Allelic Expression Imbalance in the Human Retinal Transcriptome and Potential Impact on Inherited Retinal Diseases

Abstract

Inherited retinal diseases (IRDs) are often associated with variable clinical expressivity (VE) and incomplete penetrance (IP). Underlying mechanisms may include environmental, epigenetic, and genetic factors. Cis-acting expression quantitative trait loci (cis-eQTLs) can be implicated in the regulation of genes by favoring or hampering the expression of one allele over the other. Thus, the presence of such loci elicits allelic expression imbalance (AEI) that can be traced by massive parallel sequencing techniques. In this study, we performed an AEI analysis on RNA-sequencing (RNA-seq) data, from 52 healthy retina donors, that identified 194 imbalanced single nucleotide polymorphisms(SNPs) in 67 IRD genes. Focusing on SNPs displaying AEI at a frequency higher than 10%, we found evidence of AEI in several IRD genes regularly associated with IP and VE (BEST1, RP1, PROM1, and PRPH2). Based on these SNPs commonly undergoing AEI, we performed pyrosequencing in an independent sample set of 17 healthy retina donors in order to confirm our findings. Indeed, we were able to validate CDHR1, BEST1, and PROM1 to be subjected to cis-acting regulation. With this work, we aim to shed light on differentially expressed alleles in the human retina transcriptome that, in the context of autosomal dominant IRD cases, could help to explain IP or VE.

Keywords: Inherited retinal diseases; allelic expression imbalance; expressivity; penetrance; retina.

Figure 1

RNA-seq allelic expression imbalance (AEI) analysis workflow. Detailed are all steps contained in the pipeline (see also 2.4. Allelic expression imbalance (AEI) RNA-sequencing analysis). This process is subcategorized into three major blocks: Data cleanup, variant discovery, and selection of AEI single nucleotide polymorphism (SNPs). QC – quality control, GATK - Genome Analysis Toolkit, Indel – insertion/deletion, REF – reference, ALT – alternative, Qual –quality, IRD – inherited retinal disease.

Figure 2

Representation of the relative allelic percentage (mean ± standard error of the mean or SEM). Shown are those SNPs in inherited retinal diseases (IRD) genes displaying allelic expression imbalance (AEI) frequencies higher than 10% in the 52 RNA-seq sample-set. Genes associated with autosomal dominant inherited IRDs are marked with an asterisk. Allelic balance is delimited within the 40–60% range.

Figure 3

Relative allele percentages for SNPs rs149698 and rs1800009in BEST1 at the DNA and RNA levels. Allelic balance is delimited within the 40–60% range. Error bars correspond to the standard of the mean (SEM) of allele A. (A) BEST1 rs149698 pyrosequencing results. Allele percentages at the DNA level remained close to 50%. At the RNA level, there was a bias in HAS6 and HAS13 in favor of allele T. (B) BEST1 rs1800009 pyrosequencing results. Similar to rs149698, the DNA levels remained balanced, whereas at the RNA level again HAS6 and HAS13 allele T were overrepresented.