Loss of RNA expression and allele-specific expression associated with congenital heart disease

Abstract

Congenital heart disease (CHD), a prevalent birth defect occurring in 1% of newborns, likely results from aberrant expression of cardiac developmental genes. Mutations in a variety of cardiac transcription factors, developmental signalling molecules and molecules that modify chromatin cause at least 20% of disease, but most CHD remains unexplained. We employ RNAseq analyses to assess allele-specific expression (ASE) and biallelic loss-of-expression (LOE) in 172 tissue samples from 144 surgically repaired CHD subjects. Here we show that only 5% of known imprinted genes with paternal allele silencing are monoallelic versus 56% with paternal allele expression-this cardiac-specific phenomenon seems unrelated to CHD. Further, compared with control subjects, CHD subjects have a significant burden of both LOE genes and ASE events associated with altered gene expression. These studies identify FGFBP2, LBH, RBFOX2, SGSM1 and ZBTB16 as candidate CHD genes because of significantly altered transcriptional expression.

Figure 1. Identification of extreme ASE genes in subjects with CHD.

Shown are both alleles of a gene that differ by the SNP haploblocks ‘CC' (blue) and ‘GT' (red), as identified by WES, WGS or SNP-array genotyping. RNAseq analysis (read counts at heterozygous positions) reveals the expression of both alleles (biallelic RNA expression) or the disproportionate expression of one allele over another (ASE). RNAseq expression analyses (comparing each sample to the average of all other samples within a tissue group) identify relative loss and gain of expression. Variant analysis, in conjunction with RNAseq analysis, can further identify LOF mutations in the expressed allele (*).

Figure 2. Extreme ASE genes preferentially identified in WGS subjects.

Shown in a are the number of genes with a minimum expression of 2 r.p.m. (grey bars), and the number of expressed genes that contain heterozygous SNPs (black bars) for CHD WGS (n=30 tissues) and CHD WES probands (n=142 tissues), GTEx donors (n=113 tissues) and mouse C57Bl6/Castaneus F1 hybrids (n=7 tissues). s.d. is indicated. b The distribution of extreme ASE events per subject by genotyping platform. Extreme ASE events were identified in >70% of WGS subjects (n=14). However, extreme ASE events were identified in only ∼20% of WES subjects (n=130) and ∼45% of GTEx donors (n=95).