Genetic variants in telomerase reverse transcriptase (TERT) and telomerase-associated protein 1 (TEP1) and the risk of male infertility

Gene 534 (2014) 139–143

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Genetic variants in telomerase reverse transcriptase (TERT) and telomerase-associated protein 1 (TEP1) and the risk of male infertility Lifeng Yan a, Shengmin Wu b, Shenghu Zhang b, Guixiang Ji b,⁎, Aihua Gu a,⁎⁎ a b

State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing 210029, China Nanjing Institute of Environmental Sciences/Key Laboratory of Pesticide Environmental Assessment and Pollution Control, Ministry of Environmental Protection, Nanjing 210042, China

a r t i c l e

i n f o

Article history: Accepted 1 November 2013 Available online 20 November 2013 Keywords: TERT TEP1 SNPs Male infertility

a b s t r a c t Telomeres are critical in maintaining genomic stability and integrity, and telomerase expression in spermatogonial stem cells is responsible for the maintenance of telomere length in the human male germline. Genetic variants in telomere-associated pathway genes might affect telomere length and chromosomal stability, and subsequently disease susceptibility. Thus, we hypothesize that single nucleotide polymorphisms (SNPs) in this pathway could contribute to male infertility risk. In a case–control study of 580 male infertility cases and 580 matched controls, 8 common SNPs in telomerase reverse transcriptase (TERT) and telomeraseassociated protein 1 (TEP1) were genotyped. Overall, we found that TERT rs2736100 was inversely associated with male infertility risk (adjusted odds ratio (OR) = 0.66, 95% confidence interval (CI): 0.47– 0.92; P trend = 0.011), whereas TEP1 rs1713449 was positively associated with risk of male infertility (adjusted OR = 1.39, 95% CI: 1.20–1.62; P trend b 0.001). In addition, subjects carrying risk genotypes of these both loci had a two-fold (95% CI: 1.34–3.15) increase in the risk of male infertility, indicating a significant gene–gene interaction between these two loci (P for multiplicative interaction = 0.009). Moreover, linear regression analysis showed that individuals carrying the TEP1 rs1713419 variants have significantly higher levels of sperm DNA fragmentation (β = 2.243, P = 0.016). In conclusion, our results give the first evidence that genetic variations of TERT rs2736100 and TEP1 rs1713449 were associated with susceptibility to male infertility. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Telomeres, specialized DNA–protein structures that cap the ends of linear eukaryotic chromosomes, are critical for genomic stability and integrity by protecting the chromosomal termini against nucleolytic degradation, end-to-end fusion, and irregular recombination (de Lange, 2005; McEachern et al., 2000). They consist of repetitive DNA (TTAGGG nucleotide repeats) with a single-stranded overhang and the associated telomere protein complex (de Lange, 2005). The number of the telomere repeats decreases with each cell division, and eventually reaches a critical state, at which time cellular senescence and/or apoptosis is normally triggered (Rodier et al., 2005). Therefore, the length of telomere repeats is essential for telomere maintenance and short telomere length decreases the cell's integrity. Telomerase is a ribonucleoprotein that adds TTAGGG repeats de novo after each cell division and stabilizes telomere length (Dahse Abbreviations: TERT, telomerase reverse transcriptase; TEP1, telomerase-associated protein 1; SNP, single nucleotide polymorphism; OR, odds ratio; LD, linkage disequilibrium; FDR, false discovery rate; TUNEL, terminal-deoxynucleoitidyl transferase mediated nick end labeling; BMI, body mass index; TERC, telomerase RNA component; MAF, minor allele frequency; CI, confidence interval; DSB, DNA double-strand break. ⁎ Corresponding author. Tel.: +86 25 85287201. ⁎⁎ Corresponding author. Tel.: +86 25 86862834. E-mail addresses: [email protected] (G. Ji), [email protected] (A. Gu). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.11.008

