Association between single nucleotide polymorphism of BRCA1-interacting protein C-terminal helicase 1 and early-onset breast cancer in Uygur and Han women in Xinjiang of China

Abstract

The study investigated the association between single nucleotide polymorphisms (SNPs) of BRCA1-interacting protein C-terminal helicase 1 (BACH1) gene and early-onset breast cancer in Uygur and Han women in Xinjiang. SNPs of BACH1 gene exons were detected by direct sequencing and snapshot technique in 80 Uygur and 80 Han patients with breast cancer and 240 healthy Uygur and 240 healthy Han women (all younger than 40 years old). In Uygur and Han breast cancer patients, the variant C allele of rs4986764 SNP can reduce the risk of breast cancer. The protective effect of this locus is more obvious in Han breast cancer patients without tumor family history (P = 0.001, OR = 0.079). In Uygur breast cancer patients, the GG genotype of rs4986765 (OR = 5.617) and the G genotypes (AG + GG) in the dominant model (OR = 4.254) and the AG of c.587A > G SNP (OR = 7.590) and G genotypes (AG + GG) in the dominant model (OR = 7.590) significantly increased the risk of breast cancer. However, they did not significantly increase the risk of breast cancer in Han population. The present study demonstrates that the rs4986764 mutation of BACH1 gene may reduce the risk of early-onset breast cancer in Uygur and Han populations in Xinjiang. The protective effect was more obvious in Han population without family history of cancer. The change of rs4986765 and c.587A > G sites both increased the risk of breast cancer for Uygur. However, there was no significant correlation with early-onset breast cancer in Han population.

Keywords:

• BRIPI gene
• SNP (Single nucleotide polymorphism)
• genetic susceptibility
• early-onset breast cancer

Introduction

Breast cancer has the highest incidence among all malignant tumors in women [1], and its occurrence and development are a process of multiple factors and stages [2]. Invasion and metastasis of malignant tumors are complex and multistage processes [3], and up-regulation of BRCA1-interacting protein C-terminal helicase 1 (BACH1) can promote the growth and metastasis of tumors [4]. BRCA1 is a breast cancer susceptibility gene discovered in recent years, which is highly correlated with familial/early-onset breast cancer. The main roles of BRCA1 include DNA damage repair, transcriptional regulation in many important pathways and maintenance of genome stability. The incidence of breast cancer in women with BRCA1 mutation is as high as 60–80% in their lifetime. BACH1 is a low-level exogenous gene, which can act on important functional regions of the BRCA1 gene and form a complex with BRCA1. Therefore, BACH1 may play an important role in the etiology of breast cancer [5]. BACH1 is a member of the DEAH-box family of DNA helicases [5]. It is reported that the interaction between BACH1 and BRCA1 depends on the phosphorylation status of BACH1, and this phosphorylation-specific interaction may be involved in the double strand repair function of BACH1 [6]. BACH1 can act as a DNA-dependent ATPase and DNA helicase and play an important role in the repair of DNA double strand breaks [7]. When the amino acid residues required in the reaction between BACH1 and BRCA1 are mutated, the repair function of BRCA1 on the DNA double strand is interrupted [8]. Based on the role of BACH1 in maintaining cell integrity, BACH1 may be a susceptibility gene for breast cancer [9].

Reportedly, BRCA1 interacting protein C-terminal helicase 1 (BRIP1) gene mutation is associated with the risk of breast cancer [10]. In addition, the occurrence and prognosis of breast cancer and other malignant tumors are related to single nucleotide polymorphisms (SNPs) [11]. In the present study, we investigate whether SNPs of the BACH1 gene are associated with the risk of early-onset breast cancer in Uygur and Han populations of Xinjiang Uygur Autonomous Region through sequencing of the exons of the BACH1 gene.

