Scientific paper
Genetic Polymorphisms in Base Excision Repair in Healthy Slovenian Population and Their Influence
on DNA Damage
Nina Erculj, Maja Zadel and Vita Dolžan*
University of Ljubljana, Faculty of Medicine, Institute of Biochemistry, Vrazov trg 2, 1000 Ljubljana, Slovenia,
* Corresponding author: E-mail: vita.dolzan@mf.uni-lj.si, Tel: +38615437670, fax: +38615437641
Received: 08-06-2009
Abstract
Genetic polymorphisms in DNA repair genes may result in altered DNA damage. The aim of our study was to determine the frequencies of common functional single nucleotide polymorphisms (SNPs) in specific base-excision DNA repair (BER) genes in healthy Slovenian population and evaluate their influence on DNA damage established by comet assay. In total 141 unrelated healthy subjects were genotyped for hOGG1 Ser326Cys, XRCC1 Arg194Trp and Arg399Gln polymorphisms by real-time TaqMan assay. The frequencies of the hOGG1 326Ser/Ser, 326Ser/Cys and 326Cys/Cys genotypes were 63.8, 29.8, and 6.4%, respectively. The frequency distribution of XRCC1 polymorphism was 88.7% for 194Arg/Arg, 9.2% for 194Arg/Trp, 2.1% for 194Trp/Trp and 46.8% for 399Arg/Arg 40.4% for 399Arg/Gln, 12.8% for 399Gln/Gln. The influence of selected BER polymorphisms on the percentage of comet tail DNA (% TD) was determined in a subgroup of 26 subjects. We found that% TD was significantly increased among individuals with hOGG1 326Ser/Cys heterozygous variant genotype as compared to 326Ser/Ser wild-type genotype (% TD = 8.9 ± 4.2 vs. % TD = 6.9 ± 1.4, P = 0.017). No significant associations between XRCC1 Arg194Trp and Arg399Gln polymorphisms and% TD were found. Our results confirmed that DNA damage is modulated by hOGG1 Ser326Cys polymorphism.
Keywords: DNA repair genes, base excision repair, genetic polymorphism, comet assay
1. Introduction
The large genomes of mammalian cells are vulnerable to an array of DNA-damaging agents, of both endogenous and environmental origin. If DNA damage is recognized by cell machinery, several responses may occur to prevent replication in the presence of genetic errors and to repair the damage. A deficiency in repair capacity due to the defects in genes involved in DNA repair is a major threat to the genetic integrity of cells and can lead to car-cinogenesis. Several pathways of DNA repair exist that recognise and repair specific types of DNA damage and each pathway involves numerous enzymes.1,2
Three forms of excision repair are distinguished: mismatch repair (MMR), nucleotide excision repair (NER), and base excision repair (BER).2 MMR corrects post-replicative errors caused by insufficient proof-reading activity of DNA polymerase.3 The NER pathway mainly removes bulky adducts caused by environmental
agents. One of its most important functions in humans is to repair UV-induced DNA damage.4 The BER proteins excise and replace damaged bases and some of these enzymes are also involved in single-strand breaks (SSB) repair.5 Repair of double-strand breaks (DSB) is intrinsically more difficult than other types of DNA damage because no undamaged template is available. Therefore DSB repair pathway utilizes either the non-homologous end-joining or homologous recombination.6
Continuous exposure of the cellular DNA to a multitude of agents in our living and working environment can cause damage to cellular macromolecules.710 However, the majority of DNA damage has an endogenous origin and it is mainly due to oxidation of DNA by reactive oxygen species (ROS), which are generated in the cells by normal aerobic metabolism.11 Oxidative stress may lead to DNA lesions, such as oxidized DNA bases, apuri-nic/apyrimidinic (AP) sites, SSBs, and DSBs. The BER pathway is the most important mechanism involved in re-
pair of oxidative lesions, as it recognises and repairs base modifications, as well as AP sites and DNA SSBs.12,13
