Splicing QTL analysis focusing on coding sequences reveals pathogenicity of disease susceptibility loci

Abstract

Splicing QTL (sQTL) are one of the major causal mechanisms in GWAS loci, but their role in disease pathogenesis is poorly understood. One reason is the huge complexity of alternative splicing events producing many unknown isoforms. Here, we proposed two novel approaches, namely integration and selection, for this complexity by focusing on protein-structure of isoforms. First, we integrated isoforms with the same coding sequence (CDS) and identified 369-601 integrated-isoform ratio QTLs (i2-rQTLs), which altered protein-structure, in six immune subsets. Second, we selected CDS incomplete isoforms annotated in GENCODE and identified 175-337 isoform-ratio QTL (i-rQTL). By comprehensive long-read capture RNA-seq among these incomplete isoforms, we revealed 29 full-length isoforms with novel CDSs associated with GWAS traits. Furthermore, we have shown that disease-causal sQTL genes can be identified by evaluating their trans-eQTL effects. Our approaches highlight the understudied role of protein-altering sQTLs and are broadly applicable to other tissues and diseases.

Long-read sequencing reveals novel isoform-specific eQTLs and regulatory mechanisms of isoform expression

“…The common group showed an intermediate pattern between the gene eQTL-specific group and ieQTL-specific group. These observed patterns are consistent with findings from a prior study [17].…”

“…The expression patterns of isoforms vary among tissues and between cancerous and non-cancerous tissues [14,15,16,17,18]. These variations likely lead to functional changes in genes and regulate diverse biological processes [14,15,16,17,32].…”

“…The expression patterns of isoforms vary among tissues and between cancerous and non-cancerous tissues [14,15,16,17,18]. These variations likely lead to functional changes in genes and regulate diverse biological processes [14,15,16,17,32]. Despite the intriguing nature of the regulatory mechanisms governing isoform expression, this area remains understudied.…”

“…The recent advent of long-read sequencing technologies enables us to analyze full-length transcripts. The utilization of long-read sequencing for full-length transcript analysis enables a comprehensive observation of different isoforms within genes, significantly contributing to the understanding of gene expression [17,18]. While previous studies have utilized long reads to detect eQTLs, these efforts typically employed an allele-specific expression analysis on a limited number of samples or specific genes.…”

“…While previous studies have utilized long reads to detect eQTLs, these efforts typically employed an allele-specific expression analysis on a limited number of samples or specific genes. Notably, a comprehensive eQTL analysis covering all isoforms across individuals within a population has yet to be conducted [17,18]. We propose that an expression analysis using long reads will unveil novel eQTLs, shed light on new regulatory mechanisms of gene expression, and facilitate the prediction of functional effects of GWAS variants.…”