Abstract Background: Ten to 20% percent of all breast cancers occur in a familial context and in 20-30% of these cases a mutation in the BRCA1 or BRCA2, CHEK2 genes or, more rarely, in PALB2 can be found. The remaining cases remain routinely undiagnosed with regard to a possible genetic cause. We have examined a cohort of undiagnosed probands using exome germline sequencing in order to identify other potential breast cancer predisposition genes. Methods: In total, 63 BRCA1/2-negative high risk familial BC cases (BRCAX) were considered for pair-end whole exome germline DNA sequencing on a HiSeq1500 (Illumina). High quality reads were mapped (BWA-MEM) to the reference genome (hg19) and variants were called according to GATK best practice guideline. The variants detected within the panel of 372 cancer associated genes were annoted with ANNOVAR. Synonymous variant as well as variants with MAF>1% were discarded. In a first phase protein truncating variants were validated using another NGS method. Subsequent validation of non-synonymous missense mutations and non-frameshift indels is planned. Patients signed a multilayered informed consent also covering disclosure or not of different types of incidental findings. Results: For each exome, the mean breath of coverage was about 96 % at 10X or more and the mean depth of coverage for targeted region was about 126X. In total, 3570709 SNPs (∼56678 SNPs/sample) and 477801 INDELs (7584 INDELs/sample) were called. Of them, 20829 SNPs (331 SNPs/sample) and 16071 INDELs (255 INDELs/sample) passed quality filter. In total, 445 variants were found in the publicly available cancer genes panel. Twenty-seven stop-gain/loss, frame-shift insertion/deletion and splice site variants were considered for validation with 454 Roch Junior, among which 22 variants in genes ABCC11, AFP, BARD1, BBS10, CD96, CYP1A1, DNAH11, ESCO2, EXO1, FANCI, FLCN, FLT4, HPS6, MYH8, NME8, PALB2, PDE11A, RECQL4, TTC8 were validated as true positive. Some of these genes have been found earlier to be associated with breast cancer and/other cancer types. Functional prioritization of the remaining 416 non-synonymous and non-frameshift insertion/deletions was also done in-silico before further sequencing validation, which is ongoing. The mutations found are further clinically validated by examining other affected and non-affected family members and mining the literature. Some of the mutations in known cancer predisposing genes are considered for prudent application in clinical counseling. Genotype-phenotype correlations are being examined. Conclusion: Next-generation sequencing enabled us to detect variants with high/low penetrance in known cancer predisposing genes in > 35% of BRAX families, in addition to many novel variants in many other genes not yet tied to cancer predisposition occurring singly or in combination with known cancer gene mutations. Validated variants are further examined in the families for co-segregation with the disease and potential application in counseling. Citation Format: Shahi RB, Caljon B, De Brakeleer S, Decoster L, Fontaine C, Vanacker L, Vanhoeij M, Pauwels I, Bonduelle M, Vandooren S, Croes D, Teugels E, De Greve J. Exome based germline mutation detection in a panel of 372 cancer associated genes in BRCA1/2-negative familial breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD7-04.