http://informahealthcare.com/bmk ISSN: 1354-750X (print), 1366-5804 (electronic) Biomarkers, 2014; 19(6): 509–516 ! 2014 Informa UK Ltd. DOI: 10.3109/1354750X.2014.943291

RESEARCH ARTICLE

Polymorphism of DNA repair genes OGG1, XRCC1, XPD and ERCC6 in bladder cancer in Belarus Volha P. Ramaniuk1#, Nataliya V. Nikitchenko1, Nataliya V. Savina1, Tatyana D. Kuzhir1#, Alexander I. Rolevich2, Sergei A. Krasny2, Vadim E. Sushinsky3, and Roza I. Goncharova1 Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Minsk, Republic of Belarus, 2N.N. Alexandrov National Cancer Centre, Minsk, Republic of Belarus, and 3Belarusian Medical Academy of Postgraduate Education, Minsk, Republic of Belarus

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1

Abstract

Keywords

Context: The study of DNA base and nucleotide excision repair gene polymorphisms in bladder cancer seems to have a predictive value because of the evident relationship between the DNA damage response induced by environmental mutagens and cancer predisposition. Objective: The objective was to determine OGG1 Ser326Cys, XRCC1 Arg399Gln, XPD Asp312Asn, and ERCC6 Met1097Val polymorphisms in bladder cancer patients as compared to controls. Methods: Both groups were predominantly represented by Belarusians and Eastern Slavs. DNA samples from 336 patients and 370 controls were genotyped using a PCR-RFLP method. Results: The genotype distributions were in agreement with the Hardy–Weinberg equilibrium. The minor allele frequencies in the control population were in the range of those in Caucasians in contrast to Asians. The OGG1 326 Ser/Cys and XPD 312 Asp/Asn heterozygous genotypes were inversely associated with cancer risk (OR [95% CI] ¼ 0.69 [0.50–0.95] and 1.35 [1.0–1.82], respectively). The contrasting effects of these genotypes were potentiated due to their interactions with smoking habit or age. Conclusions: Among four DNA repair gene polymorphisms, the OGG1 326 Ser/Cys and XPD 312 Asp/Asn heterozygous genotypes might be recognized as potential genetic markers modifying susceptibility to bladder cancer in Belarus.

Bladder cancer, DNA repair gene polymorphisms, ERCC6, OGG1, XPD, XRCC1

Introduction Bladder cancer is one of the most common malignancies of the urinary tract in Western Europe and United States as compared to Asian countries (Jankovic´ & Radosavljevic´, 2007; Kakehi et al., 2010). The annual incidence rate is about 1000 in Belarus (Polyakov et al., 2011). Epidemiologic data indicate that the occurrence frequency of bladder cancer among men is three–four times as high as among women, and it is strongly associated with age (Jankovic´ & Radosavljevic´, 2007; Kakehi et al., 2010). The risk factors for bladder cancer include occupational chemical exposures and tobacco smoking. Industrial pollutions cause about 10% of bladder cancer incidence in Western countries, whereas tobacco smoking, at least among men, causes almost 50% (Jankovic´ & Radosavljevic´, 2007). Accumulation of DNA lesions induced by environmental mutagens initiates genome instability that, as a possible outcome, may result in cell malignant

#Volha P. Ramaniuk and Tatyana D. Kuzhir are responsible for statistical design/analysis. E-mail: [email protected] (V. P. Ramaniuk); [email protected] (T. D. Kuzhir) Address for correspondence: Tatyana D. Kuzhir, Institute of Genetics and Cytology, National Academy of Sciences of Belarus, 27, Akademicheskaya St., Minsk 220072, Republic of Belarus. Tel: +375-17-294-91-79. Fax: +375-17-284-19-17. E-mail: T.Kuzhir@ igc.bas-net.by

History Received 28 May 2014 Revised 7 July 2014 Accepted 7 July 2014 Published online 4 August 2014