Subjects and methods

Patients

A total of 80 Uygur and 80 Han women with breast cancer who received their first treatment at Affiliated Tumor Hospital of Xinjiang Medical University between January 2010 and June 2013 were included in the present study. The age range of the patients was 30–40 years. Before surgical resection, none of the patients had received radiotherapy or chemotherapy, or had any other types of malignant tumors. In addition, 240 Uygur and 240 Han age-matched healthy women who undertook physical examinations at our hospital in the same time period were included in the control group.

Ethics statement

All procedures performed in this study were approved by the Ethics Committee of Xinjiang Medical University. Written informed consent was obtained from all patients or their families.

DNA extraction

Fasting peripheral blood (5 mL) was collected from all subjects. Blood genome DNA extraction system (DP319-02; Tiangen, Beijing, China) was used to extract DNA according to the manufacturer’s manual. Sample quality and concentration were examined by ultraviolet spectrophotometry (Nanodrop ND2000, Thermo Scientific, Waltham, MA, USA).

DNA sequencing (patient group)

A total of 26 pairs of primers were designed to amplify 26 fragments, covering 20 coding exons of the gene. The primers were designed by Primer3 software (http://primer3.sourceforge.net/) and synthesized by Genesky (Shanghai, China). Sequencing was performed using a BigDye3.1 kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s manual. The sequencing document was analyzed by Polyphred (v6.18; https://droog.gs.washington.edu/polyphred/). Details about the primers are shown in Supplemental Tables S1 and S2.

Snapshot typing (control group)

According to the test results of the case group, the case group sites were screened according to the high frequency site and SNP substitution principle. Three sites, MUTATION (c.587A > G), rs4986765 and rs4986764, were selected, and these sites were examined for the control group.

Two pairs of PCR primers were designed to amplify fragments containing the three sites, and 3 extended primers close to the SNP sites were designed for single base extension. The primers were designed using Primer3 software (http://primer3.sourceforge.net/). PCR products were obtained by multiple PCR using HotStarTaq (Qiagen, Hilden, Germany) according to the manufacturer’s manual. After purification, PCR products were extended by SNaPshot Multiplex kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s manual. Then, the purified product was sequenced by ABI3730XL DNA Analyzer and analyzed using GeneMapper4.1 software (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s manual. Details about the primers are shown in Supplemental Tables S1 and S2. Polyphen and SIFT software were used for prediction of protein functions.

Statistical analysis

Statistical analyses were performed by SPSS 20.0 software (IBM, Armonk, NY, USA). Chi-square (χ²) test was used to compare quantitative data for demographic differences between cases and controls (bicategorized variables). Comparison of demographic characteristics (measurement data) between cases and controls was performed by Student’s t-test (paired t-test). If data variables did not conform to normal distribution, the rank sum test was used for analysis. Multivariate logistic regression analysis was used to calculate the relative risk of genetic polymorphism and breast cancer correlation and interaction. P < 0.05 meant statistically significant difference in the bilateral test. Chi-square goodness-of-fit test was used to calculate whether the genotype distribution of the control group conformed to a Hardy–Weinberg equilibrium.

Results and discussion

Direct sequencing - DNA sequencing map

The ‘A’ base at position 587 in c.587A > G of exon 6 of the BACH1 gene was replaced by ‘G’, and aspartate at position 196 was replaced by serine (Asn196Ser), which is a missense mutation. The ‘A’ base at position 2637 in rs4986765 of exon 19 of the BACH1 gene was replaced by ‘G’ and glutamate at position 879 was not changed (Glu879Glu), which is a synonymous mutation. The ‘T’ base at position 2755 in rs4986764 of exon 19 of the BACH1 gene was replaced by ‘C’, and serine at position 919 was replaced by proline (Ser919Pro), which is a missense mutation.

