Targeted Long-Read sequencing provides functional validation of variants predicted to alter splicing

Amp-LRS is a sensitive and cost-effective method for validating non-coding variants that alter splicing, potentially enhancing molecular diagnosis in rare neurological diseases.

Summary and findings

This study applied targeted amplicon-based long-read RNA sequencing (Amp-LRS) to validate candidate non-coding variants predicted to alter splicing in five patients with neurological disorders. The variants were identified in genes such as POLR3A and OPA1, and the study reported various aberrant splicing events. No therapeutic claims are made.

Abstract

<h4>ABSTRACT</h4> <h4>Background</h4> Whole-genome sequencing (WGS) has improved the diagnosis of rare genetic disorders, yet interpretation of non-coding variants that affect splicing remains challenging. In silico predictions alone are insufficient, and short-read RNA sequencing may fail to capture complex or low-abundance splicing events. Targeted amplicon-based long-read RNA sequencing (Amp-LRS) offers a cost-effective approach for functional validation of candidate splice-altering variants. <h4>Methods</h4> We applied Amp-LRS to five patients with neurological disorders (central nervous system, peripheral nervous system, or muscle) harbouring candidate non-coding variants predicted to alter splicing. RNA was extracted from fibroblasts or peripheral blood, and full-length transcript amplicons were sequenced using Oxford Nanopore Technologies. Nonsense-mediated decay (NMD) inhibition was performed on fibroblast cultures using cycloheximide. <h4>Results</h4> Amp-LRS validated all five candidate variants, including intronic and UTR variants in POLR3A, OPA1, PYROXD1, GDAP1 , and SPG11 . Aberrant splicing events included exon skipping, intron retention, cryptic splice site activation, and pseudoexon inclusion, often resulting in frameshifts and premature termination codons. For POLR3A and OPA1 , multiple abnormal isoforms arose from single variants, highlighting the complexity of splicing disruption. Some pathogenic effects were detectable only in a minority of reads and variably enriched by NMD inhibition, consistent with being hypomorphic. The approach was successfully applied using accessible tissues and enabled multiplexed sequencing at low per-sample cost. <h4>Conclusions</h4> Amp-LRS is a sensitive, versatile, and cost-effective method for functional assessment of non-coding splice-altering variants identified by WGS. By enabling full-length transcript analysis from accessible tissues, this approach improves interpretation of variants of uncertain significance and could enhance molecular diagnosis in rare neurological diseases.

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