Association Between Human Telomerase Reverse Transcriptase Gene Variations and Risk of Developing Breast Cancer

Abstract

Background: Despite a reduction in the number of deaths from cancers made possible by the development of early detection tests, improvements in treatment, changes in the age distribution of the population, and changes of personal behaviors as a result of awareness, breast cancer remains a major health problem worldwide. Breast cancer is the most common cancer and second leading cause of cancer death in women. Several genetic and environmental factors are known to be involved in breast cancer pathogenesis, but its exact etiology is complicated and is not clearly identified. The structure and integrity of telomeres are pivotal for genome stability, and telomere length is maintained by the expression of the telomerase enzyme. The human telomerase reverse transcriptase (hTERT) gene is a principal functional subunit of the telomerase. Several recent studies have provided evidence that hTERT gene variants may have an important role in cancer development. Methods: Three hTERT variants (rs2736100, rs2736098, and rs2853669) were genotyped for 107 breast cancer patients and 110 healthy controls to determine their effect on breast cancer susceptibility. Results: It was observed that hTERT rs2736098 was associated with breast cancer risk (odds ratio [OR] = 1.88; p = 0.034), while rs2736100 and rs2853669 did not significantly differ between the groups. Conclusions: These findings are the first description of hTERT allele distributions in the Turkish population and may contribute to our understanding of breast cancer development. Nevertheless, further large-scale population studies are needed to understand the role of the hTERT polymorphisms and haplotypes in the development of breast cancer.

Introduction

Breast cancer is the most prevalent malignancy in females worldwide, occurring in both hereditary and sporadic forms. It is estimated that more than 1 million women are diagnosed with breast cancer every year (Aloraifi et al., 2015; Sheikh et al., 2015).

In recent years, several common low-penetrance genes, such as human telomerase reverse transcriptase (hTERT), have been identified as potential cancer susceptibility genes (Qi et al., 2012; Pellatt et al., 2013). The ends of linear eukaryotic chromosomes are formed by a special heterochromatic structure, known as the telomere, which protects chromosomes from degradation and, therefore, plays an important role in chromosome stability and integrity (Blackburn, 2001; Blasco, 2003). The telomerase is an RNA-dependent DNA polymerase that is responsible for telomeric DNA synthesis. The telomerase consists of two essential components: (1) a catalytic subunit with the reverse transcriptase activity; and (2) an essential structural RNA component with a sequence complementary to the telomere sequence (Cong et al., 1999; Mocellin et al., 2013). DNA replication cannot effectively copy the ends of chromosomes, and telomeres become progressively shorter as a function of age (i.e., with each cell division). This leads telomeres to generate a DNA damage signal, inducing a stop in cell division that forces cell senescence or cell death. The shortening of the telomere regulates the number of cell divisions for each cell before entering senescence, which has been suggested as being a protection mechanism against cancer development (Callén and Surralles, 2004; Wright and Shay, 2005; Ding et al., 2013). High levels of telomerase activity and maintained telomere length are present in 90% of tumors from all cancer types, thus reactivation of telomerase is critical in human carcinogenicity (Harley, 2008; Vinagre et al., 2014). In addition, several studies have reported that cigarette smoking, oxidative stress, and chronic inflammation might cause telomere shortening (Valdes et al., 2005; Machiela et al., 2015). Individual genetic differences with shorter telomeres are observed with an increased risk of human cancers, including lung and bladder cancers (McGrath et al., 2007; Jang et al., 2008).

Epidemiological studies have reported that some hTERT gene variants are associated with the risk of cancer (e.g., breast cancer) and that chromosome instability is present very early in carcinogenesis. Nevertheless, the molecular mechanisms of this association are unclear and findings are inconclusive (Savage et al., 2007; Choi et al., 2009; Varadi et al., 2009a, 2009b; Haiman et al., 2012; Shadrina et al., 2015). To obtain a better understanding on the hTERT gene polymorphisms in cancer development, comprehensive gene-gene and gene-environment interactions in the related metabolic pathway should be researched in further studies. In this study, we evaluated the association of the common hTERT variants (rs2853669, rs2736100, and rs2736098) with the risk of breast cancer.