BACH1 gene mutation sites are distinct in Han and Uygur breast cancer patients

To examine the mutations in the patients and in the control group of Uygur and Han populations, DNA sequencing was performed. In the group of Uygur patients, 4 mutation sites were identified, including 2 missense mutations and 2 synonymous mutations distributed at exon 6, exon 19 and exon 20 (Table 1). Analysis by Polyphen and SIFT software showed that the mutation at rs4986764 might affect the functions of the BACH1 protein (Table 1). In the Uygur control group, missense mutation, synonymous mutation and missense mutation occurred at MUTATION (c.587A > G), rs4986765 and rs4986764 sites, respectively. MAF for the three mutations were 0.004, 0.192 and 0.371, respectively (Table 1). In the group of Han patients, 9 mutation sites were identified, including 5 missense mutations, 2 synonymous mutations and 2 deletions distributed at exon 6, exon 9, exon 19 and exon 20. In addition, rs4986765, rs4986764 and rs4986763 were high frequency sites, with MAF values of 0.14, 0.30 and 0.30, respectively. The other sites were low frequency sites, with MAF values of 0.01. Analysis by Polyphen and SIFT software showed that c.2593C > T and rs4986764 might affect the BACH1 protein function (Table 2). In the Han control group, c.587A > G, rs4986765 and rs4986764 sites had missense mutation, synonymous mutation and missense mutations with MAF values of 0.008, 0.105 and 0.294, respectively. The other sites had no mutations (Table 2). These results suggest that the BACH1 gene mutation sites were distinct in the Han and Uygur breast cancer patients included in this study.

Table 1. Mutations in BACH1 gene exons and functional prediction for Uygur women.

Group	Nucleotide change	SNP loci in database	Position	Amino acid change	Variant type	SIFT protein change	SIFT prediction score	Polyphen prediction score	CEU database MAF	MAF in the present study
BC/N	c.587A > G	MUTATION	EXON6	Asn196Ser	M	Neutral	0.64	0.009	—	0.01/0.004
BC/N	c.2637A > G	rs4986765	EXON19	Glu879Glu	S	No influence	—	—	0.36	0.21/0.192
BC/N	c.2755T > C	rs4986764	EXON19	Ser919Pro	M	Impair	0.00	0.001	0.44	0.43/0.371
BC	c.3411T > C	rs4986763	EXON20	Tyr1137Tyr	S	No influence	—	—	0.44	0.43

Note: Polyphen prediction score > 0.9 means that the protein function is likely to be affected; SIFT prediction score < 0.05 means that the protein function is likely to be affected. BC, breast cancer patients; N, normal women (control group); S, synonymous mutation; M, missense mutation; F, frameshift mutation. CEU is European population; MAF, minimum allele frequency.

Table 2. Mutations in BACH1 gene exons and functional prediction for Han women.

Group	Nucleotide change	SNP loci in database	Position	Amino acid change	Variant type	SIFT protein change	SIFT prediction score	Polyphen prediction score	CHB database MAF	MAF in the present study
BC/N	c.587A > G	MUTATION	EXON6	Asn196Ser	M	Neutral	0.64	0.009	—	0.01/0.008
BC/N	c.2637A > G	rs4986765	EXON19	Glu879Glu	S	No influence	—	—	0.12	0.14/0.105
BC/N	c.2755T > C	rs4986764	EXON19	Ser919Pro	M	Impair	0.00	0.001	0.28	0.30/0.294
BC	c.3411T > C	rs4986763	EXON20	Tyr1137Tyr	S	No influence	—	—	0.28	0.30
BC	c.2593C > T	MUTATION	EXON19	Arg865Trp	M	Impair	0.00	1.000	—	0.01
BC	c.1225A > G	MUTATION	EXON9	Ser409Gly	M	Neutral	0.17	0.022	—	0.01
BC	c.2830C > G	rs140233356	EXON19	Gln944Glu	M	Neutral	0.05	0.281	0.00	0.01
BC	c.2990_2993del	MUTATION	EXON20	Thr997ArgfsX61	F	—	—	—	—	0.01
BC	c.2992_2995del	MUTATION	EXON20	Lys998GlufsX60	F	—	—	—	—	0.01

Note: Polyphen prediction score > 0.9 means that the protein function is likely to be affected; SIFT prediction score < 0.05 means that the protein function is likely to be affected. BC, breast cancer patients; N, normal women (control group); S, synonymous mutation; M, missense mutation; F, frameshift mutation. CHB is northern Chinese Han population; MAF, minimum allele frequency.

