Abstract Breast cancer (BC) is the most common malignancy in women and has a major heritable component that is driven by both common SNPs and rarer variants in susceptibility genes. However, the risks associated with most rare susceptibility alleles are not well estimated. We sequenced the coding regions of ATM, CHEK2, PALB2, and XRCC2 in 13,087 BC cases and 5,488 controls from East Anglia to characterize their role in BC risk in the largest sequencing study of these genes to date. We performed multiplexed microfluidic PCR target enrichment of 221 amplicons, covering 99.3% of the coding bases. Amplified DNA was barcoded, pooled, and sequenced on the Illumina HiSeq2000. Reads were demultiplexed, trimmed, and aligned to the hg19 reference sequence. Variants were called with GATK UnifiedGenotyper and filtered for missing data. Retained variants were annotated with CADD and VEP. Truncating variants, missense alleles, and other functional variant groups were assessed for association with BC risk using logistic regression. Truncating variants in ATM, CHEK2, and PALB2 increased overall BC risk, whereas XRCC2 variants did not (Table 1). The estimated relative risk was higher for PALB2 than ATM or CHEK2. While 88% of CHEK2 variant alleles were attributable to CHEK2*1100delC, other truncating alleles appeared to confer similar risks. CHEK2 truncating alleles were more strongly associated with risk of estrogen receptor (ER) positive disease than ER negative or triple negative disease. In contrast, PALB2 protein-truncating variants conferred similar relative risks for all subtypes. Analysis of rare missense variants suggested that specific subset of alleles in ATM (P = 0.012), CHEK2 (P = 0.009), and PALB2 (P = 0.017) may contribute to risk. Localization within known functional domains were a predictor of association among these variants. These results confirm that truncating variants in ATM, CHEK2, and PALB2 are associated with moderate BC risks, and exclude a large effect on risk from XRCC2 variants. Truncating Variant RiskATMATMCHEK2CHEK2PALB2PALB2XRCC2XRCC2ORP-ValueORP-ValueORP-ValueORP-ValueSEARCH Cases3.262.11E-053.345.18E-115.424.45E-080.940.9232ER Positive3.191.04E-043.719.11E-124.322.73E-050.870.8419ER Negative1.280.67431.560.23655.583.62E-040.880.9102Triple Negative2.850.14880.420.32217.500.0022NANA Citation Format: Brennan Decker, Jamie Allen, Craig Luccarini, Karen A. Pooley, Paul PD Pharoah, Alison M. Dunning, Douglas F. Easton. Rare protein truncating and missense variants in ATM, CHEK2, PALB2, but not XRCC2, confer increased breast cancer risks. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4309.