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

Genetic variations linked to changes in gene expression are known as expression quantitative loci (eQTLs). The identification of eQTLs provides a profound understanding of the mechanisms governing gene expression. However, prior studies have primarily utilized short-read sequencing techniques, and the analysis of eQTLs on isoforms has been relatively limited. In this study, we employed long-read sequencing technology (Oxford Nanopore) on B cells from 67 healthy Japanese individuals to explore genetic variations associated with isoform expression levels, referred to as isoform eQTLs (ieQTLs). Our analysis revealed 33,928 ieQTLs, with 69.0% remaining undetected by a gene-level analysis. Additionally, we identified ieQTLs that have significantly different effects on isoform expression levels within a gene. A functional feature analysis demonstrated a significant enrichment of ieQTLs at splice sites and specific histone marks, such as H3K36me3, H3K4me1, H3K4me3, and H3K79me3. Through an experimental validation using genome editing, we observed that a distant genomic region can modulate isoform-specific expression. Moreover, an ieQTL analysis and minigene splicing assays unveiled functionally crucial variants in splicing, which software-based predictions failed to anticipate. A comparison with GWAS data revealed a higher number of colocalizations between ieQTLs and GWAS findings compared to gene eQTLs. These findings highlight the substantial contribution of ieQTLs identified through long-read analysis in our understanding of the functional implications of genetic variations and the regulatory mechanisms governing isoforms. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was supported by AMED under Grant Number JP21km0908001 (A.F.) and Takeda Science Foundation (A.F.). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Institutional review boards (IRBs) at the University of Tokyo approved this work (2022121Ge). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript