Breast cancer (BC) is a serious health problem that women face around the world. In 154 of 185 countries, BC is the most frequently diagnosed cancer according to the GLOBOCAN 2018 and is the highest mortality cause in women in more than 100 countries . The most rapid increase in incidence rates of BC shows an incline in countries that have been historically relatively low like South America, Africa, and Asia . BC pathogenesis is affected by several factors, including genetic, family history, lifestyle, endocrine factors, as well as social and economic development . However, there is a compelling need to clarify its exact etiology.
Telomeres are (TTAGGG)n repetitive sequences of DNA that can be found at the ends of chromosomes. Telomeres function is to maintain genetic stability by protecting chromosome during repetitive DNA replication cycles . Due to the end-replication problem, telomeres are shortened in most somatic tissues, and are proposed physiological markers of aging and age-related pathology as well as cancer . The function of telomeres and its role in cancer development is mainly determined by telomere length (TL) . Although shortened telomeres can induce apoptosis, a critical length threshold is required. Otherwise, cells may bypass such signals and lead to what is known as telomere crisis. During telomere crisis, genomes experience an extensively unstable state that is a typical event in cancer progression  .
Telomeres are maintained by the telomerase complex, which consists of the Telomerase reverse transcriptase (TERT) catalytic subunit encoded by gene positioned at chromosome 5p15.33, and the Telomerase RNA component (TERC) encoded by the gene positioned on chromosomal region 3q26, with several other associated proteins required for proper telomerase assembly and recruitment to chromosomes . While TERC is widely expressed, hTERT is downregulated in most human somatic cells leading to progressive telomere shortening .
Given the key role of hTERT in maintaining telomere stability, several common functional single nucleotide polymorphisms (SNPs) of the hTERT gene promoter region may impact the risk of different types of cancers  . The relationship between hTERT polymorphisms and BC risk has been investigated in a number of studies   . Downstream of the hTERT gene, a polymorphic region known as the MNS16A minisatellite region was also found to be linked with the hTERT activity in many types of cancers   , however, the results were inconclusive.
The association of hTERT genomic variation with tumor biology draws attention to the need to verify this relation in different populations with diverse ancestries. In this case-control study, we genotyped a number of hTERT variants (rs2736098G > A, rs2735940C > T and MNS16A) since the previous studies reached paradoxical conclusions regarding their association with cancer risk. We also measured the leukocyte telomere length and analyzed their association with BC risk in a sample of Egyptian females.
This case-control study enrolled 218 Egyptian breast cancer female patients who were referred to the Medical Research Institute and Ayadi Al-Mostakbal hospitals (Egypt) and 178 age-matched healthy females; they were unrelated to the patients and had no history of any type of cancer. Ethical approvals for recruitment were obtained from the local Ethics Committee of Medical Research Institute, University of Alexandria, and informed consent was obtained from each patient and healthy individual.
2.2. DNA Extraction
Whole blood samples were collected from breast cancer patients and healthy controls into EDTA-coated Vacutainer tubes and stored at −80˚C till genomic DNA was extracted. Genomic DNA was extracted using Invitrogen PureLink™ Genomic DNA Mini Kit (Thermo Fischer Scientific, USA) according to the manufacturer’s instructions. The purity and concentration of DNA were evaluated by NanoDrop® ND-1000 UV-Visible Spectrophotometer (Thermo Fischer Scientific, USA). The extracted DNA was stored at −20˚C for further use.
2.3. hTERT Genotyping
Polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) analysis was performed for genotyping of hTERT rs2736098G > A and rs2735940C > T polymorphisms. The primers’ sequences, length of amplified DNA fragments, restriction pattern, and restriction enzymes used are presented in Table 1. PCR reactions were performed in a final volume of 25 µl contained 12.5 µl of 2x Dream Taq green master mix with dual dye (Thermo Fischer Scientific, USA), 100 ng genomic DNA, 0.2 μmol of each appropriate primer. The PCR cycling conditions were: a denaturation step of 5 min at 95˚C; 30 cycles of 30 s at 94˚C, 30 s at 57˚C (for rs2736098G > A) or 56˚C (for rs2735940C > T), and 30 s at 72˚C; a final elongation step of 7 min at 72˚C. Each PCR product was digested using 10 U of the corresponding restriction enzyme Bsp120I at 37˚C for 1 hour then the reaction was stopped by incubation at 80˚C for 10 minutes. After digestion, then the products were electrophoresed on 3% agarose gel containing 0.5 μg/mL ethidium bromide and visualized under UV illumination.