conversion. Several DNA repair pathways controlled by over than 130 genes maintain genome integrity and are active in the cellular responses to DNA damage (Wood et al., 2001). In the context of cancer prevention, error-free excision repair systems are of particular interest, as they repair lesions in a ‘‘cut-and-patch’’ manner, excising the damaged segment and filling the single-stranded gap by using the intact complementary strand as a template (Hoeijmakers, 2001). The base excision repair (BER) pathway is involved in the removal of damaged (e.g. oxidised) DNA bases and singlestrand breaks (Parsons & Dianov, 2012). The 8-oxo-guanine DNA glycosylase (OGG1) and X-ray repair cross-complementing protein 1 (XRCC1) are among the functionally important proteins of this repair system. The OGG1 enzyme is responsible for the elimination of highly mutagenic DNA lesion, 8-oxo-7,8-dihydroguanine (8-oxoGua) (Boiteux & Radicella, 2000; Hazra et al., 2007), whereas the XRCC1 protein interacts with DNA glycosylases, AP endonuclease-1 (APE-1), DNA polymerase b (POLb), DNA ligase III (Lig III), poly (ADP-ribose) polymerase 1 (PARP-1), polynucleotide kinase (PNK) at the damaged site, by modulating their activities and coordinating the subsequent enzymatic steps of BER (Campalans et al., 2005; Hanssen-Bauer et al., 2011). The majority of environmental agents, including cigarette smoke constituents, is known to induce DNA damage through a reactive oxygen species (ROS)-mediated mechanism

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(Vadhanam et al., 2012). Current findings indicate both a trigger role of the oxidatively generated DNA damage in cell malignancy and their accumulation in tumors. Therefore, the impairment of the catalytic function of the OGG1 DNA glycosylase is assumed to affect susceptibility to cancer as well as to other oxidative pathologies (Dizdaroglu, 2012; Simonelli et al., 2013). Functional studies reveal the impact of certain polymorphic variants of the XRCC1 gene on the cellular genotoxic response and BER efficiency (AbdelRahman & El-Zein, 2000; Takanami et al., 2005). Both the polymorphisms at the 399 codon of XRCC1 and at the 326 codon of OGG1 have been reported to decrease the capacity of human blood lymphocytes to repair radiation- and oxidantinduced DNA damage (Vodicka et al., 2007), suggesting their potential association with cancer risk. Widespread polycyclic aromatic hydrocarbons induce bulky adducts, which are commonly considered to be associated with cancer development (Veglia et al., 2003, 2008). They are classical substrates for the nucleotide excision repair (NER) pathway that operates globally throughout the genome (global genome, GG-NER) or during transcription (transcription coupled, TC-NER); both subsets differ only in their initial recognition of the helixdistorting DNA damage (Hoeijmakers, 2001; Shuck et al., 2008). In this multi-step repair process, DNA helicases unwind the double helix, thereby promoting the recruitment of other repair enzymes to the site of lesion (Kuper & Kisker, 2013). The XPD helicase, mutated in the cancer-prone xeroderma pigmentosum (XP), is part of the TFIIH complex that is essential for signaling events triggering transcription, cell cycle checkpoints and DNA repair (Fuss & Tainer, 2011). Apart from point mutations critical for some rare genetic disorders, variations (single nucleotide polymorphisms, SNPs) in the XPD sequence are found among the general population; some of them might be associated with a reduced repair capacity (Benhamou & Sarasin, 2002). TC-NER requires specific factors, including Cockayne syndrome protein B (CSB) or excision repair cross-complementing rodent repair deficiency, complementation group 6 (ERCC6). The latter belongs to both the helicase superfamily 2 and the SWI/SNF complex maintaining and remodeling chromatin structure (Lake & Fan, 2013; Newman et al., 2006). This protein has recently been reported to remove transcriptionblocking lesions and to act as a ‘‘regulator’’ at the crossroads of transcriptional networks (Fousteri & Mullenders, 2008; Ve´lez-Cruz & Egly, 2013). Additionally, it is involved in the elimination of oxidatively generated DNA damage through the BER pathway (Menoni et al., 2012). The important biological functions of both helicases suggest a possible association of their polymorphic variants with increased cancer risk (Benhamou & Sarasin, 2002; Hoeijmakers, 2001). Genetic variation in DNA repair genes and their potential impact on carcinogenesis have been intensively investigated during the last decade, but these studies have yielded frequently conflicting results. Nevertheless, analysis of the currently available data allows some general trends to be revealed: (1) the genotype distribution as well as association of certain genotypes/alleles with cancer risk is likely to depend on ethnicity of populations; (2) the DNA repair polymorphisms may be associated with specific types of

