Am J Cancer Res 2020;10(7):2160-2173 www.ajcr.us /ISSN:2156-6976/ajcr0116772 Original Article Variants in SNAI1, AMDHD1 and CUBN in vitamin D pathway genes are associated with breast cancer risk: a large-scale analysis of 14 GWASs in the DRIVE study Haijiao Wang1,2,3*, Lingling Zhao2,3,4*, Hongliang Liu2,3, Sheng Luo5, Tomi Akinyemiju3, Shelley Hwang6, Qingyi Wei2,3,7 1Department of Gynecology Oncology, The First Hospital of Jilin University, Changchun 130021, Jilin, China; 2Duke Cancer Institute, Duke University Medical Center, Durham 27710, NC, USA; 3Department of Population Health Sciences, Duke University School of Medicine, Durham 27710, NC, USA; 4Cancer Center, The First Hospital of Jilin University, Changchun 130021, Jilin, China; 5Department of Biostatistics and Bioinformatics, Duke University School of Medicine, Durham 27710, NC, USA; 6Department of Surgery, Duke University School of Medicine, Durham 27710, NC, USA; 7Department of Medicine, Duke University School of Medicine, Durham 27710, NC, USA. *Equal contributors. Received June 22, 2020; Accepted June 30, 2020; Epub July 1, 2020; Published July 15, 2020 Abstract: Vitamin D has a potential anticarcinogenic role, possibly through regulation of cell proliferation and differ- entiation, stimulation of apoptosis, immune modulation and regulation of estrogen receptor levels. Because breast cancer (BC) risk varies among individuals exposed to similar risk factors, we hypothesize that genetic variants in the vitamin D pathway genes are associated with BC risk. To test this hypothesis, we performed a larger meta-analysis using 14 published GWAS datasets in the Discovery, Biology, and Risk of Inherited Variants in Breast Cancer (DRIVE) Study. We assessed associations between 2,994 (237 genotyped in the DRIVE study and 2,757 imputed from the 1000 Genomes Project) single nucleotide polymorphisms (SNPs) in 33 vitamin D pathway genes and BC risk. In unconditional logistic regression analysis, we found 11 noteworthy SNPs to be associated with BC risk after multiple comparison correction by the Bayesian false-discovery probability method (<0.80). In stepwise logistic regression analysis, with adjustment for age, principal components and previously published SNPs in the same study popula- tions, we identified three independent SNPs (SNAI1 rs1047920 C>T, AMDHD1 rs11826 C>T and CUBN rs3914238 C>T) to be associated with BC risk (P = 0.0014, 0.0020 and 0.0022, respectively). Additional expression quantita- tive trait loci analysis revealed that the rs73276407 A allele, in a high LD with the rs1047920 T allele, was associ- ated with decreased SNAI1 mRNA expression levels, while the rs11826 T allele was significantly associated with elevated AMDHD1 mRNA expression levels. Once replicated by other investigators and additional mechanistic stud- ies, these genetic variants may serve as new biomarkers for susceptibility to BC. Keywords: Breast cancer susceptibility, single nucleotide polymorphism, Vitamin D, expression quantitative trait loci analysis Introduction treatments, the incidence rate is still rising each year (https://seer.cancer.gov/statfacts/ Breast cancer (BC) is the most common malig- html/breast.html). Therefore, it is urgent to nancy and the second leading cause of cancer search for additional risk factors to identify indi- deaths in women in the United States [1]. In viduals who are at high risk of BC for early 2019, there were about 268,600 cases esti- screening and prevention that will reduce the mated to be diagnosed with BC in the United incidence and mortality of BC. States, accounting for 15.2% of all new cancer cases, while an estimated 41,760 individuals There are several risk factors for developing died from BC, accounting for 6.9% of all cancer female BC, including aging, unhealthy lifestyle, deaths [2]. Although the mortality of BC has estrogen status, germ-line mutations and fami- declined due to early detection and advanced ly history [3]. Genetic variation, such as single SNPs in vitamin D pathway and breast cancer risk nucleotide polymorphisms (SNPs) as reported genes are associated with BC risk, and we test- by genome-wide association studies (GWASs) is ed this hypothesis in a larger meta-analysis of believed to contribute to BC risk. However, the 53,107 BC case-control study subjects with risk contribution of individual SNPs is consid- genotyping datasets from 14 previously pub- ered small [4] as identified in the hypothesis- lished GWAS datasets in the DRIVE study. free GWASs, which have always focused on the most important SNPs with a rigorous P value Materials and methods after the most stringent multiple test correc- tion. Furthermore, few of the GWAS-identified Study subjects SNPs are found to be functionally annotated. In the post-GWAS