Abstract Background: Hereditary cancer confirmed by germline pathogenic variant has been increased with increasing next generation sequencing (NGS) panel tests in recent years. However, some cases who were suspected hereditary cancer represented having variant of uncertain significance (VUS) or no variants even though they had family members with many cancers. The aim of this study was to investigate variants function especially in splicing variants and to explore other candidate genes by whole genome sequencing (WGS) in suspected hereditary cancer patients. Methods: Total of 44 patients who clinically suspected hereditary cancer without defined pathogenic variants after NGS panel test were enrolled. Among them 11 patients had intronic variants and their variant effects were predicted by SpliceAI, a deep learning-based tool. To define alternative splicing, total RNA was extracted from patients buffy coats and cDNA was synthesized using oligo dT. Then the cDNA was amplified by specific primers and performed sequencing. Also, WGS was performed in 15 patients then comprehensive analysis encompassing 392 unique genes was performed including 282 well-established hereditary cancer predisposition genes and 228 DNA repair-related genes. We confirmed the variant by various type of studies and predicted impact on protein by analysis. Results: We analyzed intronic variants of 11 patients; ATM c.4611+3A>T(n=1). BRCA1 c.547+30A>G (n=2), BRCA1 c.5152+6T>C(n=1), MSH2 c.2635-7G>A(n=1), MUTYH c.934-2A>G(n=2), NF1 c.1527+4A>C(n=1), NF1 c.3871-14G>T(n=1), PALB2 c.3350+5G>A(n=1), and SMARCA4 c.3546+1G>A(n=1). Though two intronic variants (ATM c.4611+3A>T and SMARCA4:c.3546+1G>A) were observed a likelihood of aberrant splicing aggregate SpliceAI score more than 0.8, we observed three intronic variants induce alternative splicing (ATM c.4611+3A>T, NF1 c.1527+4A>C, and PALB2 c.3350+5G>A).In addition, WGS analysis demonstrated 6 out of 15 patients have germline variants in relevant genes. Four single nucleotide variants (SMARCAL1:c.1632delA, SETMAR:c.441dup, FAN1:c.590C>A, and PRF1:c.445G>A) were validated by sequencing and those were likely pathogenic variant (LPV) by ACMG/AMP criteria of ClinGen. Copy number variations were identified in 2 patients (PMS2 heterozygous deletion and RAD50 duplication) and structure variant of MSH2 was identified in one patient. Conclusion: Our analysis showed in silico analysis results might be different from functional analysis and rare variants in cancer predisposition genes could be confirmed WGS. However, large number of patients study would be needed to confirm necessity of WGS for hereditary cancer. This work was supported by National Cancer Center, Korea (No_NCC-2110181). Citation Format: Hyeji Kim, Jong Eun Park, Gi Yeon Lee, Jung-Ah Hwang, Jin-Sun Ryu, Ye-Ryeong Jung, Seeyoun Lee, Kum Hei Ryu, Dong Ock Lee, So-Youn Jung, Sang-Yoon Park, Wonyoung Choi, Sun-Young Kong. Exploring the impact of cancer predisposition gene variants by functional analysis and whole genome sequencing analysis in hereditary cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7336.