Biomarkers, 2014; 19(6): 509–516

tumor; (3) gene–environment interactions seem to modify the cancer risk. For example, the OGG1 326Cys variant is proposed as a marker for susceptibility to lung cancer rather than to other types of tumors (Wei et al., 2011). At the same time, this polymorphism is closely associated with cancer risk in Asian populations, in contrast to Caucasians (Guan et al., 2011). As earlier reported, ever smokers with allelic combinations in three NER genes, including the ERCC6 1097Val variant allele, have an almost 30-fold increased risk of bladder cancer (Chen et al., 2007), whereas Pro-carriers of the ERCC6 Arg1230Pro polymorphism show a decreased risk for laryngeal cancer (OR ¼ 0.53, 95% CI 0.34–0.85), strongest in heavy smokers (Abbasi et al., 2009). The present study deals with the detection of genetic variations in DNA repair genes in the residents of Belarus. The frequencies of OGG1 Ser326Cys (rs 1052133), XRCC1 Arg399Gln (rs 25487), XPD Asp312Asn (rs 1799793) and ERCC6 Met1097Val (rs 2228526) polymorphisms were compared between clinically healthy individuals and bladder cancer patients in order to elucidate the potential contribution of these variants to predisposition to bladder cancer in Belarus.

Materials and methods Study subjects The case group comprised 336 bladder cancer (BC) patients who were treated at the Department of Urology of N.N. Alexandrov National Cancer Centre of Belarus during 2011– 2013. All urothelial carcinoma diagnoses were verified histologically after transurethral resection of bladder tumors. Blood samples (3–5 ml) collected by venal puncture were accompanied with information on clinical and histological parameters of tumors. Three-hundred seventy individuals were randomly recruited among healthy volunteers involved in blood donation at the Republic Research and Production Center for Transfusiology and Medical Biotechnologies (Minsk), as well as among seniors who were examined at the Department of Gerontology and Geriatrics at the Belarusian Medical Academy of Postgraduate Education. They had no positive cancer history or acute diseases and should be considered as the population-based controls matched to the cases by the recruitment period, the ethnic origin and age, as both the control and the patient groups included middle-aged and older adults, all of them were residents of Belarus (predominantly Belarusians or other Eastern Slavs). Informed consent was obtained from each participant included in the study before the collection of blood samples. All participants were interviewed to complete a questionnaire covering medical, residential and occupational history as well as age, gender and lifestyle habits (the tobacco smoking status). The smoking status was summarized as ‘‘smoker’’ (combining current smokers and ex-smokers) or ‘‘nonsmoker’’ (never smoking persons). Genotyping DNA for genotyping procedures was extracted using the traditional phenol-chloroform technique. SNPs in the chosen DNA repair genes were determined by the PCR-RFLP method under conditions described elsewhere (Arizono et al., 2008;

Met/Met: 123 + 78; Met/Val: 201 + 123 + 78; Val/Val: 201 35 cycles: 94  C – 30 s, 55  C – 30 s, 72  C – 30 s. 2228526 ERCC6/CSB

10q11.23

Met1097Val

A!G

(F) 50 -CCT GCT T CT AAC ATA TCT GT-30 (R) 50 - AAT CAC TGA CAA CTC TTC TG-30

Nla III

Asp/Asp: 507 + 244; Asp/Asn: 507 + 474 + 244 + 33; Asn/Asn: 474 + 244 + 33 34 cycles: 94  C – 30 s, 64  C – 30 s, 72  C – 60 s.

StyI

511

(F) 50 -CTG TTG GTG GGT GCC CGT ATC TGT TGG TCT-30 (R) 50 -TAA TAT CGG GGC TCA CCC TGC AGC ACT TCC T-30 1799793 G!A

(F) 50 -GGA CTG TCA CCG CAT GCG TCG G-30 (R) 50 -GGC TGG GAC CAC CTG TGT T-30 25487 G!A Arg399Gln

Asp312Asn

Arg/Arg: 115 + 34; Arg/Gln: 149 + 115 + 34; Gln/Gln: 149 MspI

MboI

32 cycles: 94  C – 40 s, 60  C – 40 s, 72  C – 30 s. 33 cycles: 94  C – 40 s; 62  C – 40 s; 72  C – 30 s. (F) 50 -CTG TTC AGT GCC GAC CTG CGC CGA-30 (R) 50 -ATC TTG TTG TGC AAA CTG AC-30 1052133 Ser326Cys

SNP

C!G

19q13.2

Nucleotide excision repair ERCC2/XPD 19q13.2–13.3

Before characterizing the genotypic/allelic distributions of DNA repair genes among BC patients as compared to controls, it should be noted that the genotype frequencies completely conformed to the Hardy–Weinberg equilibrium in both groups, p values being as follows: p ¼ 0.37 in controls

XRCC1

Genotypic and allelic distributions in bladder cancer as compared to the control population