Materials and Methods

Subjects

We evaluate the influence of three hTERT variants on the susceptibility to breast cancer in 107 Turkish female patients and 110 ethnic- and age-matched controls. These 107 patients were operated at Acibadem Maslak Hospital Breast Health Center or admitted for follow-up after breast cancer surgery. The diagnosis of breast cancer in the patients was based on clinical examination in combination with imaging and confirmed by pathological assessment. Healthy control volunteers, who never had any type of cancer, were in-patients with various diagnoses (e.g., eye diseases, pulmonary diseases, cardiovascular diseases, and neurological disorders) at the Hospital of Istanbul University. All samples were collected between 2012 and 2015. Demographic and anthropometric factors (ethnicity, age, body mass index [BMI, kg/cm²], and smoking status) were assessed by a short questionnaire. The pathological types of patients were categorized as invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), or ductal carcinoma in situ (DCIS). We also evaluated the association between patient genotypes and the status of the estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor 2 receptor (HER2). All participants provided informed consent and the study was approved by the Ethics Committees of Istanbul and Acibadem University (2011/87-555; 2015/2-9).

Genotyping

Genomic DNA was extracted from whole blood using standard phenol chloroform extraction protocols. Genotyping of hTERT rs2853669 (4814 T>C) variant was performed on Roche LightCycler 480 real-time polymerase chain reaction (PCR) platform. Required DNA purification ensured by High Pure PCR Product Purification Kit and single nucleotide polymorphism (SNP) analysis was performed using LightCycler FastStart DNA Master HybProbe and custom designed LightSNiP assay probe (Roche). Genotyping of hTERT rs2736100 (13647 G>T) and rs2736098 (6077 G>A) variants was performed by PCR restriction fragment length polymorphism (RFLP) methods. The temperature was controlled by a programmable heat block (GeneAmp PCR System 9700; Applied Biosystems). Restriction enzymes were obtained from New England Biolabs and Fermentas. All the other molecular biological chemicals were obtained from Fermentas and Sigma-Aldrich.

For rs2736100 G>T, primers were 5′-gCTgTTTTCCCTgCTgACTT-3′ and 5′-AgAACCACgCAAAggACAAg-3′. The following PCR protocol was used: 94°C for 5 min; 35 cycles of 94°C for 30 s, 59.9°C for 30 s, 72°C for 30 s; and 72°C for 7 min. The PCR product was directly digested with SfcI restriction enzyme at 37°C overnight. Digestion with SfcI produced an uncut 194-bp fragment from the wild-type allele (T) and 101- and 93-bp fragments from the mutant allele (G). For rs2736098 G>A, primers were 5′-gTgACCgTggTTTCTgTgTg-3′ and 5′-AgAggAAgTgCTTggTCTCg-3′. The following PCR protocol was used: 94°C for 5 min; 35 cycles of 94°C for 30 s, 59.8°C for 30 s, 72°C for 30 s; and 72°C for 7 min. The PCR product was directly digested with ApaI restriction enzyme at 37°C overnight. Digestion with ApaI produced an uncut 201-bp fragment from the mutant allele (A) and 123- and 78-bp fragments from the wild-type allele (G). Genotyping was performed blinded to case-control status. A 10% random sample was genotyped twice for quality assurance. In addition, to confirm the genotyping results of the variants, the selected PCR amplified DNA samples (n = 2, for each genotype in the cases and controls) were examined by DNA sequencing. The results were also 100% concordant.

Statistical analyses

Hardy-Weinberg equilibrium analysis was performed using the χ² test. For the analyses of the genotype frequencies, the wild-type category (chosen either as the most common wild-type frequency or arbitrarily if the two alleles showed similar frequencies) was used as the reference group. To evaluate the association between the hTERT genotype frequencies and breast cancer, odds ratios (ORs) and 95% confidence interval (95% CI) were estimated. The haplotypes and their frequencies were estimated by PHASE (Version 2; Stephens and Donnelly, 2003). The distribution of haplotypes in the cases and controls was compared using the χ² test. All statistical analyses were performed using Statistical Package for Social Sciences (SPSS) software (Version 17). A two-sided p-value <0.05 was considered to be statistically significant.