et al., 1997). Normally, telomerase is active in specific germ line cells, proliferative stem cells of renewal tissues, and immortal cancer cells, while inactive in normal human somatic cells (Jiang et al., 1999; Shay and Bacchetti, 1997; Wright et al., 1996). Worthy to mention, telomerase remains active in germline cells during spermatogenesis to ensure the transmission of full-length chromosomes to progeny. By examining the telomerase activity during the development of the rat testis from birth to adulthood, this activity is found in meiotic spermatocytes with high expression levels in the type A spermatogonial stem cells, but is down-regulated during spermatogenesis, and is absent in the differentiated spermatozoa (Ravindranath et al., 1997). Previous studies have shown that telomere dysfunction may lead to fertility defects (Hemann et al., 2001) and sperm DNA fragmentation (Rodriguez et al., 2005), and telomerase deficiency on germ cells can cause meiotic telomere dysfunction (Liu et al., 2002). Telomerase reverse transcriptase (TERT), a catalytic component of telomerase, is the rate-limiting catalytic subunit of the telomerase enzyme and hence, is responsible for the maintenance of telomere DNA length, chromosomal stability, and cellular immortality (Cheung and Deng, 2008; Cong et al., 1999). The TERT gene is subdivided into 16 exons and 15 introns located at 5p15.33, which is the third region associated with the multiple cancers besides the other two loci: 8q24 and 9p21 (Turnbull et al., 2010; Wick et al., 1999). The introduction of TERT gene into normal somatic diploid cells would result in an induced

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telomerase activity and significantly lengthened telomeres (de Lange, 1998), which confirms the determinant role of TERT in telomerase activity. Telomerase-associated protein 1 (TEP1) is another component of the telomerase ribonucleoprotein complex and is responsible for catalyzing the addition of new telomeres to chromosomes. TEP1 contains a region homologous to the Tetrahymena telomerase protein p80, which is sufficient for its interaction with the telomerase, a putative NTPase motif and a region of WD-40 repeats (Poderycki et al., 2005). Though TEP1deficient mice showed no apparent change in telomere length or telomerase activity (Liu et al., 2000), immunoprecipitates of TEP1 possess telomerase activity and TEP1 is associated with TERT and telomerase RNA component (TERC) (Nakayama et al., 1997). Thus, we hypothesized that common genetic variation (minor allele frequency (MAF) greater than 5%) in the form of single nucleotide polymorphisms (SNPs) in TERT and TEP1 could affect susceptibility to male infertility. To test the hypothesis, the association between 8 genetic variations in both genes, including TERT rs2735940 (CNT), TERT rs2736100 (TNG), TERT rs2736098 (GNA), TEP1 rs1713419 (TNC), TEP1 rs938886 (GNC), TEP1 rs1713449 (CNT), TEP1 rs1760904 (CNT), TEP1 rs1760903 (TNC), and male infertility risk was evaluated in a case–control study in ethnic Han-Chinese.

Table 1 Primary information for 8 genotyped SNPs in TERT and TEP1. Genotyped SNPs

Region

MAF for Chinese in databasea

P value for HWE test

TERT: rs2735940 CNT TERT: rs2736100 TNG TERT: rs2736098 GNA TEP1: rs1713419 TNC TEP1: rs938886 GNC TEP1: rs1713449 CNT TEP1: rs1760904 CNT TEP1: rs1760903 TNC

Promoter Intron 2 nsSNP/A305A 3′ UTR nsSNP/M2486I nsSNP/V2214I nsSNP/P1195S nsSNP/C1055R

0.447 0.407 0.411 0.407 0.389 0.384 0.372 0.378

0.767 0.509 0.296 0.363 0.934 0.908 0.574 0.370

Abbreviations: MAF, minor allele frequency; HWE, Hardy–Weinberg equilibrium. a Minor allele frequency in the Chinese (CHB, Han Chinese in Beijing, China) population, as reported in dbSNP database.

(Applied Biosystems), which employs a chip-based Taq-Man genotyping technology. Genotyping was conducted according to the manufacturer's protocol, and genotypes were identified by the OpenArray SNP Genotyping Analysis Soft-ware version 1.0.3. For quality control, 10% of the samples were randomly genotyped again, and 5% of the selected PCR-amplified DNA samples, randomly chosen among wild-type, heterozygous and mutant subjects, were examined by direct sequencing. The results were 100% concordant.