Location

The data summarized in Table 2 show significant differences between two groups with respect to gender and the smoking status with prevalence of males and smokers among BC patients as compared to non-cancer subjects. These results represented the specificity of bladder cancer as a gender- and smoking-related disease. Although individuals of similar age (BC patients aged from 31 to 88 years and non-cancer individuals aged from 31 to 94 years) were included in the present study, the groups slightly differed from each other in the average and median age. The previous study demonstrated no differences between the randomly recruited populationbased controls and strongly age- and gender-matched controls in the allele frequencies and genotype distributions for XPD Asp312Asn, XRCC1 Arg399Gln and OGG1 Ser326Cys polymorphisms (Ramaniuk et al., 2013). These findings allowed us to use the described control group to estimate adequately the genetic variations in the clinically healthy middle-aged and older Belarusian population.

Gene

Population characteristics

Base substitution

Results

Reference SNP ID number

Primer sequences

PCR conditions

The Pearson’s 2 test (or the Fisher’s exact test when necessary) was used to verify the significance of differences between the groups of BC patients and control individuals in genotype/allele frequencies, as well as in distribution of gender and smoking status. The Student’s t test was used for the comparison of groups by age and other continuous variables. The statistical significance for deviation from the Hardy–Weinberg equilibrium was determined using the 2 test. The p  0.05 values were considered significant. The relative risk was estimated as odds ratio (OR) with 95% confidence intervals (CI). Gene–lifestyle interaction was assessed after stratification of subjects based on the smoking status by comparing the genotypic/allelic frequencies in BC patients and controls among smokers and non-smokers.

Table 1. Characteristics of allelic variants and conditions for their detection.

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Statistical analysis

Base excision repair OGG1 3p26.2

Restriction enzyme

PCR products (bp)

Chiu et al., 2008; Lo´pez-Cima et al., 2007) and are presented in Table 1. The PCR reaction mixture (50 ml) contained 10 mol of each primer, 2.0 mM MgCl2, 200 mM of each dNTP, 1 unit of Taq DNA polymerase and 100–300 ng of extracted DNA. The definite temperature conditions were fulfilled after pre-incubation of the reaction mixture for 5 min at 94  Q. The PCR products were digested with restriction enzymes and electrophoresed through 3% agarose gels containing ethidium bromide. The digested PCR products were visualized under UV light. The genotypes of OGG1 Ser326Cys, XRCC1 Arg399Gln, XPD Asp312Asn and ERCC6 Met1097Val were determined by the length of the DNA fragments, shown in Table 1.

Ser/Ser: 224 + 23; Ser/Cys: 247 + 224 + 23; Cys/Cys: 247

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Table 2. Cases/controls characteristics.

Variable Gender Male Female Age (years) Min Max Mean ± SD Median Tobacco smoking Smokers Non-smokers unknown

Table 3. Distribution of allelic variations of DNA repair genes in the group of BC patients as compared to controls.

BC patients (n ¼ 336)

Controls (n ¼ 370)

n

n

Frequency (%)

Frequency (%) Genotypes/minor alleles

a

275 61

81.9 18.1a

31 88

254 116 31 94

67.0 ± 10.7 68 231 97 8

68.8 28.9 2.3

114 229 27

a

68.6 31.4a

64.5 ± 13.5b 59 30.8c 61.9c 7.3

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a

Significant differences between cases and controls with respect to sex ratio (2 ¼ 16.325, p ¼ 0.00005). b Average age (t ¼ 2.81, p ¼ 0.005). c Frequencies of smokers/nonsmokers (2 ¼ 92.84, p ¼ 0.0).