Abnormal expression of BACH1 is associated with breast cancer risks [12]. In addition, single allele mutations of the BACH1 gene have been proven to be the main factor leading to over-expression of BACH1, and these mutations may increase the susceptibility of hereditary breast cancer [13]. A meta-analysis shows that the BACH1 919Ser polymorphism (rs4986764) may reduce the risk of breast cancer in Caucasian women, especially postmenopausal women with family history of breast cancer or without BRCA1/2 mutations [14]. A study on 319 breast cancer patients and 306 healthy women in the Han population shows that the rs4986764 allele (exon 18) is weakly correlated with breast cancer, and the frequency of the C allele at rs4986764 in the patients was significantly higher than that in the control group [15]. By contrast, another meta-analysis indicates that the rs4986764 polymorphism is not a susceptible allele for breast cancer [16].

Risk factors for breast cancer in Uygur and Han populations

Next, we analyzed the risk factors for breast cancer in Uygur and Han populations. The t-test indicated that in the Uygur population, the age of menarche in the group of patients was significantly lower than that in the control group (P < 0.05), and the age at first birth in the group of patients was significantly higher than that in the control group (P < 0.05) (Table 3). In the Han population, the percentage of subjects with family history of malignant tumors in the group of patients was significantly higher than that in the control group (P < 0.05) (Table 3). These results suggest that lower age of menarche and higher age at first birth may be risk factors for breast cancer in the Uygur population, and family history of malignant tumors is a risk factor for breast cancer in the Han population.

Table 3. Risk factors for breast cancer in Uygur and Han populations.

	Uygur		P value	Han		P value
	Case group (N = 80)	Control group (N = 240)		Case group (N = 80)	Control group (N = 240)
BMI ($\overline{x}\pm s$)	24.16 ± 3.92	24.16 ± 3.57	0.731	23.02 ± 3.92	23.02 ± 3.65	0.963
Age of menarche ($\overline{x}\pm s$)	13.15 ± 1.64	13.78 ± 1.66	0.003	13.28 ± 1.59	12.98 ± 1.28	0.099
Age at first birth ($\overline{x}\pm s$)	26.93 ± 2.63	24.20 ± 4.38	0.000	27.25 ± 3.77	27.33 ± 3.24	0.864
Number of pregnancies
0–1	16 (20.0)	64 (26.7)		34 (42.5)	98 (40.8)
≥ 2	64 (80.0)	176 (73.3)	0.233	46 (57.5)	142 (59.2)	0.793
Family history of tumors
Yes	4 (5.0)	22 (9.2)		18 (22.5)	24 (10.0)
No	76 (95.0)	218 (90.8)	0.237	62 (77.5)	216 (90.0)	0.006

Note: BMI, body mass index.

Polymorphism of the BACH1 gene is closely related to early onset of breast cancer in Uygur women