The MNS16A variable number of tandem repeat polymorphisms was genotyped using PCR as Wang et al.  reported previously. The primers’ sequences and length of amplified DNA fragments were also mentioned in Table 1. The PCR conditions started with an initial denaturation step of 5 min at 95˚C; 30
Table 1. Primer sequences, DNA fragment length and restriction enzyme and restriction pattern (if available) for hTERT rs2736098, rs2735940 and MNS16A polymorphisms.
cycles of 30 s at 94˚C, 30 s at 57˚C, and 30 s at 72˚C; a final elongation step of 7 min at 72˚C. The PCR product was then electrophoresed on 3% agarose gel containing 0.5 μg/mL ethidium bromide and visualized under UV illumination.
2.4. Telomer Length Determination
Leukocyte telomere length measurement was carried out according to the quantitative PCR method reported previously by Gil et al. . The rhodopsin gene was selected as single copy gene to normalize the telomere PCR. The primer sequences for both telomere length and rhodopsin were as follows: Telomere Length 1 5’-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3’; Telomere Length 2 5’-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3’ Rhodopsin Forward 5’-CGTGGCCTGGTACATCTTCA-3’ and Rhodopsin Reverse 5’-ACGCCGGCAGGGTCAT-3’. All samples were run in an Applied Biosystems Real-Time PCR System (Applied Biosystems, Foster City, CA). PCR reactions were performed in a final volume of 25 µl contained 12.5 µl of SYBR Green master mix (Thermo Fischer Scientific, USA), 100 ng genomic DNA, 2.5 μl primers, 8 μl DNase-free water. The PCR cycling conditions were a denaturation step of 10 min at 95˚C followed by 25 cycles of 15 s at 95˚C, 120 s at 54˚C. The Ct was calculated for both telomere and Rhodopsin were calculated. Telomere length was represented as a T/S ratio where (T) represented the telomere repeat copy number and (S) represented the single copy gene number according to the equation reported previously .
2.5. Statistical Analysis
Statistical analysis was performed using the Statistical Package for Social Sciences Software (SPSS 21; SPSS Inc.; Chicago, IL, USA). The frequencies of genotypes, alleles between breast cancer patients and healthy control as well as the clinicopathological characteristics were analyzed using Person chi-square test. The association between genotypes and alleles and the risk of breast cancer were assessed by computing the odds ratio (OR) and 95% confidence intervals (95% CI) from unconditional logistic regression analyses. The statistical analysis of telomere length was performed using Mann-Whitney U nonparametric test. A p value of <0.05 was considered statistically significant. The Hardy-Weinberg equilibrium was tested for all polymorphisms.
3.1. Characteristics of Study Population
The age of breast cancer patients and healthy controls enrolled in the study are presented in Table 2. There was no significant difference between patients and controls regarding age (
p = 0.464). Table 2 also presents the distribution of clinical characteristics of breast cancer patients including histological grade, axillary lymph node involvement and receptors’ status.
Table 2. Description of the Clinicopathological Characteristics of Breast Cancer Patients.
3.2. The Impact of hTERT rs2736098G > A, rs2735940C > T and MNS16A on Breast Cancer Risk
The different genotypes of studied hTERT polymorphisms are illustrated in Figure 1. The genotype and allele frequencies of hTERT polymorphisms for breast cancer cases and controls as well as their association with BC risk are presented in Table 3. The statistical analysis of rs2736098G > A frequency distribution showed that the GG increased the risk of breast cancer (OR = 7.484, 95% CI = 2.593 - 21.597, p ≤ 0.001 and OR = 1.146, 95% CI = 1.080 - 1.216, p ≤ 0.001 respectively) compared to AA genotype which significantly lowered the risk of BC (OR = 0.332, 95% CI = 0.195 - 0.563, p ≤ 0.001). Our results also indicated that the G allele was associated with higher BC risk compared to the A allele (OR = 1.743, 95% CI = 1.168 - 2.600, p = 0.006). The rs2735940C > T genotyping showed that the TT genotype was associated with a significant increase in the risk of BC compared to the rest of genotypes (OR = 1.519, 95% CI = 1.008 - 2.289, p = 0.045). However, neither the MNS16A genotypes nor its alleles showed any association with BC risk or protection. The association between the studied hTERT polymorphisms and BC risk was further examined by stratifying the subjects according to tumor histology (Table 4).