Results

We performed a genetic association study on three common and functional SNPs of hTERT (rs2853669, rs2736100, and rs2736098) with breast cancer risk in Turkish women. There were no significant differences for age (52.37 ± 12.5 vs. 44.4 ± 14.2 years) or BMI (27.9 ± 5.3 vs. 24.5 ± 4.5 kg/cm²) between the breast cancer and control groups, respectively, suggesting that the matching based on these two variables was adequate. The genotypic distributions did not significantly deviate from the Hardy-Weinberg equilibrium in either the patient or control groups for any of the examined SNPs (Table 1). We then analyzed the differences between cases and controls in the distribution of genotype. The hTERT rs2853669 C, rs2736100 T, and rs2736098 G recessive allele frequencies were 0.444, 0.402, and 0.411 in the cancer cases compared with 0.464, 0.391, and 0.464 in the controls, respectively. hTERT rs2736098 was significantly associated with increased risk of breast cancer (p = 0.034). Patients carrying the GG genotype had a higher risk of disease compared to patients carrying the A allele (OR = 1.88; 95% CI = 1.04-3.40). rs2736100 and rs2853669 showed no association with breast cancer (p ≥ 0.632) (Table 1). Compared with the GG genotype, the hTERT rs2736100 T allele was less frequent in the IDC breast cancer type patients (OR = 5.9; 95% CI = 1.3-28.5; p = 0.014). No significant difference in the receptor status was found with the hTERT variants (Table 2).

Table 1.

Allele Frequencies of hTERT Polymorphisms in Cases with Breast Cancer and Healthy Subjects

		Frequency
SNP	Genotype	Case (n = 107)	Control (n = 110)	OR (95% CI)	p
rs2853669 T<C	TT	36	31	TT vs. any C 1.26 (0.71-2.25)	0.435
	TC	47	52
	CC	24	25
rs2736100 G<T	GG	40	37	GG vs.any T 1.15(0.66-2.00)	0.632
	GT	48	56
	TT	19	15
rs2736098 G<A	GG	40	26	GG vs.any A 1.88(1.04-3.40)	0.034
	GA	52	62
	AA	15	20

ORs were adjusted for age, sex, and BMI.

95% CI, 95% confidence interval; BMI, body mass index; hTERT, human telomerase reverse transcriptase; OR, odds ratio; SNP, single nucleotide polymorphism.

Table 2.

Associations Between Clinical/Pathologic Characteristics and hTERT Polymorphisms

		T/C (rs2853669)			G/A (rs2736100)			G/T (rs2736098)
Variables	Patients, n (%)	TT	TC	CC	GG	GT	TT	GG	GA	AA
Pathological type
IDC	63 (58.9)	27	21	15	29	26	8	23	32	8
ILC	8 (7.5)	4	2	2	0	6	2	4	2	2
DCIS	8 (7.5)	1	5	2	2	2	4	3	4	1
Unknown	28 (26.2)	4	19	5	9	14	5	10	14	4
ER status
Positive	55 (51.4)	23	19	13	20	23	12	23	27	5
Negative	20 (18.7)	8	8	4	9	11	0	4	12	4
Unknown	32 (29.9)	5	20	7	11	14	7	13	13	6
PgR status
Positive	38 (35.5)	16	14	8	12	18	8	15	17	6
Negative	38 (35.5)	15	13	10	17	17	4	13	22	3
Unknown	31 (29.0)	5	20	6	11	13	7	12	13	6
HER2 status
Positive	17 (15.9)	6	7	4	9	6	2	6	10	1
Negative	56 (52.3)	23	18	15	20	27	9	21	27	8
Unknown	34 (31.8)	7	22	5	11	15	8	13	15	6

Bold value represents OR = 5.9 (95% CI = 1.3-28.5) and p-value = 0.014 for GG genotype versus any T allele.

DCIS, ductal carcinoma in situ; ER, estrogen receptor; HER, human epidermal growth factor 2 receptor; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; PgR, progesterone receptor.