and 0.23 in cases (2 ¼ 0.82 and 1.47, respectively) for OGG1 codon 326 (rs 1052133); p ¼ 0.87 in controls and 0.73 in cases (2 ¼ 0.03 and 0.12, respectively) for XRCC1 codon 399 (rs 25487); p ¼ 0.43 in controls and 0.27 in cases (2 ¼ 0.63 and 1.22, respectively) for XPD codon 312 (rs 1799793); p ¼ 0.96 in controls and 0.47 in cases (2 ¼ 0.00 and 0.53, respectively) for ERCC6 codon 1097 (rs 2228526). The results of genotyping DNA samples are presented in Table 3. According to these data, marginal differences were observed between distributions of the OGG1 Ser326Cys genotypes (p ¼ 0.05) due to the significantly increased frequency of the Ser/Cys heterozygous genotype carriers among controls as compared to BC patients (p ¼ 0.023) that indicated decreased likelihood of disease development. On the contrary, the higher frequency of the XPD 312 Asp/Asn heterozygous genotype was found among BC patients as compared to controls (p ¼ 0.046), suggesting a slightly increased risk of bladder cancer in their carriers. The carriers of XPD 312 Asp/Asn heterozygous genotype occurred more frequently among patients over 80 years than among controls of the same age (62.96% as opposed to 38.57%; 2 ¼ 4.68, p ¼ 0.03). The genotype/allele frequencies for the XRCC1 Arg399Gln and ERCC6 Met1097Val polymorphisms were rather similar between BC patients and controls, and their distributions as well as those for OGG1 Ser326Cys polymorphisms, were not affected by age. Thus, the OGG1 326 Ser/Cys and the XPD 312 Asp/Asn heterozygous genotypes might serve as potential markers inversely associated with incidence of bladder cancer in Belarus. Gene–lifestyle interactions Taking into account the crucial role of tobacco smoking in etiology of bladder cancer, the relationship between the polymorphisms in OGG1 (rs 1052133), XRCC1 (rs 25487), XPD (rs 1799793) and ERCC6 (rs 2228526) and the smokingrelated cancer risk was evaluated. The individuals with the unknown smoking status were excluded from the analysis. The interaction between tobacco smoking and OGG1 Ser326Cys or XPD Asp312Asn polymorphisms was of

BC cases

Controls

n

n

%

%

p

OR

95% CI

OGG1 Ser326Cys (nBC/Control ¼ 335/366) Ser/Ser 223 66.57 221 Ser/Cys 94 28.06 132 Cys/Cys 18 5.37 13 Ser/Cys + Cys/Cys 112 33.43 145 Cys 130 19.40 158

60.38 0.05c 36.07 3.55 39.62 0.09 21.58 0.31

1.31 0.69d 1.54 0.77 0.87

0.96–1.78 0.50–0.95a 0.74–3.20 0.56–1.04 0.67–1.13

XRCC1 Arg399Gln (nBC/Control ¼ 332/364) Arg/Arg 141 42.47 151 Arg/Gln 154 46.39 165 Gln/Gln 37 11.14 48 Arg/Gln + Gln/Gln 191 57.53 213 Gln 228 34.34 261

41.48 0.71c 45.33 13.19 58.52 0.15 35.85 0.55

1.04 1.04 0.83 0.96 0.94

0.77–1.41 0.77–1.41 0.52–1.30 0.71–1.30 0.75–1.17

XPD Asp312Asn (nBC/Control ¼ 333/364) Asp/Asp 99 29.73 128 Asp/Asn 178 53.45 169 Asn/Asn 56 16.82 71 Asp/Asn + Asn/Asn 234 70.27 240 Asn 290 43.54 311

34.78 0.14c 45.92 19.29 65.22 0.15 42.26 0.76

0.79 1.35d 0.85 1.26 1.05

0.58–1.09 1.00–1.82b 0.57–1.24 0.92–1.73 0.85–1.30

0.96 1.08 0.87 1.04 1.00

0.72–1.29 0.80–1.46 0.50–1.52 0.77–1.40 0.79–1.26

ERCC6 Met1097Val (nBC/Control ¼ 336/369) Met/Met 167 49.70 187 50.68 0.81c Met/Val 145 43.15 152 41.19 Val/Val 24 7.14 30 8.13 Met/Val + Val/Val 169 50.30 182 49.32 0.79 Val 193 28.72 212 28.73 1.0

Significant according to Pearson’s 2 criterion (a2 ¼ 5.12; p ¼ 0.024; b 2  ¼ 3.96, p ¼ 0.047). c p Values concern the distribution of all genotypes. d OR values describe a heterozygous genotype as a protective or risk factor for developing bladder cancer.

interest (Table 4); genotypic/allelic distributions of either XRCC1 Arg399Gln or ERCC6 Met1097Val did not depend on the smoking status in the groups of both patients and controls (not shown). Among non-smokers, the genotypic/allelic distribution at the OGG1 locus in BC patients was similar to that in the controls. A significant difference was observed between the two groups in the genotypic distributions of the XPD Asp312Asn (p ¼ 0.02) apparently due to a decreased frequency of homozygous carriers of the XPD 312Asn variant allele among BC patients as compared to controls (p ¼ 0.007). Among smokers, the genotypic/allelic distributions at the XPD locus were comparable in both groups, while the frequency of the OGG1 326 Ser/Ser genotype was significantly higher and the proportion of the OGG1 326 Ser/Cys genotype was lower in the BC patients than in the controls. A trend towards the protection against carcinogenesis provided by the OGG1 326 Ser/Cys heterozygous genotype in the total population (Table 3) was stronger in smokers (Table 4). Another approach (when comparing the subgroups of smokers and non-smokers with each other) yielded unexpected results concerning the XPD Asp312Asn polymorphism. In the control group, the significant differences between smokers and non-smokers were observed with respect to Asp/ Asn and Asp/Asn + Asn/Asn genotype frequencies (2 ¼ 4.76, p ¼ 0.029 and 2 ¼ 7.19, p ¼ 0.007, respectively), and to the proportion of a XPD 312Asn variant allele (2 ¼ 4.42, p ¼ 0.035). The analogous trends were found in the group