The BER pathway is a multi-step process that is initiated with recognition and removal of the altered base by a DNA glycosilase, which catalyses the cleavage of the N-glycosil bond between the modified base and sugar moiety, thus generating an AP site.14,15 After base excision an AP endonuclease (APE) nicks the phosphodiester backbone 5' to the AP site and also generates 3'-hydroxyl termini at SSBs,16,17 which is necessary for gap-filling repair synthesis by a DNA polymerase P (POLB).18 The final step is the sealing of the nicked DNA by DNA ligase I or III.19
The human oxoguanine glycosylase 1 (hOGG1) and X-ray repair cross-complementing protein 1 (XRCC1) are key enzymes in the BER pathway, and they may play an important role in maintaining DNA repair capacity (DRC) and preventing cancer occurrence.20 The hOGG1 directly removes 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged DNA. 8-oxoG is one of the most stable deleterious products of oxidative DNA damage and represents a highly mutagenic miscoding lesion that can lead to G:C to T:A transversion mutations.21 The XRCC1 protein serves as a scaffold protein for other proteins involved in DNA repair, such as DNA ligase III and POLB.22,23 It is also involved in SSBs repair through interaction with the poly-ADP-ri-bose polymerase (PARP).24
As single nucleotide polymorphisms (SNPs) in the DNA repair enzymes alter enzymes' activity or its expression, they may modify the inter-individual variability in their DRCs, and can be consequently associated with the altered cancer risk. The aim of our study was to determine the frequencies of polymorphisms in hOGG1 and XRCC1 genes in healthy Slovenian population and to evaluate their influence on DNA damage established by comet assay.
2. Experimental
2. 1. Patients
The study population consisted of 141 unrelated healthy Central European Caucasian. A subgroup of 26 subjects was selected randomly and blind to any genoty-ping data for participation in comet assay. All subjects in the subgroup were asked to refrain of physical activities for at least 2 days before venepuncture. The study was approved by the Slovenian Ethics Committee for Research in Medicine and was carried out according to the Helsinki Declaration. Written informed consent was obtained from all individuals prior to participation.
2. 2. Genotyping
Genomic DNA was isolated from peripheral blood leukocytes using a standard salting-out procedure and a Qiagen FlexiGene kit (Qiagen GmbH, Germany).25 To determine the hOGG1 Ser326Cys (rs1052133), XRCC1
Arg194Trp (rs1799782) and Arg399Gln (rs25487) polymorphisms TaqMan SNP genotyping assays were used (C_3095552_1, C_11463404_10 and C_622564_10, respectively, AB assay, Applied Biosystems, Foster City, California, USA). Real time PCR was performed under universal conditions on ABI 7500 Real-Time PCR System (Applied Biosystems) and results were analysed with its System SDS software version 1.2.3. To ensure the data quality, 20% of samples were re-genotyped and genotypes for each subject were also checked manually.
2. 3. Comet Assay
Comet assay is an alkaline single-cell gel electrop-horesis assay that has been optimized to analyse single-and double-stranded DNA damage in human lymphocytes. Cells were isolated from 1 ml of fresh whole blood26 and separated in two aliquots; one aliquot was used immediately for comet assay and the other was processed for freezing. Cells were mixed with freezing mix (FCS:DM-SO = 9:1), aliquoted in 200 pl per tube and slowly frozen in a cell freezing box with iso-propanol and stored at -70 °C. For the comet assay the cells were thawed at room temperature, pelleted by centrifugation at 3300 rpm for 15 min at 20 °C and washed with 1,5 ml of washing mix (RP-MI :1 00 mM PBS = 2 : 1) per tube. Cells were again pelleted at 2800 rpm for 10 min at 4 °C and resuspended in 1% Low Melting Point (LMP) agarose.
