The association of CASC16 variants with breast Cancer risk in a northwest Chinese female population

Purpose Genetic variants play a critical role in the development of breast cancer. This investigation aimed to explore the association between CASC16 polymorphisms and breast cancer susceptibility. Methods We conducted a case-control study of 681 patients and 680 healthy individuals to investigate the correlation of five SNPs with breast cancer in a Northwest Chinese female population. Odds ratios (OR) and 95% confidence intervals (CIs) were used to assess the association. Results Our study found that rs4784227 and rs12922061 were significantly related to an increased susceptibility to breast cancer (OR 1.22, p = 0.022; OR 1.21, p = 0.026). While rs3803662 was a protective role in breast cancer risk (OR 0.69, p = 0.042). Stratified analyses indicated that rs4784227 and rs12922061 would increase breast cancer susceptibility at age >  50 years. Rs3803662 was a reduced factor of breast cancer risk by age ≤ 50 years. Rs4784227 was significantly increased risk of breast cancer in stage III/IV. The rs45544231 and rs3112612 had a protective effect on breast cancer with tumor size > 2 cm. Rs4784227 and rs12922061 could enhance breast cancer risk in lymph node metastasis positive individuals. CASC16 rs12922061 and rs4784227 polymorphisms correlated with an increased risk of breast cancer in BMI >  24 kg/m2. Haplotype analyses revealed that Grs45544231 Trs12922061 Ars3112612 and Grs45544231 Crs12922061 Ars3112612 haplotypes decreased breast cancer risk. Conclusion Our study revealed that CASC16 genetic variants were significantly related to breast cancer susceptibility, which might give scientific evidence for exploring the molecular mechanism of breast cancer.


Introduction
Breast cancer (BC) is one of the common malignant tumors in women (Torre et al. 2017) and the 2nd leading cause of cancer death among females in China (Chen et al. 2016a). The China National Cancer Centre recently reported that the incidence of breast cancer is 7.33% in China, of which 6.29% is in the northwest. Breast cancer showed a high mortality (2.70%) and the highest incidence (5.70%) rates in women of Northwest China. As of 2014, the newly increased incidence rates were 25.33, 24.47, and 11.28% among those aged 15-44 years, 45-59 years and 60-79 years, respectively (Wanqing et al. 2014;F B, et al. 2018;Wan-qing et al. 2019). As a kind of multifactorial disease, BC is due to complex non-genetic and genetic factors (Rudolph 2016). Although non-genetic factors such as age, age of menarche, body mass index (BMI), procreative, and menstrual history were associated with an increased susceptibility to breast cancer (Anderson et al. 2004;Islam et al. 2013;Nelson et al. 2012;Zarco et al. 2012). Many recent studies have established that genetic factor also had a vital role in progression of breast cancer (Bray et al. 2013;Sehrawat et al. 2011;Ruiz-Narvaez et al. 2013;Han et al. 2011), and there were 27% of the breast cancer risk influenced by genetic variants (Lichtenstein et al. 2000). In addition, a number of genes including BRCA1, BRCA2, PTEN, TP53, CYP17 and other different genes have demonstrated that their polymorphisms were associated with risk of breast cancer (Nelson et al. 2012;Liao et al. 2018;Walsh and King 2007;Han et al. 2016a;Wang et al. 2016;Lilyquist et al. 2018;He et al. 2014;Chen et al. 2016b;Yang et al. 2018).
Cancer-susceptibility candidate 16 gene (CASC16), also termed LOC643714, is a kind of long non-protein coding RNA and located at chromosome 16q12.1. Data from one study showed that CASC16 gene had a higher expression in breast cancer cells compared with normal cells (Han et al. 2016b). Furthermore, several studies had revealed a correlation between LOC643714 gene and BC (He et al. 2014;Ruiz-Narvaez et al. 2010;Low et al. 2013), but the functions of this gene are still unknown. Liao et al. found that rs12922061 polymorphism of the CASC16 gene was significantly increased susceptibility to breast cancer in southern China population (Liao et al. 2018). And the rs3803662 and rs12922061 also could increase the risk of breast cancer in a Japanese population (Low et al. 2013). However, another study indicated that rs4784227 of LOC643714 could improve BC risk, but rs3803662 and rs3112612 haven't observed a significant association in a southern Chinese population (He et al. 2014). The rs3803662 of LOC643714 also had no significant association with BC risk in African-American women (Ruiz-Narvaez et al. 2010). These differences in the previous results may be due to the race, geographical location, lifestyle, and environmental exposure in specific Chinese population, which may be resulted in differences in the frequencies of genetic polymorphisms. As we all known, the Han Chinese population exhibits a complicated substructure, because the genes of northern China differ greatly from those of Southern China. However, the previous studies mainly focused on rs3803662, rs12922061, and rs3112612 polymorphisms in CASC16 association with breast cancer risk in a Southern Chinese population. The correlation between these three SNPs and breast cancer hadn't been identified in the Northwest Chinese population.
In this case-control study, we selected five SNPs (rs3803662, rs4784227, rs45544231, rs12922061, and rs3112612) in the CASC16 gene according to the previous studies and the 1000 genomes project. We further investigated the association between CASC16 genetic variants and BC susceptibility in a Northwest Chinese female population.  Our findings would give available information for prevention and management of breast cancer.

