Many human genes produce multiple mRNA isoforms, and changes in splicing or expression can strongly influence disease risk. Yet most clinical genetic tests focus on DNA sequence alone and often miss how variants affect RNA, leaving many results uncertain.

Researchers at the Medizinisch Genetisches Zentrum (MGZ) set out to close this gap by developing a practical way to examine RNA isoforms directly in patient samples. Their approach, called CAPLRseq, combines targeted RNA capture with ultradeep long-read sequencing using Oxford Nanopore technology. This strategy enriches specific disease-related transcripts before sequencing, overcoming the lower sensitivity that often limits long-read RNA sequencing.

Outline of capture and ultradeep long-read RNA sequencing

The different experimental steps and approximate times are shown for a representative mRNA (see text for details). ONT, Oxford Nanopore Technology.

The team applied CAPLRseq to 123 transcripts linked to hereditary cancer, with a focus on genes involved in Lynch syndrome. By reading full-length RNA molecules, the method clearly revealed abnormal splicing patterns and loss of allele-specific expression in key mismatch repair genes, including MLH1, PMS2, MSH2, and MSH6. Using this information, the researchers confirmed 17 cases of Lynch syndrome that could be explained by RNA-level defects.

Importantly, CAPLRseq also helped resolve clinical uncertainty. Two variants previously labeled as variants of uncertain significance were reclassified as either likely pathogenic or benign based on their real impact on RNA structure and abundance. This demonstrates how transcript-focused analysis can directly inform clinical decision making.

Because CAPLRseq is automatable and adaptable, it fits well into diagnostic laboratory workflows. By combining targeted capture with long-read RNA sequencing, this method provides a clearer view of how genetic variants alter gene expression, improving the accuracy of hereditary cancer diagnostics.