2. Materials and methods 2.3. DNA fragmentation analysis 2.1. Subjects and sample collection The study was approved by the Ethics Review Board of Nanjing Medical University. All studies involving human subjects were conducted under full compliance with government policies and the Helsinki Declaration. A total of 1657 infertile patients, diagnosed with unexplained male factor infertility, were drawn from the Centre of Clinical Reproductive Medicine (NJMU infertility center) between April 2005 and March 2009. All patients had undergone complete historical and physical examinations. Those with a history of orchitis, cryptorchidism, obstruction, congenital bilateral absence of vas deferens, cytogenetic abnormalities and Y chromosome microdeletions were excluded from this study (Wu et al., 2007). In the final analysis, a total of 580 subjects ranging from 24 to 42 years old including 137 infertility patients with non-obstructive azoospermia, 125 infertility patients with oligozoospermia (sperm counts b20 × 106/ml) and 318 infertility patients with normal count (sperm counts ≥ 20 × 106/ml) were eligible for this study. The control group consisted of 580 fertile men ranging from 25 to 40 years old who had fathered at least one child without assisted reproductive technologies. All participants were ethnic Han-Chinese and completed an informed consent form. Participants completed a detailed questionnaire that included information pertaining to their age, cigarette smoking frequency, alcohol and tea consumption, vitamin regimen and abstinence time. Each subject then donated 5 ml blood that was used to extract genomic DNA and an ejaculate for routine semen analysis. The semen was analyzed for sperm concentration, motility and morphology, and the analysis was performed according to the WHO criteria (Lu et al., 2010). 2.2. SNP selection and genotyping The SNPs were selected based on the following criteria: i) they had a high frequency of the rare allele (to allow the highest statistical power to detect associations) and ii) they were of potential functional significance, i.e. those located at 5′-flaking regions, UTRs, coding regions or 3′-UTRs, according to the databases of the International HapMap Project and NCBI dbSNPs. Finally, we identified 8 potential functional polymorphisms from TERT and TEP1 (Table 1). As described in our previous study (Ji et al., 2012), genomic DNA was isolated from leukocyte pellets of the venous blood by phenol–chloroform extraction with proteinase K digestion. Genotyping was performed using the OpenArray platform

A detailed protocol of the terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay for human sperm has been described previously (Sun et al., 1997). We used flow cytometry to detect TUNEL staining of sperm from patients with sperm concentrations N5 × 106/ml. This assay has been shown to be a feasible and sensitive way to detect DNA fragmentation (Muratori et al., 2008). TUNEL labeling was carried out using a Cell Death Detection kit (APO-DIRECT kit; BD Biosciences PharMingen) according to the manufacturer's instructions. Briefly, semen samples, frozen at − 70 °C, were thawed in a 37 °C water bath and immediately diluted with buffer (0.15 M NaCl, 0.01 M Tris, 0.001 M EDTA, pH 7.4) to obtain a sperm concentration of 1–2 × 106/ml. Washed sperm were resuspended in 2% paraformaldehyde for 30 min at room temperature. After rinsing in PBS, samples were resuspended in permeabilization solution (0.2% Triton X-100, 0.1% sodium citrate) for 10 min on wet ice. TUNEL reagent (50 μl) was added to each sample. For each batch, a negative control lacking the terminal deoxynucleotidyl transferase and a positive control treated with DNase I were included to ensure assay specificity. After incubation for 1 h at 37 °C, samples were analyzed immediately by flow cytometry (FACSCalibur; BD Biosciences Pharmingen). Flow during the analysis was controlled at approximately 500 spermatozoa/s, and 10,000 cells were analyzed for each sample. The percentage of FITC-positive cells (FL1 channel) was calculated as the percentage of cells with a fluorescence intensity exceeding the threshold obtained with the negative control. 2.4. Statistical analysis The statistical analyses were performed with the Statistical Analysis System (version 9.1.3, SAS Institute, Cary, NC, USA). Differences in selected demographic variables as well as smoking and alcohol status between the cases and the controls were evaluated by the χ2-test. The Student's t-test was used to evaluate continuous variables, including age and pack-years of cigarette smoking. Hardy–Weinberg equilibrium was tested by a goodness-of-fit χ2-test. The risk of male infertility was estimated as odds ratio (OR) and 95% confidence interval (CI) using unconditional multivariate logistic regression. When appropriate, the ORs were adjusted for age, cigarette smoking and alcohol use. The false discovery rate (FDR) method was applied to the P values for trends to reduce the potential for spurious findings due to multiple testing. The