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Table 4. OGG1, XRCC1, XPD, ERCC6 genotypic/minor allelic frequencies related to the smoking status. Smoking status Non-smokers

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Smokers

Genotype/allele

BC cases n (%)

OGG1 Ser326Cys (nBC/Control ¼ 96/228) Ser/Ser 61 (63.5) Ser/Cys 31 (32.3) Cys/Cys 4 (4.2) Cys/Cys + Ser/Cys 35 (36.5) Cys 39 (20.3) XPD Asp312Asn (nBC/Control ¼ 96/229) Asp/Asp 39 (40.6) Asp/Asn 50 (52.1) Asn/Asn 7 (7.3) Asn/Asn + Asp/Asn 57 (59.4) Asn 64 (33.3) OGG1 Ser326Cys (nBC/Control ¼ 231/110) Ser/Ser 155 (67.1) Ser/Cys 62 (26.8) Cys/Cys 14 (6.1) Cys/Cys + Ser/Cys 76 (32.9) Cys 90 (19.5) XPD Asp312Asn (nBC/Control ¼ 229/112) Asp/Asp 59 (25.8) Asp/Asn 124 (54.1) Asn/Asn 46 (20.1) Asn/Asn + Asp/Asn 170 (74.2) Asn 216 (47.2)

Controls n (%) 142 80 6 86 92

(62.3) (35.1) (2.6) (37.7) (20.2)

91 94 44 138 182

(39.7) (41.1) (19.2) (60.3) (39.7)

60 44 6 50 56

(54.6) (40.0) (5.4) (45.5) (25.5)

28 60 24 84 108

(25.0) (53.6) (21.4) (75.0) (48.2)

p 0.71e 40.05 40.05 0.02e 40.05 40.05 0.09e 0.025 40.05 0.96e 40.05 40.05

OR [95% CI]f 1.06 0.88 1.61 0.95 1.01

[0.64–1.73] [0.53–1.46] [0.44–5.83] [0.58–1.55] [0.66–1.53]

1.04 1.56 0.33 0.96 0.76

[0.64–1.69] [0.97–2.52] [0.14–0.76]a [0.59–1.57] [0.53–1.08]

1.7 [1.07–2.71]b 0.55 [0.34–0.89]c 1.12 [0.42–2.99] 0.59 [0.37–0.94]d 0.71 [0.48–1.04] 1.04 1.02 0.92 0.96 0.96

[0.62–1.75] [0.65–1.61] [0.53–1.61] [0.57–1.62] [0.70–1.32]

Significant according to Pearson’s 2 criterion (a2 ¼ 7.27, p ¼ 0.007; b2 ¼ 5.04, p ¼ 0.025; c2 ¼ 6.02, p ¼ 0.014; d 2  ¼ 5.04, p ¼ 0.025). f OR values separately describe genotype/allele contribution to the risk of developing bladder cancer. e p Values relate to the distribution of all genotypes.

of BC patients: the differences were significant for Asp/ Asn + Asn/Asn genotypes (2 ¼ 7.09, p ¼ 0.008), and for a XPD 312Asn variant allele (2 ¼ 11.46, p ¼ 0.0007), rather than for the XPD 312 Asp/Asn heterozygous genotype (2 ¼ 0.12, p ¼ 0.73). Thus, genotypes containing at least one XPD 312Asn variant allele seemed to be associated with a smoking status. Accordingly, the analysis of gene–lifestyle interactions in bladder cancer showed increased cancer risk in smokers with the OGG1 326 Ser/Ser wild-type homozygous genotype and decreased cancer risk in smokers with the Ser/Cys + Cys/Cys genotypes, whereas no differences between cancer patients and controls were observed in non-smokers except for carriers of the XPD 312 Asn/Asn homozygous variant genotype. The data also suggested a possible indirect effect of a XPD 312Asn variant allele on a smoking addiction.