Comet assay was performed according to the protocol by Tice et al.27 Two slides were assayed in parallel for each sample and 50 randomly selected cells were analysed per slide within 24 hours after preparation. For visualization on epifluorescence microscope (Olympus CH 50) slides were stained with DAPI (1 pg mL-1). Observations were made using 200 x final magnifications. Microscope was connected to the computer through CCD camera (Hamamatsu Orca 1) and DNA damage was quantified using Comet 5 software (Kinetic Imaging Ltd, 2000, UK). The level of DNA damage was evaluated by two parameters: the percentage of DNA in the tail (% TD) and the Olive Tail Moment (OTM, the product of the amount of DNA in the tail and the mean distance of migration in the tail).
2. 4. Statistical Analysis
To verify that allele frequencies were in Hardy-Weinberg equilibrium a chi-square statistic was used. Multiple linear regression analysis with the adjustment of age and sex was used to determine the influence of studied genetic polymorphisms on % TD. In this analysis logarithmic transformation of non-normally distributed % TD was used and normality of its standardized residuals was evaluated by Kolmogorov-Smirnov test. Arithmetic mean was used to measure central tendency, while standard deviation was a measure of variability. The level of signifi-
cance was set to P < 0.05. All statistical analyses were done using SPSS for Windows version 14.0.1 software (Statistical Package for the Social Sciences, Chicago, IL).
3. Results and Discussion
In our study we first determined the frequencies of common functional single nucleotide polymorphisms (SNPs) in specific BER genes in healthy Slovenian population. In total, 141 subjects, 60 male (42.6%) and 81 female (57.4%), median age 23 (range 18-36) years, were analysed for hOGG1 Ser326Cys, XRCC1 Arg194Trp and Arg399Gln polymorphisms. The distribution of polymorphism frequencies is summarized in Table 1.
The frequencies of the hOGG1 326Ser/Ser, 326Ser/ Cys and 326Cys/Cys genotypes in the Slovenian popula-
Table 1 Allele frequencies of hOGG1 Ser326Cys, XRCC1 Arg194Trp and Arg399Gln polymorphisms in healthy unrelated subjects (N = 141)
Gene	Genotype	N (%)
hOGG1	326Ser/Ser	90 (63.8)
326Ser/Cys	42 (29.8)
326Cys/Cys	9 (6.4)
XRCC1	194Arg/Arg	125 (88.7)
194Arg/Trp	13 (9.2)
194Trp/Trp	3 (2.1)
XRCC1	399Arg/Arg	66 (46.8)
399Arg/Gln	57 (40.4)
399Gln/Gln	18 (12.8)
tion were 63.8, 29.8, and 6.4%, respectively. The frequency distribution of XRCC1 polymorphism was 88.7% for 194Arg/Arg, 9.2% for 194Arg/Trp, 2.1% for 194Trp/ Trp and 46.8% for 399Arg/Arg 40.4% for 399Arg/Gln, 12.8% for 399Gln/Gln. The genotype distributions for investigated polymorphisms, except for XRCC1 194Arg/ Trp (P = 0.007), were consistent with Hardy-Weinberg equilibrium.
Ethnic and inter-individual differences in BER genetic polymorphisms have been observed in various populations. As compared to other studies, we found that the observed genotype frequencies of hOGG1 Ser326Cys polymorphism were mostly in agreement with values published for other Caucasian populations (Table 2).
The variations in the distribution of XRCC1 Arg399Gln polymorphism showed higher prevalence of the XRCC1 399Arg allele in Slovenian population in comparison to other Caucasian populations (Table 3).
We also observed that XRCC1194 Trp/Trp genotype was slightly but not significantly more frequent in Slovenian population than in other Caucasian population, as other studies mostly found less than 1% of subjects with Trp/Trp genotype (Table 4).
It is important to obtain data for the observed frequencies of these polymorphisms comparable to those available for Caucasian populations, since there was no information on the inter-individual variability of these polymorphisms in the Slovenian population before our study.
