Abstract 995: Leveraging RNA and long-read DNA to improve genetic etiology identification in individuals with elevated cancer risk: A pilot study in individuals with Li-Fraumeni-like phenotype

Abstract Individuals with clinical Li-Fraumeni Syndrome (LFS) are at hereditary risk of developing multiple cancers in their lifetime. Among those meeting classic LFS criteria, about 10% do not have a germline mutation identified in TP53. Long-read DNA sequencing identifies complex and structural variants missed on short-read sequencing. Clinical germline testing labs also recently began using short-read RNA-seq to characterize alternative splicing events. This project aims to explore underlying genetic etiology of those with LFS and no identifiable germline TP53 variant using long-read DNA-seq and RNA-seq. We performed germline Oxford Nanopore long-read DNA-seq and Illumina short-read RNA-seq on blood samples from 21 individuals participating in the NCI’s Li-Fraumeni Syndrome Study. Selected individuals had a personal history of cancer and/or at least 2 1st degree relatives diagnosed with cancer and no known pathogenic germline TP53 variant. Insertion-deletion variants in the long-read DNA-seq were called using Clair, and structural variants were called using Severus. Annotation was performed using OpenCravat. Variants were excluded if they were designated as benign/likely benign in ClinVar or if they were common (>1%) in GnomAD populations. Variants were prioritized if they were designated as “pathogenic” or “likely pathogenic” in Clinvar, if they had an association with disease based on genome-wide association studies, and if they were predicted to have missense / frameshift / start site-loss effects in Ensembl. We used RNA-seq to both assess the functional effects of splicing variants and to compare relative expression levels of candidate associated genes versus housekeeping genes as compared with control blood samples from the GTEx dataset. We used rMATS to calculate a Percent Spliced In (PSI) index for splicing variants. PSI, which measures the ratio of isoforms to canonical transcripts, reflects differences related to splicing that can clinically impact gene function. We prioritized 674 rare insertion-deletion variants for initial analysis. Two related individuals (one diagnosed with cancer, one not) had a TP53 variant of uncertain significance (c.845G>A). Two unrelated individuals had variants in ATM: c.9023G>A and c.8147T>C. The genes most affected by these prioritized variants were TTN, GBA, ADGRV1, ATM, FRAS1, NEB, PKHD1, and SYNE2. We did not observe any significant difference in expression levels of TP53 compared to housekeeping genes when comparing our samples to GTEx. We did not observe any family-specific PSI patterns. Long-read DNA-seq and RNA-seq can help characterize the familial cancer syndromes without established genetic etiology. In this pilot study, we are actively evaluating potential causal genes related to clinical presentations of LFS with no identifiable germline TP53 variant. Citation Format: Yewon Kim, Anvith Kakkera, Asher Bryant, Sharon A. Savage, Michael Dean, Payal Khincha, Misha Kolmogorov, Padma Rajagopal. Leveraging RNA and long-read DNA to improve genetic etiology identification in individuals with elevated cancer risk: A pilot study in individuals with Li-Fraumeni-like phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 995.