Next, we examined the association between the hTERT haplotypes (rs2853669-rs2736100-rs2736098) and risk of breast cancer. The results of haplotype-based analyses are shown in Table 2. We observed no significant haplotype effect across all haplotypes (p = 0.330). Eight haplotypes were found, with the hTERT haplotype TGG being the most common. None of the hTERT haplotypes was associated with the development of breast cancer (p < 0.306) (Table 3).

Table 3.

Haplotype Frequencies of hTERT Constructed by PHASE Algorithm and the Association with Breast Cancer Risk

Haplotype			Frequency
rs2853669 T<C	rs2736100 G<T	rs2736098 G<A	Case n = 107	Control n = 110	OR (95% CI)	p
T	G	G	0.178	0.199	1 (reference)	-
C	G	A	0.153	0.164	0.96 (0.37-2.48)	0.920
T	G	A	0.139	0.087	0.58 (0.20-1.66)	0.306
C	G	G	0.132	0.148	1.04 (0.39-2.76)	0.920
T	T	G	0.116	0.206	1.58 (0.61-4.09)	0.348
C	T	A	0.086	0.068	0.70 (0.22-2.27)	0.549
C	T	G	0.102	0.064	0.54 (0.16-1.79)	0.310
T	T	A	0.095	0.064	0.63 (0.20-2.00)	0.431

ORs were adjusted for age and BMI.

Discussion

Telomere dysfunction is an essential feature in carcinogenesis, implicating the involvement of hTERT (Feldser et al., 2003; Qi et al., 2012; Pellatt et al., 2013; Gao et al., 2014). Although up to 80% of the variation of telomere length is estimated to be due to heritable factors, association studies on hTERT SNPs and differences in telomere length and risk of cancer remain inconclusive (Baird, 2010; Bojesen et al., 2013). In recent years, the hTERT gene has been identified as a potential cancer susceptibility gene and telomere-associated gene polymorphisms have been related to breast cancer susceptibility (Savage et al., 2007; Choi et al., 2009; Shen et al., 2010; Haiman et al., 2012; Ledwoń et al., 2013). Therefore, we evaluated the association between functional and common hTERT variations (rs2853669, rs2736100, and rs2736098) and susceptibility of breast cancer in Turkish women.

In this study, we found that the hTERT rs2736098 and rs2853669 variants were associated with a reduction in risk in families with a history of breast cancer (Choi et al., 2009). Haiman et al. (2012) observed a positive association between the 5p15 locus of the hTERT gene and an increased risk of breast cancer, while among Polish women with a positive family history, Savage et al. (2007) suggested a protective effect of the SNPs in this region, including rs2736098. Savage et al. (2007) also reported that the rs2736098 T allele was associated with reduced risk of breast cancer among individuals with a family history of breast cancer. Similarly, Shen et al. (2012) found that the rs2853669 CC genotype was statistically significant (compared with the T allele) in a population-based case-control study conducted with 1067 cases and 1110 controls among Americans (OR = 0.69). In another study by Shen et al. (2012), rs2853669 was shown to be significantly associated with reduced all-cause mortality in a population-based cohort study of 1026 women diagnosed with first primary breast cancer (hazard ratio [HR] = 0.71).

The hTERT rs2736098 G allele was found to be associated with an increased risk of breast cancer in an Iranian population (OR = 1.38) (Hashemi et al., 2014). Ledwoń et al. (2013) have reported that rs2736098 was associated with a decreased risk of breast cancer in a population of Polish women (OR = 0.77). Shadrina et al. (2015) observed that the rs2853669 C allele was associated with an increased risk of prostate cancer (OR ≥1.42), whereas rs2853669 was not associated with the risk of breast cancer in a Russian population. Similar to the results of Shadrina et al. (2015), Varadi et al. (2009a, 2009b) observed no apparent association between a reduction in inherited or sporadic breast cancer risk with rs2853669 in a Swedish population.