Further on, we investigated the influence of SNPs in the genes coding for the BER enzymes on DNA damage
Table 2 The distribution of genotype frequencies for hOGG1 Ser326Cys polymorphism in different Caucasian populations
Individuals
N
Genotype frequency (%) Ser326Ser Ser326Cys Cys326Cys
References
Czech
Hungarian
German
Irish
Italian
Slovenian
Turkish
532 149 105 247 146 141 206
62 64 57 57 66 64 50
34 33 41 39 30 30 41
Pardini et al.28 Sugimura et al.29 Wikman et al.30 Ferguson et al.31 Coppede et al.32 present study Karahalil et al.33
Table 3 The distribution of genotype frequencies for XRCC1 Arg399Gln polymorphism in different Caucasian populations
Individuals
N
Genotype frequency (%) Arg399Arg Arg399Gln Gln399Gln
References
American	549	42	45	13	Applebaum et al.34
Czech	532	41	46	14	Pardini et al.28
Irish	247	40	39	14	Ferguson et al.31
Italian	121	44	50	6	Improta et al.35
Polish	124	40	43	18	Kowalski et al.36
Slovenian	141	47	40	13	present study
Table 4 The distribution of genotype frequencies for XRCC1 Arg194Trp polymorphism in different Caucasian populations
Individuals
N
Genotype frequency (%) Arg194Arg Arg194Trp Trp194Trp
References
American
Czech
Italian
Polish
Slovenian
549 532 121 124 141
88 88 86 82 89
11 11 14 18
9
Applebaum et al.34 Pardini et al.28 Improta et al.35 Kowalski et al.36 present study
level quantified as % TD and OTM, established by the comet assay in a subgroup of 26 randomly selected subjects, 6 male (23.1%) and 20 female (76.9%), mean age 24 (range 20-29) years. In its basic form the comet assay is a sensitive method for detecting DNA strand breaks. Additionally, the alkaline conditions of the comet assay are sufficient to convert the alkali-labile sites (i.e. AP-sites) to breaks, and thus it is possible to detect the inefficient activity of hOGGl.37 Consequently, variable DNA content in the comet tail may directly reflect genetic variability in DNA repair genes. For the evaluation of DNA repair by comet assay we used % TD rather than OTM, as % TD is linearly related to the DNA break frequency.26 Additionally, several studies recommended the use of % TD for regulatory studies.38,39
Selected genetic polymorphisms, age and sex were used as predictors in a multivariate analysis of the logarithmically transformed % TD and OTM. We observed that % TD was influenced by age (P = 0.01) but not by sex (P = 0.964). When we assessed the influence of hOGG1 Ser326Cys and XRCC1 Arg194Trp and Arg399Gln polymorphisms on % TD and OTM in the subgroup of 26 subjects, we found an association of hOGG1 Ser326Cys polymorphism with % TD (P = 0.032), but no significant influence of XRCC1 genotypes on % TD (data not shown). For further statistical analysis we excluded three smokers, 1 male and 2 female, from this subgroup, because some studies observed an effect of smoking on DNA migration in the comet assay.40,41 The new subgroup consisted of 23 subjects, 5 male (21.7%) and 18 female (78.3%), mean age 23 (range 20-29) years. The influence of the studied polymorphisms on% TD and OTM in this subgroup is presented in Table 5.
In the group of 23 subjects we clearly demonstrated that hOGG1 Ser326Cys polymorphism has an important impact on DNA damage. We observed that the individuals with the homozygous wild-type hOGG1 326Ser/Ser genotype had significantly lower mean % TD values as compared to the subjects with heterozygous variant 326Ser/Cys genotype (% TD = 6.9 ± 1.4 vs. % TD = 8.9 ± 4.2, P = 0.017) (Fig 1a). The XRCC1 Arg399Gln polymorphismhad no significant influence on % TD, although the subjects with XRCC1 399Arg/Arg genotype tended to have lower mean % TD values as compared to those with at least one 399Gln allele (% TD = 6.9 ± 1.4 vs. % TD = 7.7 ± 3.0, P = 0.064) (Fig 1b). We observed no significant influence of h-OGG1 Ser326Cys (P = 0.087) and XRCC1 Arg399Gln (P = 0.087) polymorphism on OTM. Only two subjects with XR-CC1 194Arg/Trp genotype were detected, so the association of this SNP with % TD and OTM was not assessed.