Study population
In this present case-control study, 681 unrelated Chinese female breast cancer patients and 680 healthy subjects were recruited from the Shaanxi Provincial Cancer Hospital. All patients were newly diagnosed with histological examination and confirmed to be BC. Patients with a history of autoimmunity, secondary tumors, severe infections diseases, other types of cancer and family history of any cancers included breast cancer were excluded. Healthy individuals were matched with the case subjects based on age and ethnicity, who were randomly selected from the cancer-free female population with a routine health examination in the same hospital. Controls with the family history of any cancers were excluded. Each study participant was informed the purpose of the sample collection and their written consent were obtained. The participants' basic information were obtained from the patients or their medical records including age, ethnicity, place of residence, tutor position, lymph node metastasis status, clinical stage, tumor size, estrogenic receptor (ER), progesterone receptor (PR) status, menopausal status, procreative times, age of menarche, and body mass index (BMI). All experiments were carried out depending on the guideline of Helsinki's declaration and our study were approved by the ethics committee of the Shaanxi Provincial Cancer Hospital.

Selection of SNPs and genotype analysis
We selected five polymorphisms of CASC16 in the present study. Of the five SNPs, three polymorphisms (rs3803662, rs12922061, and rs3112612) were chosen basing upon the published papers which they reported that these SNPs might be related to breast cancer susceptibility (He et al. 2014). While rs4784227 and rs45544231 were obtained from the 1000 Genomes Project with a minor allele frequency (MAF) > 5% for further genotype. We extracted genomic DNA from peripheral blood samples from the study participants using a blood genomic DNA extraction kit (GoldMag,  (Xia et al. 2014;Zhou et al. 2015). To validate the genotype results, 10% of samples were randomly selected, and genotypes showed 100% concordance for all SNPs according to Sanger sequencing.

Statistical analysis
The differences in demographic characteristics between the case and control group were analyzed by continuous variable independent sample t-test and category variable Pearson's chi-square test. Hardy-Weinberg equilibrium (HWE) of each SNP was tested by chi-squared test to assess genotype frequencies in controls. Comparisons of distribution in SNP allele and genotype frequencies between case and control were checked by a Pearson chi-squared test or Fisher′s exact test. The association between CASC16 SNPs and BC susceptibility were assessed by computing odds ratios (ORs) and 95% confidence intervals (CIs) in five inheritance models (allele, co-dominant, dominant, recessive, and log-additive) using logistic regression analysis with or without adjustment for age or BMI. Linkage disequilibrium (LD) was constructed by Haploview V4.2 software and haplotype was analyzed by logistic regression. Besides, we also evaluated the relationship between CASC16 polymorphisms and BC patient subgroups with stratification analyses. All statistical analyses were performed using SPSS version 17.0 software (IBM Analytics, Chicago, IL) and PLINK software. All statistical tests were two-tailed and p-value < 0.05 was considered statistical significance.

Characteristics of the study population
The basic information of the study subjects was summarized in Table 1. The average ages were 50.58 ± 9.84 years

Association between CASC16 polymorphisms and BC risk
Five SNPs in the CASC16 gene were selected and analysed in this case-control study. The distribution of allele frequencies between cases and controls was compared using chi-square test (

Haplotype analyses of CASC16 polymorphisms and breast cancer risk
We further examined the linkage disequilibrium (LD) and haplotype analyses of CASC16 polymorphisms in case and control subjects via Haploview software and logistic regression. The LD plot was shown in Fig. 1, and LD block was consisted of three SNPs including rs45544231, rs12922061 and rs3112612. The haplotype analysis revealed that G rs45544231 T rs12922061 A rs3112612 and G rs45544231 C rs12922061 A rs3112612 haplotypes in the CASC16 gene were found to reduce risk of breast cancer (OR = 0.82, 95% CI = 0.69-0.98, p = 0.025; OR = 0.85, 95% CI = 0.73-0.99, p = 0.039; respectively; Table 7).

