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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 non-coding variants by detecting genes and novel 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 remained robust in subsampling analyses to cohorts of 11 individuals and ~5 million full-length transcripts per individual. However, performance was dependent upon isoform caller choice, particularly for NMD-sensitive and novel isoforms. Among 28 previously unsolved cases, IsoRanker deprioritized most fibroblast-expressed candidate splice site variants while nominating new leads. In one individual, IsoRanker prioritized , revealing biallelic non-coding variants that together produced a partial loss-of-function and informed targeted therapy in this individual using histidine supplementation. These findings establish 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 diseases.