The interpretation of our results is biologically plausible since there are evidences that all the three polymorphisms have a functional effect. In hOGG1 enzyme, polymorphic amino acid 326Cys is located outside the conserved domain. Nevertheless, recent data showed that the variant hOGG1 326Cys protein has lower ability to prevent mutagenesis by 8-OHdG than hOGG1 326Ser in vivo. 42 This polymorphism was described to affect the glycosyla-se function due to the localization and phosphorylation status.43 The XRCC1 is a multi-domain protein that interacts with many other proteins involved in DNA repair. It was shown that XRCC1 Arg194Trp and Arg399Gln polymorphisms have impact on DRC, since amino acid 194 is in the linker region of the XRCC1 N-terminal functional domain and 399 is located within the BRCA1 C-terminus functional domain.44
Table 5 The influence of hOGG1 and XRCC1 genotypes on the mean % TD and OTM values
Gene
Genotype
N (%)a
Mean % TD ± SD
P
Mean OTM ± SD
hOGG1
XRCC1
XRCC1
326Ser/Ser
326Ser/Cys
194Arg/Arg
194Arg/Trp
399Arg/Arg
399Arg/Gln
+ Gln/Gln
17 (73.9) 6 (26.1) 21 (91.3) 2 (8.7) 9 (39.1)
6.895	± 1.392 8.862 ± 4.220 7.566 ± 2.554 5.750 ± 0.863
6.896	± 1.359
0.017
0.062
14 (60.9) 7.738 ± 3.020
0.924 ± 0.295 1.298 ± 1.073 1.034 ± 0.620 0.895 ± 0.233 0.863 ± 0.241
1.124 ± 0.730
0.087
0.087
a Genotype frequencies in the subgroup (N = 23)
P
Fig. 1 hOGG1 Ser326Cys polymorphism and % TD in 23 subjects. Subjects with hOGG1 326Ser/Ser genotype had significantly lower mean % TD as compared to subjects with hOGG1 326Ser/Cys and/or 326Cys/Cys genotype (a). XRCC1 Arg399Gln polymorphism and % TD in 23 subjects. The XRCC1 polymorphism had no significant influence on the % TD, although the subjects with XRCC1 399Arg/Arg genotype tended to have lower mean % TD values as compared to those with XRCC1 399Arg/Gln + Gln/Gln genotype (b).
Additionally, the observed influences of individual genotypes on DNA damage are in good agreement with other studies. Similar to our study, decreased activity of variant hOGGl 326Cys enzyme has already been established.20,45 Our results are also consistent with findings that XRCC1 399Arg/Arg genotype is associated with reduced DRC9,46 and increased number of chromosomal breaks per cell.20 Due to the known biological function of amino acid 194 in XRCC1 protein, an effect of this polymorphism on DNA damage is expected. So far several studies have evaluated the influence of XRCC1 Arg194Trp polymorphism on DNA integrity, yet their findings are conflicting. Some investigators observed higher values of DRC in the carriers of 194Arg allele,7 while others found no functional significance of this SNP.20,46,47 Similarly to our study, low significance of XRCC1 Arg194Trp polymorphism may be a consequence of low frequency of 194Trp allele in Caucasian population.