Discussion
In the present case-control study, 681 breast cancer patients and 680 free-cancer subjects were recruited to evaluate the correlation between CASC16 variants and BC risk in a Northwest Chinese female population. The research showed that CASC16 polymorphisms (rs4784227, rs12922061, and rs3803662) were significantly associated with BC susceptibility. Furthermore, rs4784227, rs12922061, rs3803662, rs45544231, and rs3112612 polymorphisms were associated with breast cancer patients with stratified subgroups including age, lymph node metastasis status, clinical    (Udler et al. 2010). Considerably increased association between rs3803662 in the CASC16 gene and breast cancer was studied in Japanese and Caucasian women (Low et al. 2013) (Guan et al. 2016). In contrast, our present study indicated that rs3803662 played a protective role in BC risk (OR = 0.69, p = 0.042) in a Northwest Chinese population, and the same finding was showed in patients ≤50 years (OR = 0.53, p = 0.014).
However, Edward A et al. suggested that no relationship was found between rs3803662 and breast cancer in African-American population (Ruiz-Narvaez et al. 2010). The SNP rs12922061, located in the first intron of LOC643714, was identified as a susceptibility variant of breast cancer in a Japanese GWAS (Huang et al. 2019). In our study, rs12922061 polymorphism was associated with an increased susceptibility to BC or patients with lymph node metastasis, age ≤ 50 years and BMI > 24 kg/ m 2 individuals. Data from Chen's research showed that the increased association only observed in BC patients, no significant association was found in stratified subgroups in Southeast China population (Chen et al. 2016b). In summary, these results may be due to the differences in geography, ethnicity, and region among population, which leads to genetic variants. Our study also indicated that rs3803662 and rs12922061 played crucial roles in the progression of breast cancer. Rs447842227 polymorphism in CASC16 is also a strong current candidate association with breast cancer risk. This study found that rs4784227 significantly increased susceptibility to breast cancer patients with age > 50 years, clinical stage III/IV, lymph node metastasis status, and BMI > 24 kg/m 2 . These findings were in line with that of He (2014) who confirmed that rs4784227 could increase risk of breast cancer in a Southern Chinese population, while they hadn't identified correlation under stratified analysis (He et al. 2014) due to the difference in population. In a word, our present findings revealed that rs44842227 might be associated with age, clinical stage, lymph node metastasis status, and BMI in breast cancer.
Furthermore, our study firstly revealed that rs45544231 and rs3112612 in CASC16 played protective roles in tumor size > 2 cm individuals. In addition, we also studied linkage disequilibrium (LD) and haplotype analyses of CASC16 polymorphisms in cases and controls. Haplotype analyses disclosed that G rs45544231 T rs12922061 A rs3112612 and G rs45544231 C rs12922061 A rs3112612 haplotypes reduced BC risk.
The major limitation of this study was the fact that we just studied the association between SCAC16 variants and breast cancer in a Northwest Chinese population. Further research in other areas or races in China is an essential step in supplementing the extant data. Besides, we determined the role of CASC16 SNPs in risk of breast cancer but there were still not detecting function of CASC16 in occurrence and evolution of breast cancer. Therefore, next work should focus on exploring the functions of CASC16 in breast cancer. In spite of its limitations, the study certainly adds to our understanding of the association between SNP variants and breast cancer. Moreover, our present work provided the possibility of using these SNPs to diagnose breast cancer in the future.

Conclusions
In summary, CASC16 rs4784227 and rs12922061 were significantly related to increased susceptibility to breast cancer. Stratification analysis revealed that rs4784227 and rs12922061 would increase BC susceptibility in age > 50 years. Rs3803662 was a reduced factor of BC in age ≤ 50 years. Rs4784227 was significantly improved susceptibility to BC patients in stage III/IV. The rs45544231 and rs3112612 had protective effects on BC with tumor size > 2 cm. Rs4784227 and rs12922061 could increase BC risk in lymph node metastasis positive individuals. CASC16 rs12922061 and rs4784227 polymorphisms were correlated with increased BC risk in BMI > 24 kg/m 2 . We noted that G rs45544231 T rs12922061 A rs3112612 and G rs45544231 C rs12922061 A rs3112612 haplotypes reduced BC risk. These findings would give some new insights in the molecular mechanism of breast cancer occurrence.