Low-frequency Genetic Variants Are Associated With Breast Cancer In Chinese Women

Breast cancer is the most prevalent malignant tumor and the leading cause of cancer-related deaths among women around the world. It was reported that the global burden of breast cancer was substantial, with an estimate of 1.67 million breast cancer new cases diagnosed worldwide in 2012, accounting for 25.1% of all new cancer cases. Although the incidence of breast cancer was relative low in China, it had been increasing with an average rate of 3-5% in the past decades. It was estimated that, in 2015, there were 268.6 thousands breast cancer new cases diagnosed in China, accounting for 15% of all new cancer cases. Therefore, breast cancer is a major public health problem to be solved. Early identification of high-risk individuals will be able to effectively reduce the morbidity and mortality of breast cancer, which is also a basic strategy and a vital challenge that we faces.It is well known that the etiology of breast cancer is a combination of genetic determinants and environmental factors. To date, many high penetrance susceptibility genes have been identified, including BRCA1/BRCA2, ATM and TP53, suggesting the import role of high or moderate penetrance genetic variations.To date, more than one hundred independent common loci associated with breast cancer risk have been identified through genome-wide association studies (GWASs) and large-scale replication studies. These variants are generally common in the general population (minor allele frequency [MAF] of>5%) and with modest effect in breast cancer risk (odds ratios [ORs]<1.5 for risk alleles individually). Since these loci explained only 16% of the familial risk of breast cancer, further study is necessary to clarify the missing heritability. Emerging evidence indicate that low-frequency (MAF ranging from 0.5% to 5%) or rare (MAF<0.5%) variants may have relatively large effects and contribute to the missing heritability of complex diseases. Therefore, it calls for exome-wide association study (EXWAS) to systematically identify low-frequency susceptible loci or genes associated with the risk of breast cancer.This EXWAS comprises two phases. The 1,064 cases of the discovery phase were recruited from the First Affiliated Hospital of Nanjing Medical University, the Cancer Hospital of Jiangsu Province and the Nanjing Drum Tower Hospital, from January 2004 to April 2010 and the 1,125 control subjects were randomly selected from a community-based screening program for non-communicable diseases in Jiangsu Province.1,040 cases and 1,240 controls from Jiangsu Province were included in the replication phase.In the discovery phase, the 1,064 cases and 1,125 controls were genotyped with Illumina Infinium(?) HumanExome Beadchip.247,870 variants in the chip were flow into quality control. A total of 1,032 cases and 1,063 controls with 65,282 polymorphic variants were included after the quality control of the study variables and subjects. We calculated the P values, odds ratio (OR) and 95% confidence intervals (CI) in the additive model by logistic regression. We selected low-frequency or rare variants (MAFs from 0.1% to 5%) which were nonsynonymous substitutions or located in splice junctions and whose single-variant association P values were less than or equal to 0.001 for further genotyping in the replication stage. Simultaneously, their genotyping clusters were clear and only one variant was selected if multiple variants were in linkage disequilibrium (LD; r2≥ 0.5).We further conducted gene-based analysis through both a simple burden test and a sequence kernel associationoptimal test (SKAT-O) with MAF< 0.05 in the discovery phase. A total of 13,001 genes were included.Eventually, we found strong evidence of two breast cancer susceptibility loci. The variants rs200847762 in FKBPL at 6p21.33 and rs1045012 in ARPC1B at 7q22.1 conferred to a decreased risk of breast cancer (rs200847762, G>A, OR= 0.34,95% CI=0.20-0.57, P=4.31×10-5; rs1045012, G>C, OR=0.56,95% CI= 0.43-0.74, P=4.30×10-5). In stratification analyses, we found that the protective effect of rs200847762 was stronger in ER-positive breast cancer (OR=0.18,95% CI=0.06-0.42) than that in ER-negative one (OR=0.59,95% CI=0.31-1.05). Conditional analysis between the two susceptibility loci indicated that they were independent signals of breast cancer susceptibility on 6p21.33 and 7q22.1.In conclusion, our study is the first one to utilize the exome array with breast cancer in Chinese women and serves as an extension to previous examination of common variants on breast cancer risk. It highlights the role of low-frequency genetic variants in breast carcinogenesis, addresses part of the missing heritability in breast cancer risk and provides novel insight into the etiology of breast cancer. Moreover, our newly identified susceptibility markers might be applied as the genetic information reference for screening, diagnosis and treatment of breast cancer.