L. Yan et al. / Gene 534 (2014) 139–143

potential gene–gene interactions were evaluated by logistic regression analyses and tested by comparing the changes in deviance (−2 log likelihood) between models of main effects with or without the interaction term. Sperm concentration and sperm DNA fragmentation were normalized by natural logarithm (ln) transformation. Linear regression models were used to estimate the association with ln-transformed sperm concentration and ln-transformed sperm DNA fragmentation for each SNP independently. Models were adjusted for age, smoking status, drinking status and abstinence time. FDR corrections were conducted for correction of multiple statistical tests. All P values were twosided, with P b 0.05 considered to be statistically significant.

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Table 2 Genotypes of cases and controls and their association with the risk of male infertility. Gene SNP

Genotype Cases N

TERT

rs2735940 CC CT

TT

rs2736100 TT TG

3. Results GG

3.1. Characteristics of the study population The distributions of selected characteristics between male infertile patients and controls were previously described elsewhere (Ji et al., 2013). Overall, case patients and control subjects were adequately matched on age, drinking status, body mass index (BMI) and abstinence time (P N 0.05). However, there were more smokers in the case group compared with the control group (P = 0.046). Moreover, there was a significant difference between cases and controls with respect to semen concentration (cases versus controls: 73.6 ± 49.37 × 106/ml versus 115.2 ± 76.58 × 106/m, P b 0.001) and sperm motility (37.9 ± 13.49% versus 65.3 ± 12.52%, P b 0.001).

rs2736098 GG GA

AA

TEP1

CC

3.2. Individual single nucleotide polymorphism and susceptibility to male infertility

rs938886

Primary information on the 8 functional SNPs found in the Chinese population in the HapMap database is shown in Table 1. All tested genotypes were in Hardy–Weinberg equilibrium in control groups (P N 0.05). The main effect models for all the genotypes are presented in Table 2. Overall, two SNPs exhibited associations with the risk of male infertility. Individuals with TERT rs2736100 homozygous variant genotype (GG) were significantly associated with reduced risk of male infertility (adjusted OR = 0.66, 95% CI: 0.47–0.92) when compared with the common homozygous genotype (TT). Trend χ2-test showed that the male infertility risk was significantly decreased in a dose–dependent manner (Ptrend = 0.011). Moreover, TEP1 rs1713449 variant genotypes (TT) were associated with significantly increased risk of male infertility compared with the common homozygous CC genotype (adjusted OR = 1.39, 95% CI: 1.20–1.62; Ptrend b 0.001).

3.4. Association of semen quality with gene polymorphisms Considering the essential role of telomeres in chromosomal stability by distinguishing chromosomal ends from DNA double strand breaks, we further evaluated the effects of these functional genetic variants on sperm DNA integrity, sperm concentration and sperm motility. Of the

GG GC

CC

rs1713449 CC CT

TT

rs1760904 CC CT

TT

rs1760903 TT TC

3.3. Effects of gene–gene interaction on male infertility risk Given that TERT and TEP1 genes are all components of telomerase ribonucleoprotein complex, we hypothesized that an enhanced risk could be detected when unfavorable alleles are combined in a given patient. To test this hypothesis, statistical gene–gene interactions between the TERT rs2736100 and TEP1 rs1713449 polymorphisms were examined. As shown in Table 3, 52.1% of the cases and 45.3% of the controls had risk genotypes at both loci (TERT TG/GG and TEP1 CT/TT), and the carriers of these loci had a 2.05-fold increased risk of male infertility (95% CI: 1.34–3.15) compared with those who had both favorable genotypes (TERT TT and TEP1 CC). Furthermore, significant multiplicative gene– gene interactions of these two loci were found in relation to the risk of male infertility (Pinteraction = 0.009).