To examine the correlation between the BACH1 gene SNPs and breast cancer of Uygur women, multivariate logistic regression analysis was employed. At rs4986764 loci, the TT, TC and CC genotype frequencies in the group of patients were 20.0%, 45.0% and 35.0%, respectively, being significantly different from those in the control group (P < 0.05). Using the wild-type TT genotype as control, the TC, CC and C genotypes (TC + CC) in a dominant model were all able to reduce the risks of breast cancer (for TC, OR = 0.086 and 95%CI = 0.023 - 0.324; for CC, OR = 0.044 and 95%CI = 0.012 - 0.166; for (TC + CC), OR = 0.058 and 95%CI = 0.016 - 0.213). At the rs4986765 loci, AA, AG and GG genotype frequencies in the group of patients were 5.0%, 32.5% and 62.5%, respectively, being significantly different from those in the control group (P < 0.05). GG and (AG + GG) dominant models were able to increase the risks of breast cancer (for GG, OR = 5.617 and 95%CI = 1.785 - 17.672; for (AG + GG), OR = 4.254 and 95%CI = 1.403 - 12.901). At MUTATION (c.587A > G), the AA, AG and GG genotype frequencies in the group of patients were 97.5%, 2.5% and 0%, respectively, being significantly different from those in the control group (P < 0.05). AG and (AG + GG) dominant model increased the risks of breast cancer (for AG, OR = 7.590 and 95% CI = 1.005 - 57.299; for (AG + GG), OR = 7.590 and 95% CI = 1.005 - 57.299) (Table 4). These results indicate that the polymorphism of the BACH1 gene is likely to be closely related to the early onset of breast cancer in Uygur women, and SNP changes at different loci have different risk levels. Hardy–Weinberg’s law of genetic balance test showed that the results of the mutation locus genotype in Uygur and Han control population conformed to the law of genetic balance (P > 0.05) (Tables 4 and 5).

Table 4. Correlation between frequency distribution of BACH1 polymorphism loci and risks for early-onset breast cancer in Uygur population.

	Case group (N = 80)	Control group (N = 240)	OR	95% CI	P value
rs4986764
TT	16 (20.0)	4 (1.7)	1.000	reference
TC	36 (45.0)	84 (35.0)	0.086	0.023-0.324	0.001
CC	28 (35.0)	152 (63.3)	0.044	0.012-0.166	0.001
TC + CC	64 (80.0)	236 (98.3)	0.058	0.016-0.213	0.001
rs4986765
AA	4 (5.0)	40 (16.7)	1.000	reference
AG	26 (32.5)	98 (40.8)	0.336	0.103-1.095	0.070
GG	50 (62.5)	102 (42.5)	5.617	1.785-17.672	0.003
AG + GG	76 (95.0)	200 (83.3)	4.254	1.403-12.901	0.011
MUTATION (c.587A > G)
AA	78(97.5)	238(99.2)	1.000	reference
AG	2(2.5)	2(0.8)	7.590	1.005-57.299	0.049
GG	0(0.0)	0(0.0)	—	—	—
AG + GG	2(2.5)	2(0.8)	7.590	1.005-57.299	0.049

Note: OR, odds ratio; 95% CI, 95% confidence interval; BACH1, BRCA1-interacting protein C-terminal helicase 1.

Table 5. Correlation between frequency distribution of BACH1 polymorphism loci and risks for early-onset breast cancer in Han population.

	Case group (N = 80)	Control group (N = 240)	OR	95% CI	P value
rs4986764
TT	10 (12.5)	4 (1.7)	1.000	reference	—
TC	28 (35.0)	42 (17.5)	0.298	0.083-1.065	0.062
CC	42 (52.5)	194 (80.8)	0.094	0.028-0.322	0.000
TC + CC	70 (87.5)	236 (98.3)	0.130	0.039-0.435	0.001
rs4986765
AA	0 (0.0)	16 (6.7)	1.000	reference
AG	22 (27.5)	108 (45.0)	—	—	0.998
GG	58 (72.5)	116 (48.3)	—	—	0.998
AG + GG	80 (100.0)	224 (93.3)	—	—	0.998
MUTATION（c.587A > G）
AA	78 (97.5)	236 (98.3)	1.000	reference	—
AG	2 (2.5)	4 (1.7)	1.910	0.303-12.032	0.491
GG	0 (0.0)	0 (0.0)	—	—	—
AG + GG	2 (2.5)	4 (1.7)	1.910	0.303-12.032	0.491

BACH1 gene polymorphism is closely related to early onset breast cancer in Han women