era, hypothesis-driven and The subjects in this case-control meta-analysis combined analyses of all published GWASs are were from 14 out of 17 previously published BC performed to identify cancer-risk associated GWASs from the DRIVE study (phs001265. functional SNPs in a combination of pathway v1.p1), which is different from previously us- analysis, meta-analysis, and functional analy- ed by others named the DRIVE-Genome-Wide sis. With such a hypothesis-driven approach, Association meta-analysis (phs001263.v1.p1); investigators can focus on SNPs with potential and the details of the specific differences biological functions by using available genotyp- between the two studies have been previously ing data from previously published GWAS data- described [19]. The DRIVE study we used was sets with the hope to be able to identify truly one of five projects funded by the NCI’s Gene- cancer-risk associated functional variants. tic Associations and Mechanisms in Oncology (GAME-ON) in 2010. We removed three studi- Vitamin D is a fat-soluble steroid hormone es out of the 17 GWASs: one was “Women of obtained from both dietary sources and sun African Ancestry Breast Cancer Study (WAAB- exposure to ultraviolet B radiation, and once CS)”, because it was on African ancestry study present in the body, it regulates the expression with a relatively small study population, and of genes in many types of tissue [5-7]. In addi- the other two were “The Sister Study (SISTER)” tion, vitamin D may have a potential anticarci- and “the Two Sister Study (2 SISTER)”, becau- nogenic role, by regulating cell proliferation and se they had different research designs from differentiation, apoptosis, immune modulation others and used cases’ sisters as controls. As and estrogen receptor levels [8, 9]. Therefore, a result, all the subjects of European ancestry the vitamin D pathway plays a role in regulating in 14 GWAS studies were included in the fi- cell growth and immune function, relevant to nal analysis, including 28,758 BC cases and tumor progression. For example, studies have 24,349 controls, whose characteristics are demonstrated that the vitamin D pathway has presented in Supplementary Table 1. an effect on T cell function, monocyte/macro- phage differentiation and cytokine production These 14 GWASs included Breast Oncology [10-12]. Other studies have found that vitamin Galicia Network (BREOGAN); Copenhagen Ge- D may affect the pathogenesis, prognosis and neral Population Study (CGPS); Cancer Preven- survival of BC at the cellular level [13, 14]. tion Study-II Nutrition Cohort (CPSII); European Many epidemiological studies have also atte- Prospective Investigation Into Cancer and Nu- mpted to determine associations of vitamin D trition (EPIC); Melbourne Collaborative Cohort levels with risk and mortality of various types of Study (MCCS); Multiethnic Cohort (MEC); Na- cancer [15-17]. In a Brazilian study of post- shville Breast Health Study (NBHS); Nurses’ menopausal BC patients, low vitamin D levels Health Study (NHS); Nurses’ Health Study 2 were found to be a risk factor for individuals (NHS2); NCI Polish Breast Cancer Study (PBCS); negative for estrogen receptor with a higher The Prostate, Lung, Colorectal and Ovarian rate of cell proliferation and positive axillary Cancer Screening Trial (PLCO); Study of Epi- lymph node [18]. However, few studies have demiology and Risk factors in Cancer Heredi- comprehensively investigated the effect of ge- ty (SEARCH); Swedish Mammography Cohort netic variation in vitamin D pathway genes on (SMC); and Women’s Health Initiative (WHI). BC risk. Illumina Infinium OncoArray-500k BeadChip ge- notyping platforms were used for these GWAS Considering the importance of the vitamin D datasets, and information on both sex and age pathway in cancer development, we hypothe- at interview was obtained for all the subjects. size that genetic variants in vitamin D pathway For the cases, there were three other variables 2161 Am J Cancer Res 2020;10(7):2160-2173 SNPs in vitamin D pathway and breast cancer risk including age at diagnosis, estrogen receptor SNP, we calculated odds ratios (ORs) and 95% status, and tumor histology type; for age vari- confidence intervals (CIs) with adjustment for ables, we used the age at diagnosis for the covariates (age and PCs with significant asso- cases and the age at interview for the controls. ciations) by unconditional logistic regression. A written informed consent was obtained from We also adjusted for the four SNPs we previ- subjects by each of the original studies that ously published from the DRIVE Study to iden- were approved by the Institutional Review tify additional independent SNPs. A meta-anal- Boards of the Participating institutions. ysis was further performed using the