rs1713419 TT TC

CC

Controls %

N

%

OR (95% CI)a

176 30.3 156 26.9 1.00 281 48.4 286 49.3 0.91 (0.70– 1.20) 123 21.2 138 23.8 0.83 (0.60– 1.15) 215 37.1 184 31.7 1.00 276 47.6 278 47.9 0.86 (0.67– 1.12) 89 15.3 118 20.3 0.66 (0.47– 0.92) 201 34.7 206 35.5 1.00 251 43.3 251 43.3 1.00 (0.77– 1.31) 128 22.1 123 21.2 1.05 (0.76– 1.43) 194 33.4 192 33.1 1.00 275 47.4 292 50.3 0.95 (0.73– 1.23) 111 19.1 95 16.4 1.18 (0.84– 1.66) 275 47.4 260 44.8 1.00 225 38.8 256 44.1 0.84 (0.66– 1.08) 81 14.0 64 11.0 1.21 (0.84– 1.76) 227 39.1 267 46.0 1.00 247 42.6 252 43.4 1.17 (0.91– 1.51) 106 18.3 61 10.5 1.39 (1.20– 1.62) 237 40.9 212 36.6 1.00 281 48.4 282 48.6 0.97 (0.76– 1.25) 62 10.7 85 14.7 0.71 (0.49– 1.04) 213 36.7 204 35.2 1.00 270 46.6 271 46.7 1.00 (0.78– 1.29) 97 16.7 105 18.1 0.93 (0.66– 1.30)

Pvalueb

P trend

0.363

0.149

0.040

0.011

0.953

0.692

0.427

0.551

0.143

0.921

b0.001 b0.001

0.091

0.036

0.782

0.480

Data in boldface represent P b 0.05. a Odds ratio was age, cigarette smoking, and alcohol use. b False discovery rate (FDR) corrected P-value.

Table 3 Interaction of TERT s2736100 and TEP1 rs1713449 on male infertility risk. TERT rs2736100

TEP1 rs1713449

Cases, n (%)

Controls, n (%)

OR (95% CI)a

TT TT TG/GG TG/GG Pinteractionb

CC CT/TT CC CT/TT 0.009

38 (6.6) 51 (8.8) 189 (32.6) 302 (52.1)

68 (11.7) 50 (8.6) 199 (34.3) 263 (45.3)

1.00 1.82 (1.05–3.18) 1.70 (1.09–2.64) 2.05 (1.34–3.15)

a b

Odds ratio was adjusted for age and alcohol use. P for multiplicative interaction.

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580 patients, a total of 420 eligible men (72.4% participation) provided their semen samples. The analyses for associations between polymorphisms and semen quality are summarized in Table 4. The direction of the regression coefficient (β) represents the effect of each minor allele increasing (+) or decreasing (−) the values of semen parameters. We observed that the TEP1 rs1713419 variants were positively associated with higher levels of sperm DNA fragmentation (β = 2.243 and P = 0.016). 4. Discussion Telomeres are critical in maintaining genomic stability and integrity. Genetic variants in telomere-associated pathway genes may affect telomere length and chromosomal stability by influencing gene expression or protein configuration in the telomeres, and subsequently disease susceptibility. The present study is the first case–control study to assess 8 potentially functional SNPs in 2 selected telomere-associated pathway genes (TERT and TEP1) with male infertility risk in ethnic Han-Chinese. Individuals with TERT rs2736100 variant GG genotype were significantly associated with reduced risk of male infertility (adjusted OR = 0.66, 95% CI: 0.47–0.92; Ptrend = 0.011) when compared with the common TT genotype. Moreover, TEP1 rs1713449 variant genotypes (TT) were associated with significantly increased risk of male infertility compared with CC genotype (adjusted OR = 1.39, 95% CI: 1.20–1.62; Ptrend b 0.001). In addition, subjects carrying risk genotypes of these both loci had a two-fold (95% CI: 1.34–3.15) increase in the risk of male infertility, indicating a significant gene–gene interaction between these two loci (P for multiplicative interaction = 0.009). Moreover, linear regression analysis showed that individuals carrying the TEP1 rs1713419 variants have significantly higher levels of sperm DNA fragmentation (β = 2.243, P = 0.016). The TERT gene codes for TERT enzyme, which is crucial for the maintenance of telomere DNA length, chromosomal stability, and cellular immortality during cell division (Cheung and Deng, 2008; Cong et al., 1999). Previous studies have found that TERT rs2736100 G allele was significantly associated with increased risk of glioma (Shete et al., 2009), lung cancer (Landi et al., 2009; McKay et al., 2008), specifically adenocarcinoma of the lung (Landi et al., 2009), childhood acute lymphoblastic leukemia (Sheng et al., 2013), and testicular germ cell cancer (Turnbull et al., 2010), while it was inversely associated with serous ovarian cancer (Johnatty et al., 2010), suggesting that this SNP might possess a key role in cancer development. TERT rs2736100 is located in intron 2 of TERT in the upstream recombination region on 5p15 and lies within a putative regulatory region based on the evolutionary and sequence pattern extraction through reduced representation (ESPERR) score (Taylor et al., 2006). It has been reported that TERT rs2736100 was associated with lower telomerase activity and longer telomeres (Sheng et al., 2013). In addition, rs2736100 is close to the location of mutations known to alter telomerase activity and may be in linkage

