Transcript capture and ultradeep long-read RNA sequencing (CAPLRseq) to diagnose HNPCC/Lynch syndrome.

Purpose: Whereas most human genes encode multiple mRNA isoforms with distinct function, clinical workflows for assessing this heterogeneity are not readily available. This is a substantial shortcoming, considering that up to 25% of disease-causing gene variants are suspected of disrupting mRNA splicing or mRNA abundance. Long-read sequencing can readily portray mRNA isoform diversity, but its sensitivity is relatively low due to insufficient transcriptome penetration.
Methods: We developed and applied capture-based target enrichment from patient RNA samples combined with Oxford Nanopore long-read sequencing for the analysis of 123 hereditary cancer transcripts (capture and ultradeep long-read RNA sequencing (CAPLRseq)).
Results: Validating CAPLRseq, we confirmed 17 cases of hereditary non-polyposis colorectal cancer/Lynch syndrome based on the demonstration of splicing defects and loss of allele expression of mismatch repair genes MLH1 , PMS2 , MSH2 and MSH6 . Using CAPLRseq, we reclassified two variants of uncertain significance in MSH6 and PMS2 as either likely pathogenic or benign.
Conclusion: Our data show that CAPLRseq is an automatable and adaptable workflow for effective transcriptome-based identification of disease variants in a clinical diagnostic setting.
Competing Interests: Competing interests: None declared.
(© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)