To examine the correlation between the BACH1 gene SNPs and breast cancer in Han women, multivariate logistic regression analysis was performed. At the rs4986764 loci, using the wild-type TT genotype as control, the CC and C genotypes (TC + CC) in the dominant model were significantly different between the group of patients and the control group (P = 0.000 and P = 0.001, respectively), and the CC and C genotypes (TC + CC) in the dominant model significantly reduced the risks of breast cancer (for CC, OR = 0.094 and 95%CI = 0.028 - 0.322; for (TC + CC), OR = 0.130 and 95%CI = 0.039 - 0.435) (Table 5). Moreover, this protective effect was more prominent in the patients without family history of tumors (P = 0.001 and OR = 0.079) (Table 6). At the rs4986765 and c.587A > G loci, no significant differences were observed between the control group and the group of patients (P > 0.05) (Table 5). The results indicate that the BACH1 gene polymorphism is likely to be closely related to the early onset of breast cancer in Han women, and only the SNP at the rs4986764 locus can reduce the risk of breast cancer in contrast to Uygur women.

Table 6. Correlation analysis between rs4986764 locus of BACH1 gene and family history of breast cancer in Han population.

Family history of tumors	Genotype	Case group (N = 80)	Control group (N = 240)	OR (95% CI)	P value
	rs4986764
No	TT	6 (7.5)	2 (0.8)	1.000 (reference)
	TC + CC	50 (62.5)	212 (88.3)	0.079 (0.015-0.401)	0.001
Yes	TT	10 (12.5)	2 (0.8)	1.000 (reference)
	TC + CC	14 (17.5)	24 (10.1)	0.117 (0.022-0.611)	0.005

In the present study, in silico analysis by Polyphen and SIFT showed that mutations at rs4986764 in early-onset breast cancer of Uygur and Han women may disrupt the function of the BACH1 protein. However, correlation analysis showed that SNPs at the rs4986764 locus could significantly reduce the risks of early-onset breast cancer in Uygur and Han women. This discrepancy between protein function prediction and correlation analysis may be due to differences between the protein prediction method and the experimental assays, and further analysis of protein functions is needed.

It is reported that the common variant E879E (rs4986765) exists in Chinese breast cancer patients with negative expression of BRCA1/BRCA2 [17], being consistent with previous reports [6, 18]. However, the mutations at rs4986765 and MUTATION (c.587A > G) sites are not significantly different between the group of patients and the control group in the Han population in the present study. This might be due to the limited sample size. Of note, changes at rs4986765 significantly increased the risks of breast cancer in the Uygur population. Especially, the GG and (AG + GG) dominant model increased the risks of breast cancer by 5.6-fold and 4.2-fold, respectively. Therefore, it is necessary to perform larger-scale screening at this locus, especially in the Uygur population. Moreover, mutations occur in both the group of patients and the control group at the c.587A > G locus, and the AG and (AG + GG) dominant model both increased the risk of breast cancer in the Uygur population by 7.5-fold.

In the present study, the patients were aged between 30 and 40 years, so the subjects in the control group were also age-matched, 30–40 years old. However, women younger than 40 years old may still develop breast cancer in the future. Therefore, it is necessary to enlarge the sample size of the control group and include healthy women older than 40 years. The loci reported in the present study are not yet studied at a functional level. Therefore, the present study also provides theoretical basis for further functional studies on these loci.

Conclusions

The mutation sites of the BACH1 gene in the Uygur and Han nationalities are different. The present study demonstrates that the rs4986764 mutation of BACH1 gene may be associated with reduced risk of early-onset breast cancer in Uygur and Han populations in Xinjiang. The potentially protective effect was more obvious in the Han population without family history of cancer. The polymorphisms of rs4986765 and c.587A > G sites were both associated with increased risk of breast cancer in the Uygur patients. However, there was no significant correlation with early-onset breast cancer in the Han population. It is necessary to increase the sample size to further screen for meaningful mutation sites, so as to provide more evidence for breast cancer screening at an early stage.

Disclosure statement

The authors declare that they have no competing interests.

Additional information

Funding

The present study was supported by the [Natural Science Foundation of Xinjiang Uygur Autonomous Region] under Grant [No. 2017D01C408].