disequilibrium (LD) with mutations that have yet to be identified (Landi et al., 2009). Consistent with the known functionality of the polymorphism, we found that TERT rs2736100 GG genotype was associated with reduced risk of male infertility. Further investigations are needed to explore the exact molecular mechanisms of rs2736100 for telomerase activity, telomere regulation, and male infertility. TEP1 is a component of the ribonucleoprotein complex and binds to telomerase. It is responsible for catalyzing the addition of new telomeres to chromosomes. Genetic variants of TEP1 have been previously examined in relation to cancer risk, including bladder cancer risk (Andrew et al., 2009; Chang et al., 2012), incident cardio/cerebrovascular disease risk (Zee et al., 2011), breast cancer susceptibility and prognosis (Varadi et al., 2009), and breast cancer risk (Pellatt et al., 2013; Savage et al., 2007). TEP1 rs1713449 is a non-synonymous SNP, Val2214Ile, which might have a damaging effect on the protein structure (Polyphen). Additionally, rs1713449 was in LD with TEP1 rs938886, which was associated with telomere length (Varadi et al., 2009). Thus, in this study, we found that the TEP1 rs1713449 CC genotype was associated with increased risk of male infertility. Future studies are needed to determine how this SNP affects TEP1 function, telomerase activity, telomere length, and male infertility susceptibility. Sperm DNA stability and integrity are essential for the accurate transmission of genetic information, and any form of sperm DNA damage may result in male infertility (Agarwal and Said, 2003). In our previous studies (Ji et al., 2012, 2013), we found that genetic variants in antioxidant genes and DNA double-strand break (DSB) repair pathway genes were associated with the risk of male infertility using the same 580 of cases of male infertility and 580 controls. Thus, all these findings could be a useful tool for determining sperm fertilization potential and could improve the diagnosis, the prevention of male infertility, and diagnostic implications for assisted reproduction success rates as well. Strengths of the present study should be acknowledged. All cases and controls were racially homogeneous (all Han-Chinese) and well matched with regard to age, drinking status, and BMI, which minimizes potential confounding bias. All tested SNPs were in Hardy–Weinberg equilibrium in controls. The most important limitation of this study was the sample size. The moderate sample size might not be sufficient for us to adequately detect a small effect from very low-penetrance SNPs. Additional studies are warranted to confirm the associations observed in the present study. 5. Conclusions In summary, of the 8 potential functional polymorphisms investigated here, we provide the first evidence that genetic variations of TERT rs2736100 and TEP1 rs1713449 were significantly associated with the risk of male infertility. These findings may be helpful in improving our understanding of the etiology of male infertility. However, additional studies are needed to validate our findings, and mechanistic studies

Table 4 Effects of SNPs in TERT and TEP1 on semen quality. SNP

TERT: rs2735940 CNT TERT: rs2736100 TNG TERT: rs2736098 GNA TEP1: rs1713419 TNC TEP1: rs938886 GNC TEP1: rs1713449 CNT TEP1: rs1760904 CNT TEP1: rs1760903 TNC

