The clinical significance of copy number variants (CNVs) in congenital heart disease (CHD) continues to be a challenge. Although CNVs including genes can confer disease risk, relationships between gene dosage and phenotype are still being defined. Our goal was to perform a quantitative analysis of CNVs involving 100 well-defined CHD risk genes identified through previously published human association studies in subjects with anatomically defined cardiac malformations. A novel analytical approach permitting CNV gene frequency "spectra" to be computed over prespecified regions to determine phenotype-gene dosage relationships was employed. CNVs in subjects with CHD (n = 945), subphenotyped into 40 groups and verified in accordance with the European Paediatric Cardiac Code, were compared with two control groups, a disease-free cohort (n = 2,026) and a population with coronary artery disease (n = 880). Gains (≥200 kb) and losses (≥100 kb) were determined over 100 CHD risk genes and compared using a Barnard exact test. Six subphenotypes showed significant enrichment (P ≤ 0.05), including aortic stenosis (valvar), atrioventricular canal (partial), atrioventricular septal defect with tetralogy of Fallot, subaortic stenosis, tetralogy of Fallot, and truncus arteriosus. Furthermore, CNV gene frequency spectra were enriched (P ≤ 0.05) for losses at: FKBP6, ELN, GTF2IRD1, GATA4, CRKL, TBX1, ATRX, GPC3, BCOR, ZIC3, FLNA and MID1; and gains at: PRKAB2, FMO5, CHD1L, BCL9, ACP6, GJA5, HRAS, GATA6 and RUNX1. Of CHD subjects, 14% had causal chromosomal abnormalities, and 4.3% had likely causal (significantly enriched), large, rare CNVs. CNV frequency spectra combined with precision phenotyping may lead to increased molecular understanding of etiologic pathways.

Aim: To visualize copy number associations and quantify relative risk of Copy Number Variants (CNVs) in 100 well-defined congenital heart disease (CHD) risk genes identified from literature sources. Method: CNVs in subjects with congenital heart malformations (n=741) and phenotyped in accordance with the European Paediatric Cardiac Code were compared to the frequencies of CNVs present in a control population of subjects with coronary artery disease (n=880). CNV gains (≥ 200 kb) and losses (≥ 100 kb) were determined using the Affymetrix 6.0 Gene Chip and confirmed with TaqMan copy number assays. In addition, each gene was tested using a two proportions z-test approach to determine if the gene frequency was significantly different in CHD vs control. To quantify the effect of observed CNVs in the CHD population, we collapsed the set of rare individual CNVs into a single group to test the collective frequency difference between cases and controls by adapting a previously described Cohort Allelic Sums Test (CAST). Results: The following phenotypes showed a significant enrichment of gains and losses in the CHD population: Aortic Stenosis, Coarctation of the Aorta, Ebstein's Anomaly, Tetralogy of Fallot, Truncus Arteriosus and Ventricular Septal Defect. The CNV percentage was increased in CHD subjects in the following genes (p<0.05 with a Bonferroni correction): Losses: FKBP6(-), ELN(s), GTF2IRD1(-), SOX7(-), GATA4(ns), NOTCH1(ns), MYH11(ns), SALL4(s), TBX1(s), CRKL(-), MAPK1(-); Gains: PRKAB2(-), FMO5(-), CHD1L(-), BCL9(-), ACP6(-), GJA5(ns), NPHP3(s), FOXL2(s), SEMA5A(-), NSD1(s), HOXA1(s), TBX20(ns), SOX7(-), GATA4(ns), HRAS(s), GATA6(ns), RUNX1(s), MAPK1(-), BCOR(s), ATRX(s), GPC3(s), ZIC3(ns), FLNA(s) where s is syndromic, ns is non-syndromic and (-) is not available on the CHD WIKI website ( http://www.chearted.eu ). The approach rapidly identified common, cytogenetically visible abnormalities (i.e. TBX1 in 22q11.2 deletion syndrome, n=30, and RUNX1 in Trisomy 21, n=66). Conclusion: This is the first study to establish a copy number spectrum in a CHD population. We conclude that the CAST approach shows promise for evaluating qualitative outcomes and statistical precision of prior studies which have focused on CNVs in CHD and other diseases.