Additional definition of the clinical significance of structural variants and variants affecting RNA structure in genes associated with hereditary cancers: Introduction of the method

Background: Patients with a personal or family history of hereditary cancer have a high probability

of carrying a pathogenic variant (PV) in one of the cancer susceptibility genes. In conventional genotyping approaches, which typically rely on short-read sequencing of exonic regions, PV are detected in only a minority of cases (10-25%). A proportion of PV remains undetected, particularly those involving changes that may affect the structure of the resulting RNA. By introducing methods that detect alterations influencing DNA structure and, consequently, RNA architecture, it would be possible to more accurately classify variants of uncertain clinical significance (VUS). In the future, such an approach could also enable the identification of novel variants in highly burdened patients whose previous genetic testing yielded noninformative negative results.

Patients and methods: In the study, 50 patients with a personal and/or family history of hereditary cancers will be included. The inclusion criteria will encompass: (1) individuals with a confirmed PV known to alter RNA structure, will be used for

method validation; (2) individuals with a confirmed variant of VUS, which is predicted by in silica

tools to affect RNA structure; (3) individuals who have already undergone genetic counseling and testing, but for whom the cause of their clinical presentation has not yet been definitively identified. RNA structural analysis will be performed using transcriptome sequencing or longread cDNA sequencing of genes associated with hereditary cancers. In selected cases, additional DNA analysis will be conducted, focusing on detection of structural variants.

Aim: Our goal is to clarify the clinical relevance of genetic variants of uncertain significance and to identify previously undetected pathogenic variants in patients with a personal or family history of hereditary cancer. To achieve this, we plan to introduce a new method that will enable more precise gene analysis and improve diagnostic accuracy for these patients.