Long-read transcriptome analysis using IsoRanker for identifying pathogenic variants in Mendelian conditions.

Identifying pathogenic non-coding variants that contribute to Mendelian conditions remains challenging, as the functional impact of these variants on gene function is often unknown. We present IsoRanker, a long-read transcriptome sequencing-based framework that prioritizes functionally relevant variants by detecting genes and isoforms with outlier expression, allelic imbalance, and/or nonsense-mediated decay (NMD). We generated paired cycloheximide-treated and untreated fibroblast transcriptomes from 31 individuals (3 individuals with known transcript-altering rare variants and 28 individuals with unsolved conditions) and linked transcripts to phased long-read genomes. IsoRanker successfully recovered known transcript alterations in this cohort, and exploratory subsampling analyses suggested that their prioritization was largely preserved down to cohorts of 11 individuals and ∼5 million full-length transcripts per individual. Performance was dependent upon de novo isoform caller choice, particularly for NMD-sensitive and previously unannotated isoforms. Among 28 previously unsolved cases, IsoRanker deprioritized 8 out of 10 fibroblast-expressed candidate splice-site variants while nominating 4 new leads. In one individual, IsoRanker prioritized HARS1, revealing bi-allelic non-coding variants that together produced a partial HARS1 loss of function and informed targeted therapy in this individual. These findings support long-read, NMD-aware transcriptomics with IsoRanker as an effective approach for generating isoform-level functional evidence, improving classification of non-coding variants and supporting the diagnosis of individuals with rare genetic conditions.