希少疾患診断における隠れたスプライシング異常の同定を改善する培養末梢血リンパ球のRNAシーケンス

Accurate identification of the genetic determinants of rare diseases is essential for effective recurrence-risk management and informed reproductive decision-making. Although whole-exome sequencing (WES) and whole-genome sequencing (WGS) have significantly improved diagnostic capabilities, a subset of affected families still receives no definitive molecular diagnosis. RNA sequencing (RNA-seq) has emerged as a promising complementary diagnostic tool, yet its clinical implementation in the context of preconception genetic counseling remains underexplored. We used phytohemagglutinin-activated peripheral blood cells (PHACs) as a robust RNA source and enhanced conventional RNA-seq through the integration of three analytical innovations: (1) transcript isoform distribution (TID) analysis, (2) realignment against the MANE (Matched Annotation from NCBI and EMBL-EBI) reference transcriptome, and (3) pharmacological induction-based cryptic splicing detection. This optimized pipeline was applied to 55 rare-disease families with negative WES/WGS results who were undergoing preconception genetic counseling. Based on prior evaluations, families were grouped as VUS (n = 7), suspected-gene/variant-negative (n = 10), and unsolved/no-candidate (n = 38). PHACs showed reduced interindividual variability and higher RNA integrity than fresh PBMCs (median RIN: 9.77 vs. 8.97; p < 0.0001). The optimized workflow improved diagnostic yield by 2.2-fold (20% vs. 9%). Stratified analysis revealed positive rates of 71% (VUS), 40% (suspected-gene/variant-negative), and 5.2% (unsolved/no-candidate). Among the 11 positive cases, 10 received definitive diagnoses, leading to diverse reproductive decisions. This enhanced RNA-seq workflow provides a clinically applicable and scalable strategy for improving molecular diagnostics in reproductive and preconception settings, offering a valuable model for future clinical transcriptomics.