Identification of Novel Pathogenic Variants in Familial Adenomatous Polyposis Through Whole Genome Sequencing

Familial adenomatous polyposis (FAP) is an autosomal dominant colorectal cancer predisposition syndrome most commonly caused by pathogenic variants in the APC gene. Although targeted gene panel sequencing is routinely used, some FAP cases remain genetically unresolved due to limitations in detecting structural and deep intronic variants. We investigated two unrelated kindreds with clinically evident but genetically unexplained FAP using targeted panel sequencing and whole exome sequencing. Pedigree-based whole genome sequencing (WGS) was performed to detect cryptic variants, with in silico splice prediction and transcript-level validation using RT-PCR, Sanger sequencing, and RT-qPCR of blood-derived RNA. WGS uncovered two novel pathogenic APC variants missed by prior testing. In Kindred 1, a heterozygous large deletion encompassing exon 10 was detected and confirmed to cause exon skipping, resulting in a frameshift and premature termination codon. In Kindred 2, a deep intronic complex deletion-insertion variant was identified between exons 11 and 12, predicted to create novel splice sites. Transcript analysis demonstrated aberrant pseudoexon activation with a 2-base-pair deletion, leading to a frameshift and premature termination. Both variants segregated with disease within the respective families and were classified as pathogenic according to ACMG guidelines based on loss-of-function effects and functional RNA evidence. Our findings demonstrate that cryptic pathogenic APC variants beyond the detection limits of routine genetic testing can be resolved through WGS combined with transcript-level validation. This study highlights the importance of transcript-aware variant interpretation and supports the integration of genome-wide sequencing and RNA-based analyses into the diagnostic evaluation of genetically unexplained FAP.