Sperm concentration

Sperm motility

Sperm DNA fragmentation

βa

95% CI

Pb

βa

95% CI

Pb

βa

95% CI

Pb

2.003 3.560 4.042 −6.736 9.039 4.044 0.587 −1.495

−5.434, 9.441 −4.610, 11.729 −3.802, 11.886 −14.842, 1.370 −0.258, 18.335 −3.850, 11.938 −7.192, 8.367 −9.505, 6.513

0.597 0.392 0.312 0.103 0.057 0.315 0.882 0.714

0.259 −1.765 0.983 −2.915 −1.464 0.166 0.843 1.366

−4.953, 1.334 −5.220, 1.691 −2.338, 4.304 −6.345, 0.514 −5.411, 2.482 −3.178, 3.509 2.448, 4.133 −2.020, 4.753

0.259 0.316 0.561 0.095 0.466 0.922 0.615 0.428

0.034 0.105 −0.126 2.243 −0.639 0.709 0.656 0.366

−1.677, 1.745 −1.776, 1.985 −1.932, 1.680 0.412, 4.074 −2.785, 1.507 −1.108, 2.525 −1.132, 2.444 −1.503, 2.236

0.969 0.913 0.891 0.016 0.559 0.444 0.472 0.700

β, regression coefficient; SE, standard error. Data in boldface represent P b 0.05. a All analyses were done using linear regression models, adjusted for age, smoking status, drinking status and abstinence time. b False discovery rate (FDR) corrected P-value.

L. Yan et al. / Gene 534 (2014) 139–143

are needed to elucidate the underlying molecular mechanisms of these associations. Declaration of interests The authors declare no conflict of interest that could be perceived as prejudicing the impartiality of the research reported in this paper. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 81202243, Grant No. 81172694 and Grant No. 30901210); the Natural Science Foundation of Jiangsu Province (Grant No. BK2012087); the Practice Innovation Training Program projects for the Jiangsu College students (2012JSSPTTP1018), the Jiangsu Province's Qinglan Project (JX2161015124), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Acknowledgments We thank Yongyue Wei for the statistical analysis. References Agarwal, A., Said, T.M., 2003. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum. Reprod. Update 9, 331–345. Andrew, A.S., et al., 2009. Bladder cancer SNP panel predicts susceptibility and survival. Hum. Genet. 125, 527–539. Chang, J., Dinney, C.P., Huang, M., Wu, X., Gu, J., 2012. Genetic variants in telomeremaintenance genes and bladder cancer risk. PLoS One 7, e30665. Cheung, A.L., Deng, W., 2008. Telomere dysfunction, genome instability and cancer. Front. Biosci. 13, 2075–2090. Cong, Y.S., Wen, J., Bacchetti, S., 1999. The human telomerase catalytic subunit hTERT: organization of the gene and characterization of the promoter. Hum. Mol. Genet. 8, 137–142. Dahse, R., Fiedler, W., Ernst, G., 1997. Telomeres and telomerase: biological and clinical importance. Clin. Chem. 43, 708–714. de Lange, T., 1998. Telomeres and senescence: ending the debate. Science 279, 334–335. de Lange, T., 2005. Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev. 19, 2100–2110. Hemann, M.T., Rudolph, K.L., Strong, M.A., DePinho, R.A., Chin, L., Greider, C.W., 2001. Telomere dysfunction triggers developmentally regulated germ cell apoptosis. Mol. Biol. Cell 12, 2023–2030. Ji, G., et al., 2012. Genetic variants in antioxidant genes are associated with sperm DNA damage and risk of male infertility in a Chinese population. Free Radic. Biol. Med. 52, 775–780. Ji, G., Yan, L., Liu, W., Huang, C., Gu, A., Wang, X., 2013. Polymorphisms in double-strand breaks repair genes are associated with impaired fertility in Chinese population. Reproduction 145, 463–470. Jiang, X.R., et al., 1999. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat. Genet. 21, 111–114. Johnatty, S.E., et al., 2010. Evaluation of candidate stromal epithelial cross-talk genes identifies association between risk of serous ovarian cancer and TERT, a cancer susceptibility “hot-spot”. PLoS Genet. 6, e1001016.

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