Pellatt et al. (2013) observed that hTERT rs2736100 was marginally associated with longer telomere length (p = 0.055) and associated with risk only in postmenopausal women (OR = 1.20). In the present study, we observed that rs2853669 and rs2736100 showed no association with breast cancer (p = 0.435 and p = 0.632, respectively), while rs2736098 was significantly associated with increased risk of breast cancer (p = 0.034) in our population of Turkish women.

As it has been reported by some researchers, hTERT variations, especially rs2736100 and rs2736098, could be an attractive candidate gene that influences the development of cancer (lung, bladder, colon cancers, etc.) with the association between shorter telomere length and lifestyle factors, including smoking and chemically induced oxidative stress (Valdes et al., 2005; Rafnar et al., 2009; Li et al., 2013; Machiela et al., 2015; Wei et al., 2015). In the present study, we could not observe the possible effects of hTERT genetic polymorphisms related to smoking on breast cancer risk (data not shown). Similar to our results, Shen et al. (2010) indicated no effect modifications between hTERT variants and breast cancer risk for subgroups stratified by cigarette smoking, BMI status, and family history.

Several researchers indicated that HER2, ER, epidermal growth factor receptor (EGFR), and leptin caused the hTERT and telomerase inductions in breast cancer patients (Kunimura et al., 1998; Misiti et al., 2000; Goueli and Janknecht, 2004; Vageli et al., 2009; Ren et al., 2010). As it is known, the receptors are ligand-dependent transcription factors capable of direct interaction between the hormone-receptor complex and estrogen responsive elements and contribute to breast cancer development, diagnosis, and prognosis (Gladych et al., 2011). Kyo et al. (1999); reported estrogens were shown to activate telomerase through direct and indirect effects on the hTERT promoter and suggested hormonal control of telomerase activity, cellular senescence, and aging, as well as estrogen-induced carcinogenesis. Similarly, Wei et al. (2015) reported that there was a strong interaction between hTERT and EGFR signaling at the molecular level in etiology of lung cancer. However, we could not observe any association between the status of ER, PgR, and HER2 and the hTERT variants.

In a meta-analysis conducted by Ma et al. (2011), it showed significant associations between shorter telomeres and overall cancer risk. They indicated that the association between shorter telomeres and cancer risk was significant in cancers, including bladder, lung and smoking-related cancers, and cancers in the digestive and urogenital systems in the stratification analysis by tumor type. However, it was not observed in this association in breast cancer. They explained that different biological pathways (such as metabolisms of hormone, tobacco carcinogens, and repair of DNA damage) could interact with telomere length, resulting in different effects on cancer susceptibility. The difference in hormones, particularly estrogen, might affect telomere dynamics through its antioxidant attributes and its ability to stimulate telomerase (Blackburn, 2001). By some researchers, it was indicated that the breast cancer risk might be affected by telomere length among premenopausal women or women with low dietary intake of antioxidants or antioxidant supplements (Shen et al., 2009), but not significant association in postmenopausal breast cancer risk (De Vivo et al., 2009).

Although haplotype analysis is considered an effective method for the identification of functional genetic variations in complex diseases (including breast cancer), we observed no significant haplotype effect across all haplotypes (p = 0.33) and none of the eight hTERT variants identified in this study was associated with the development of breast cancer (p < 0.306). Controversially, Bojesen et al. (2013) showed that a haplotype composed of rs2736107, rs2736108, and rs2736109, which are in linkage disequilibrium with rs2853669, virtually abolished promoter activity in reporter assays. These authors reported that the alleles of this haplotype (that abolished promoter activity) were associated with increased telomere length and that rs2853669 in linkage disequilibrium with the variants could be a proxy for protective effect on survival and recurrence.

In conclusion, these findings are the first reported results of hTERT allele distributions in Turkish women. We found that hTERT rs2736098 might show a protective effect for Turkish women against breast cancer. These data should be useful for understanding breast cancer etiology and in identifying individuals at risk of developing breast cancer. However, our results were obtained with a limited sample size, and therefore, only preliminary conclusions can be drawn. Larger sample sizes and functional assays will be required to confirm these findings.

Footnotes

Acknowledgment

This work was supported by the Research Fund of Istanbul University UDP53631/ONAP52253.

Author Disclosure Statement

No competing financial interests exist.

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