The main limitation of our study was that it included a relatively small number of individuals, albeit comparable to some other studies.9,48-50 Another bias was that not all the polymorphic genes involved in the BER were tested. Although studies of polymorphisms in the BER enzymes are mostly focused on the SNPs in the hOGG1 and XR-CC1 gene, other SNPs in the enzymes of BER pathway, that might have an effect on DRC, were previously studied but not investigated in our study. In addition, our study was not biased by genetic heterogeneity, since all the subjects were recruited in a geographic area with an ethnically homogeneous population.51
To our knowledge, no study on the association of genetic polymorphisms in BER pathway with DNA repair in
healthy individuals, unexposed to any agents in working environment, has been reported to date. Other similar studies have been performed on individuals, who had been occupationally exposed to various DNA-damaging agents, such as pesticide,8 styrene,9 mineral fibres,52 cobalt, hard metal dust,49 and polycyclic aromatic hydrocar-bons.10 Although, our study was established on relatively small group of healthy subject we managed to show a major impact of hOGG1 Ser326Cys polymorphism on % TD. However, further prospective studies with larger sample sizes are required to support our observations.
4. Conclusions
Our results confirmed that DNA damage is modulated by hOGG1 Ser326Cys polymorphism. This study may thus contribute to the interpretation of the association between functional DNA repair gene polymorphisms and cancer risk, cancer progression and treatment response in Slovenian cancer patients.
5. Acknowledgements
The authors wish to acknowledge Prof. Emilio Martínez de Victoria Muñoz, PhD, Maria del Carmen Ramirez Tortosa, PhD and Jose L Quiles, PhD from the Institute of Nutrition and Food Technology (Instituto de Nutrición y Tecnología de los Alimentos, INYTA), University of Granada, Spain for their kind help with setting up the comet assay. The authors wish to thank Prof. Romana Ma-
rinsek Logar, PhD from University of Ljubljana, Biotechnical Faculty for providing time on their Olympus CH 50 microscope and Petra Bohanec Grabar from University of Ljubljana, Faculty of Medicine for statistical analysis.
This work was financially supported by The Slovenian Research Agency (bilateral project BI-ES/04-05-016 and ARRS Grant No. P0-0503-0381).
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Povzetek
Genetski polimorfizmi mehanizmov za popravljanje DNA lahko vplivajo na stopnjo poškodb DNA. Namen raziskave je bil določiti pogostnost polimorfizmov posameznih nukleotidov (SNP) v izbranih genih mehanizma za popravljanje DNA z izrezovanjem baz (BER) v zdravi slovenski populaciji ter opredeliti njihov vpliv na stopnjo poškodb DNA, ki je bila določena s kometnim testom. Polimorfizme hOGG1 Ser326Cys, XRCC1 Arg194Trp in Arg399Gln smo pri 141 ne-sorodnih zdravih odraslih osebah določali z metodami genotipizacije, ki temeljijo na verižni reakciji s polimerazo (PCR) v realnem času. Frekvence genotipov hOGG1 326Ser/Ser, 326Ser/Cys in 326Cys/Cys so bile 63,8 %, 29,8 % in 6,4 %. Porazdelitev genotipov XRCC1 je bila: 194Arg/Arg 88,7 %, 194Arg/Trp 9,2 %, 194Trp/Trp 2,1 % in 399Arg/Arg 46,8 %, 399Arg/Gln 40,4 %, 399Gln/Gln 12,8 %. Na skupini 26 preiskovancev smo določili vpliv izbranih polimorfizmov BER na odstotek DNA v kometnem repu ( % TD). Ugotovili smo, da imajo heterozigoti hOGG1 326Ser/Cys statistično značilno višjo vrednost % TD kot osebe z genotipom 326Ser/Ser ( % TD = 8.9 ± 4.2 proti % TD = 6.9 ± 1.4, P = 0.017). Polimorfizma XRCC1 Arg194Trp in Arg399Gln nista imela vpliva na % TD. Rezultati naše raziskave potrjujejo vpliv polimorfizma hOGG1 Ser326Cys na stopnjo poškodb DNA.