Discussion The genotypic distributions and allelic frequencies of three excision repair genes (OGG1 Ser326Cys, XRCC1 Arg399Gln and XPD Asp312Asn) were previously studied in the clinically healthy population of Belarus (Ramaniuk et al., 2013). Briefly, the frequencies of the minor alleles were in the range of values in Caucasians from Western and Eastern Europe and America, but significantly differed from those in Asians. In the present study, the genotypic/allelic distributions among controls were similar to the prior data, with proportions of the minor alleles of OGG1 codon 326, XRCC1 codon 399 and XPD codon 312 being equal to 0.22, 0.36 and 0.42, respectively. Unlike the genes mentioned, ethnic specificity in

the ERCC6 Met1097Val polymorphism and its possible role in carcinogenesis have been less studied, with reported frequencies of the ERCC6 1097Val minor allele being equal to 0.19–0.23 in USA Caucasians (Berndt et al., 2006; Pan et al., 2009; Wang et al., 2007), 0.14 in African Americans (Zheng et al., 2010), 0.24 in Turkmens from the Golestan Province of Iran (Etemadi et al., 2013) and 0.05–0.09 in the populations of China and Taiwan (Chang et al., 2009; Ma et al., 2009). We have found that the frequency of this allele is 0.29 in Belarus, i.e. it slightly exceeds the revealed rate for Caucasians, but is significantly higher than in Asians. Among genetic variations in DNA repair genes investigated, OGG1 Ser326Cys and XPD Asp312Asn polymorphisms have shown to contribute to bladder cancer predisposition. Due to a meta-analysis of worldwide studies, the importance of OGG1 Ser326Cys polymorphism has recently been demonstrated concerning susceptibility to various tumor types in Asian countries (Wei et al., 2011). The Ser ! Cys substitution was associated with lung cancers in both Asians and Caucasians (Guan et al., 2011; Weiss et al., 2005; Wei et al., 2011; Xu et al., 2013), while the association between the OGG1 326Cys variant and the risk of hepatocellular carcinomas, prostate, breast, colorectal and gastric cancers was limited or quite absent (Gu et al., 2010; Guo et al., 2012; Lavender et al., 2010; Li et al., 2012; Wang et al., 2013). The OGG1 Ser326Cys polymorphism was neutral with respect to bladder cancer risk regardless of ethnicities (Zhong et al., 2012), whereas according to other authors, it was the clear genetic risk factor in populations of Japan and North India (Arizono et al., 2008; Mittal et al., 2012) as well as in nonsmokers (Ji et al., 2012).

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Figure 1. Inverse effects of heterozygous genotypes of OGG1 (codon 326) and XPD (codon 312) on bladder cancer risk potentiated by means of their interaction with smoking (a) and age (b). For the OGG1 326 Ser/Cys genotype, OR [95% CI] ¼ 0.69 [0.50–0.95] (p ¼ 0.024) and 0.55 [0.34–0.89] (p ¼ 0.014) in the total population and smokers, respectively. For the XPD 312 Asp/Asn genotype, OR [95% CI] ¼ 1.35 [1.00–1.82] (p ¼ 0.047) and 3.34 [1.35–8.28] (p ¼ 0.009) in the total population and the elderly, respectively.

The OGG1 326 Cys/Cys homozygous genotype was earlier shown to inhibit the enzymatic activity of 8-oxoguanine-DNA glycosylase, thereby impairing the cellular capacity to repair the oxidative DNA adducts (Hill & Evans, 2006; Vodicka et al., 2007). Nevertheless, the present results have indicated the protective effect of the OGG1 326 Ser/Cys heterozygous genotype on tumorigenesis, chances of developing bladder cancer being greatly reduced in smokers (Table 4, Figure 1a). Our findings are in line with some other data. In American populations, a reduced bladder cancer risk was revealed in smokers carrying Ser/Cys + Cys/Cys genotypes (Huang et al., 2007). The OGG1 326 Ser/Cys genotype significantly reduced the risk of developing lung cancer [De Ruyck et al., 2007], and an OGG1 326Cys variant allele was reported to play a role in the prevention of breast cancer in European women (Yuan et al., 2010), although the latter conclusion was challenged by other researchers (Ding et al., 2011). Single-nucleotide substitution in the XPD gene was supposed to serve as the marker of susceptibility to bladder cancer in the American populations, but multivariate interactions between genes involved in the NER pathway and the cell-cycle control possessed a greater predictive power (Wu et al., 2006). A meta-analysis of large-scale investigations in ethnically diverse populations indicated the significant contribution of a XPD 312Asn allele to bladder cancer predisposition (Li et al., 2010). However, Li et al. (2010) pointed out the difference between the results of genotyping in Americans and Europeans. Our data concerning the XPD Asp312Asn polymorphism were consistent with the observations demonstrating a small, but statistically significant impact of the Asp/Asn heterozygous genotype on bladder cancer risk based on the meta-analysis (OR [95% CI] ¼ 1.24 [1.06–1.46]) (Wang et al., 2008). In another study, a 312Asn variant allele was shown to increase the lung cancer risk in Asians in contrast to Europeans, and in smokers as opposed to nonsmokers (Zhan et al., 2010). We have found an age-related increase in bladder cancer susceptibility in residents of Belarus with the XPD 312 Asp/Asn heterozygous genotype (Figure 1b) that allows this genotype to be considered an additional nonspecific predictor of developing bladder cancer

