Long-read DNA and RNA sequencing for inherited polyposis and colorectal cancer: cryptic intronic variants and multiple mutational mechanisms.

Background: Molecular genetic diagnoses are critical to prevention and treatment of inherited polyposis and colorectal cancer. 19 genes responsible for these conditions are known, but many severely affected patients and families remain unsolved. Cryptic intronic variants that alter splicing of these genes and incomplete characterisation of recessive predisposition contribute to these diagnostic gaps.

Methods: Adaptive sampling long-read DNA sequencing targeted to 19 colon cancer genes, paired with direct long-read RNA whole-transcriptome sequencing, was undertaken for four patients referred for deficiency of mismatch repair proteins and/or familial polyposis, for whom multigene panel testing yielded negative or uncertain germline results.

Results: Genetic diagnoses were obtained for all four patients. Each patient carried a cryptic intronic germline variant in a different colon cancer gene. The variants abrogated splicing by various mechanisms, all leading to loss of gene function. Patient 1 was heterozygous for intronic insertion into MSH2 of an Alu element, leading to extension of transcription into the affected intron and a stop. Patient 2 was heterozygous for deep intronic insertion into APC of a Long Interspersed Nuclear Element (LINE), creating a pseudoexon and a stop. Patient 3 was compound heterozygous at MLH3, including a cryptic intronic substitution leading to exon skipping and a stop. Patient 4 was compound heterozygous at MUTYH, including a deep intronic deletion yielding an extremely short intron and transcriptional loss of an exon encoding a critical protein domain.

Conclusion: Paired long-read DNA and RNA sequencing can enhance diagnostic yield through detection of cryptic intronic variants that impact cancer predisposition.