3.3. Telomere Length
Subjects in both BC and control groups were stratified into two subgroups according to their age with cut-off 40 years of age. For estimating the leukocyte telomere length, the telomere/single copy gene (T/S) ratio was calculated for all subjects. The statistical analysis revealed that the T/S ratio of BC patients with age less than 40 years showed a significant reduction in TL compared with controls with matched age, however, the TL in both patients and controls with age older than 40 years did not show a significant difference (p ≤ 0.001 and 0.963 respectively) (Figure 2). In our study, the mean TL changed with different hTERT polymorphisms genotypes. For rs2736098G > A, the TL decreased with every G allele, and the T/S ratio was 1.14 for AA, 0.71 for AG, and 0.40 for GG, respectively. The GG genotype was significantly shorter than the AA genotype (p = 0.045). For the rs2735940C > T polymorphism, and T/S ratio was 1.22 for CC, 0.88 for TC, and 0.56 for TT. Both CT and TT genotypes were significantly shorter than the CC genotype (p = 0.002 and < 0.001 respectively). Regarding the MNS16A tandem repeat, the mean T/S ratio was 1.62 for LL, 0.83 for LS and 0.54 for SS genotypes. The LS and SS genotypes were also significantly shorter than the LL genotype (p = 0.012 and 0.062 respectively) (Figure 3).
Given the fact that telomeres are maintained by telomerase complex, it’s been proposed that genetic polymorphisms in its catalytic subunit hTERT can have a significant influence on the predisposition to developing cancer . In the current case-control study, we investigated the association of three SNPs in the hTERT gene with the risk of BC in a sample of Egyptian females. Our results indicated that the GG genotype and G allele of hTERT rs2736098G > A and TT genotype of rs2735940C > T were associated with increased risk for BC, while MNS16A didn’t show any significant association.
Table 3. Genotype and allele frequencies distribution of hTERT polymorphisms (rs2736098, rs2735940 and MNS16A) in breast cancer patients and healthy control groups and their association with breast cancer risk.
Table 4. Association of the hTERT polymorphisms (rs2736098, rs2735940 and MNS16A) genotypes with clinicopathological characteristics.
(1a) (1b) (1c)
Figure 1. Photographs of DNA gel electrophoresis for the detection of hTERT polymorphisms. 1a: rs2736098. Lane 1, AA; Lane 2, AG; lane 3, GG. 1b: rs2735940. Lane 1, TT; lane 2, CC, lane 3, TC. 1c: MNS16A. Lane 1 LL; lane 2 LS; lane 3 SS. L: 50 bp DNA ladder.
Figure 2. Statistical comparison between telomere in BC patients and control groups according to their age group (p < 0.05).
(3a) (3b) (3c)
Figure 3. Mean telomere length of BC patients stratified according to (3a) rs2736098, (3b) rs2735940 and (3c) MNS16A genotypes respectively.
One of the most commonly studied SNPs in the TERT gene is the rs2736098G > A polymorphism, it is associated with the risk of many types of cancer . Although this SNP results in silent or synonymous mutation, thus the encoded amino acid remains unchanged (Asn305Asn), it may affect both telomerase activity and TL because of its location within the gene regulatory elements resulting in alteration of transcription factor binding .
The results of the present work are supported by many previous studies, Oztas et al.  , reported that rs2736098 GG genotype is associated with increased risk of BC among Turkish women (OR = 1.88). Furthermore, G allele of hTERT rs2736098G > A is reported to be associated with BC risk in Iranian population (OR = 1.38). Hashemi et al.  reported that the hTERT rs2736098 G > A variant is associated with BC risk. Their results showed that GA as well as GG and GA + GG increased the risk of BC compared with the AA genotype. The rs2736098 G allele increased the risk of BC compared with the A allele. A meta-analysis released in 2018 indicated the association between BC susceptibility and rs2736098 variant, where A allele had a protective role against BC in allelic, homozygous, and recessive models . Nevertheless, the association of hTERT rs2736098 variant with BC risk is inconclusive in some populations. In the Polish population, Savage et al.  genotyped different SNPs including rs2736098G > A in the hTERT genomic region, and no significant association with BC risk was found. Although, his results suggested that hTERT variants may be associated with the risk in women having a positive family history of BC. On the other hand, in women of African descent, no association was reported .