in the elderly. Moreover, a potential association of the XPD 312Asn variant allele with a tobacco-smoking habit has been revealed. In this connection, the fact that smokers prevailed among BC patients (69% versus 31% in the control group) could bias the results. However, there are at least three lines of evidence in support of the contribution of the polymorphisms mentioned to bladder cancer susceptibility as an independent event: (1) genetic variation at the XPD 312 codon rather than at the OGG1 326 codon affected the smoking status per se, but both polymorphisms modified bladder cancer susceptibility; (2) only the XPD 312 Asp/Asn heterozygous genotype was associated with an increased cancer risk, whereas the frequency of the genotypes containing even one XPD 312 Asn variant allele was higher among smokers in contrast to nonsmokers; (3) in the subgroup of patients aged over 80 years, the odd ratio indicated a higher likelihood of developing cancer in carriers of the XPD 312 Asp/Asn heterozygous genotype, although the proportion of smokers was 46% among the elderly, i.e. it was significantly lower (p ¼ 0.0089) than in a total population of patients. As to other DNA repair genes, XRCC1 Arg399Gln polymorphism was reported to modify bladder cancer risk in the US and Italian populations (Andrew et al., 2008) and to be a risk factor for the Japanese population (Arizono et al., 2008). Based on the meta-analysis of 297 studies including 93 941 cases and 121 480 controls, a significant increase in a common cancer risk affected by XRCC1 polymorphisms has recently been shown (Yi et al., 2013). It has been specified due to stratification by ethnic descent and cancer types that XRCC1 Arg399Gln polymorphism is responsible for susceptibility to hepatocellular cancer in Asians and breast cancer in Indians. The analysis of allelic variations in the ERCC6 gene has shown that no substitutions at a single locus affect the lung cancer incidence, whereas their combinations are associated with susceptibility to this disease in China (Ma et al., 2009). The association between some polymorphic variants (but not ERCC6 Met1097Val) and increased cancer risk has been found with regard to non-melanoma skin cancer (basal cell carcinoma) in USA (Wheless et al., 2012) and bladder cancer in Taiwan (Chang et al., 2009). And vice

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DOI: 10.3109/1354750X.2014.943291

versa, carriers of some ERCC6 polymorphisms have a decreased risk of laryngeal cancer in the German population (Abbasi et al., 2009) as well as head and neck cancer in African Americans (Wyss et al., 2013). No association of both XRCC1 codon 399 and ERCC6 codon 1097 polymorphisms with bladder cancer incidence in Belarus has been revealed. Our results fit into a current pool of knowledge indicating the importance of ethnicity and tumor types in genotypic/ allelic distributions of genes responsible for error-free DNA repair pathways and involved in BER and NER. The present study has demonstrated the association of the XPD 312 Asp/ Asn heterozygous genotype with an increased risk of bladder cancer and the protective effect of the OGG1 326 Ser/Cys heterozygous genotype. The observed effects were potentiated due to the interaction of these genotypes with age and the smoking status, respectively. Finally, these heterozygous genotypes are likely to be capable of modifying the susceptibility of their carriers to bladder cancer.

Acknowledgements The authors are sincerely grateful to Professor Irena Szumiel (Centre for Radiobiology & Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Poland) and Professor Aksana Kucher (Laboratory of Molecular Genetics, Research Institute of Medical Genetics, Siberian Branch, Russian Academy of Medical Sciences, Russia) for previous critical reading of the manuscript, discussion of the results and helpful comments.

Declaration of interest Authors have no competing interests.

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