNVs on 15q11.2 encompassing CYFIP1, NIPA1, and NIPA2 and single de novo CNVs encompassing DUSP1, JUN, JUP, MED15, MED9, PTPRE SREBF1, TOP2A, and ZEB2, genes that interact with established CHD proteins NKX2-5 and GATA4. Integrating de novo variants in whole exome sequencing and CNV data suggests that ETS1 is the pathogenic gene altered by 11q24.2-q25 deletions in Jacobsen syndrome and that CTBP2 is the pathogenic gene in 10q subtelomeric deletions., Conclusions: We demonstrate a significantly increased frequency of rare de novo CNVs in CHD patients compared with healthy controls and suggest several novel genetic loci for CHD., (© 2014 American Heart Association, Inc.)

Digital droplet PCR (ddPCR) is an assay that combines state-of-the-art microfluidics technology with TaqMan-based PCR to achieve precise target DNA quantification at high levels of sensitivity and specificity. Because quantification is achieved without the need for standard assays in an easy to interpret, unambiguous digital readout, ddPCR is far simpler, faster, and less error prone than real-time qPCR. The basic protocol can be modified with minor adjustments to suit a wide range of applications, such as CNV analysis, rare variant detection, SNP genotyping, and transcript quantification. This unit describes the ddPCR workflow in detail for the Bio-Rad QX100 system, but the theory and data interpretation are generalizable to any ddPCR system., (Copyright © 2014 John Wiley & Sons, Inc.)