Human gene copy number spectra analysis in congenital heart malformations

Abstract

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.

Fig. 1.

CNV analysis flowchart from sample to statistics. Blue figures represent software used or a process/task performed. Red figures represent data files.

Fig. 2.

Sample size (n) and copy number variant (CNV) proportion (fraction), required to detect difference from 0.0196 (CHOP control CNV fraction) at an alpha = 0.05, power at least 80%. CHOP, Children's Hospital of Philadelphia.

Fig. 3.

Total CNV burden by cohort. Standard box-and-whiskers plot for the distribution of large rare CNV segment count per subject in each of 4 cases: congenital heart disease (CHD) vs. Milwaukee Family Heart Study (MFHS) and gains vs. losses. Boxes represent the 1st and 3rd quartiles of each distribution, thick horizontal lines represent the median value, circles represent outliers, or the CHD cohort, major syndromes would significantly skew the distribution, so those subjects were excluded, leaving 810 syndrome-free subjects. Trisomy 21 and 18, Turner, 22qDS, William's and XXX chromosomal abnormalities were therefore excluded.

Fig. 4.

CNV frequency spectrum. Note that any gene showing 0% CNV frequency in all 3 cohorts was omitted from this figure due to space considerations. Vertical error bars drawn represent 1 SD from the mean in the estimated sampling distribution. From this visualization it is clear that gains over gene FKBP6 on chromosome 7 occur in all 3 cohorts, while losses of the same gene are only seen in the CHD cohort, implying a loss could cause CHD.

Fig. 5.

CNV frequency spectra of significantly enriched phenotypes. Note that any gene showing 0% CNV frequency in all 3 cohorts was omitted from this figure due to space considerations. Vertical error bars drawn represent 1 SD in the estimated sampling distribution. Significantly enriched phenotypes included: aortic stenosis (valvar), atrioventricular canal (partial), atrioventricular septal defect (AVSD) with tetralogy of Fallot (TOF), subaortic stenosis, TOF, and truncus arteriosus.

Fig. 6.

CNV frequency spectra of collapsed phenotypes by syndrome. Note that any gene showing 0% CNV frequency in all 3 cohorts was omitted from this figure due to space considerations. Vertical error bars drawn represent 1 SD in the estimated sampling distribution. Turner phenotypes, T21 phenotypes, and 22qDS phenotypes.

Fig. 7.

Distribution of CNVs in CHD cohort. Type A represents cytogenetically visible chromosomal abnormalities (≥3 Mbp), type B are those subjects with a CNV over a syndromic-associated CHD gene as reported by the CHD WIKI portal, type C are those recognized through CHD WIKI as nonsyndromic, type D are CNVs with an unknown category, and type E represents subjects with no CNV over our predefined 100 CHD-associated genes. An individual can only fit into 1 category where D>A>B or C. Numbers are rounded to the nearest percentage.