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

Loss of function of APC through cryptic variants missed by targeted sequencing - a deletion encompassing exon 10 causing exon skipping, and a deep intronic deletion-insertion activating a pseudoexon - both leading to a frameshift and a premature termination codon.

Familial adenomatous polyposis is an autosomal dominant colorectal cancer predisposition syndrome, most often caused by pathogenic APC variants, yet some cases remain genetically unresolved because structural and deep intronic variants escape routine detection. The authors studied two unrelated kindreds with clinically evident but unexplained polyposis after targeted panel and exome sequencing, using pedigree-based whole genome sequencing (WGS) with in silico splice prediction and transcript-level validation by RT-PCR, Sanger sequencing and RT-qPCR on blood-derived RNA. In the first kindred, a heterozygous deletion encompassing exon 10 was confirmed to cause exon skipping, a frameshift and a premature termination codon. In the second, a deep intronic complex deletion-insertion between exons 11 and 12 created novel splice sites and activated a pseudoexon carrying a 2-base-pair deletion, again producing a frameshift. Both variants segregated with disease in their families and were classified as pathogenic under ACMG criteria on the basis of loss of function and functional RNA evidence.

Synthesis written by Geno'X. For the full original abstract, please refer to the source publication.

The useful result is not WGS as such - exome had already been performed and the genome covers these regions anyway - but the demonstration that transcript-aware interpretation changes the report: without blood RNA, the intronic deletion-insertion would have remained a variant of uncertain significance and the exon skipping would not have been proven. The limitation is sample size, two families, which says nothing about the proportion of unexplained polyposis this approach can resolve or about the cost per additional diagnosis. For a family that has waited years, the practical line is clear: revisit APC-negative cases with an analysis covering non-coding regions plus RNA validation, provided a usable sample is available.

Analysis by Dr Thibaut Benquey