The association between hTERT rs2735940C > T polymorphism and cancer susceptibility is contentious. Pande et al. and Walsh et al. reported that either T allele or TT genotype of hTERT rs2735940C > T polymorphism may be significantly associated with the risk of developing lung cancer in ethnic Koreans, non-Hispanic white population and subjects of African–American ethnicity  . In addition, TT genotype was linked with the risk of childhood acute lymphoblastic leukemia in Chinese population . Moreover, Bayram et al.  indicated that carriers of TT genotype and/or T allele of hTERT rs2735940C > T polymorphism are at higher risk of developing gastric cancer in codominant, dominant and log-additive inheritance models. In accordance with the aforementioned data, a meta-analysis involved 19,385 cancer patients and 17,558 healthy subjects indicated that cancer risk significantly increased for T allele and/or TT genotype of hTERT rs2735940 polymorphism . On the other hand, few studies reported different results. In Caucasian population, hTERT rs2735940C > T polymorphism has no role in colorectal cancer etiology . Iizuka et al. have reported the T allele of hTERT rs2735940C > T polymorphism may be associated with a decreased risk of epithelial cancer, particularly prostate, lung and prostate cancer .
The MNS16A minisatellite region polymorphism in our study didn’t show any correlation with neither the risk of developing BC. However, the long allele of MNS16A was found to be significantly associated with negative family history and ER+/PR+ receptor pattern. Although MNS16A found to be linked with the hTERT activity in many types of cancers, a meta-analysis suggests that MNS16A is associated with the risk of developing gliomas, not other types of cancer including lung, colorectal, nasopharyngeal and breast cancer in Asian and Caucasian population . This variation may be attributed to the differences in microenvironment of tumors of different sites. This may lead to different consequences for the same polymorphism. Collectively, the results of our chosen polymorphisms are comparable to those reported by Hashemi et al. in Iranian population. However, the MNS16A short allele not genotype was significantly associated with cancer risk .
Quantitative measurement of leukocyte TL of cancer patients with age less than 40 years showed a significant reduction in TL compared with controls with matched age, however, the TL in both patients and controls with age older than 40 years did not show a significant difference. There was a significant association between hTERT polymorphisms and telomere length. Telomere plays an essential part in protecting chromosomes and their progressive shortening in somatic cells induces chromosomal instability  . Telomere shortening is associated with diverse age-related diseases  , yet there are inconsistent reports on the relationship between telomere length and cancer incident. Liu et al. have reported that short leukocyte TL contributes to increased susceptibility of gastric cardia adenocarcinoma and, along with lifestyle risk factors, can be considered as a promising prognostic marker . Moreover, the risk of developing lung cancer risks was related to short telomeres lengths in patients with breathing problems, with age, sex, and smoking adjustment . In 2018, Pavanello et al. proposed that telomeres length was shorter in BC patients than age-adjusted controls however their study failed to link it with the risk of hereditary BC . Excessive telomeres shortening drive too many cells into senescence. Senescent cells acquire a senescence-associated secretory phenotype which has deleterious effects on the tissue microenvironment and promotes tumor progression through proinflammatory signaling . Still, other studies showed inconsistent results. Julin et al. findings suggested an association between the risk of overall prostate cancer, including more aggressive disease, with longer leukocyte TL in men with a positive family history of prostate cancer . Similarly, there is an association between longer leukocyte TL and colorectal carcinoma, but not for its premalignant precursors . In light of the results of these studies, further investigations were required to evaluate the possibility of using TL as a biomarker to evaluate the cancer risk.
From the results of the current study, we can conclude that hTERT rs2736098 and rs27365940, not MNS16A may be associated with increased risk of developing BC in Egyptian females. To our knowledge, this is the first study to investigate these polymorphisms in Egyptian population and their role in breast cancer risk. Also, we can conclude that BC patients younger than 40 years old have shorter leukocyte TL when compared to age-matched controls. Thus, telomere length can be a promising screening marker of breast cancer especially in young population.