A Plot Twist: When RNA Yields Unexpected Findings in Paired DNA-RNA Germline Genetic Testing

Germline genetic variants impacting splicing are a frequent cause of disease. The clinical interpretation of such variants is challenging for many reasons including the immense complexity of splicing mechanisms. While recent advances in splicing algorithms have improved the accuracy of splice prediction, predicting the nature and abundance of aberrant splicing remains challenging. As RNA testing becomes more mainstream in the clinical diagnostic setting, the complexities of interpretation are coming to light.

Data from patients undergoing concurrent DNA and RNA testing were retrospectively reviewed for unusual splicing impacts to underscore some of these complexities and serve as exemplars in how to avoid pitfalls in the interpretation of sequence variants.

Seven rare variants with unusual splicing impacts are presented: a variant at a consensus donor nucleotide position lacking a splice impact (NF1 c.888+2T>C); a mid-exonic missense variant creating a novel donor site and a cryptic acceptor site resulting in pseudo-intronization (BRIP1 c.727ALZTR1 c.2232G>A p.Ala744Ala); two variants that would be expected to result in nonsense-mediated-mRNA-decay triggering splicing impacts that obviated nonsense-mediated-decay (APC c.1042C>T p.Arg348Ter and BRCA2 c.6762del; c.6816_6841+1534del); and two variants causing splicing impacts through pyrimidine tract optimization (NF1 c.5750-184_5750-178dup and ATM c.3480G>T p.Val1160Val).

Paired DNA and RNA testing revealed unexpected splice events altering variant interpretation, expanding our knowledge of clinically important splicing mechanisms and highlighting the benefit of RNA testing.