Association of the XRCC1 and XRCC3 Gene Variants with Type 2 Diabetes Mellitus
- Home
- Journals
- 2017
- Association of the XRCC1 and XRCC3 Gene Variants with Type 2 Diabetes Mellitus...
2Department of Internal Medicine, Faculty of Medicine, Istanbul University, Istanbul, Turkey
ABSTRACT
Background and aims: Type 2 diabetes mellitus (T2DM), isthe most common metabolic disease of the adult population and characterized with disorders in insulin secretion and activity. The hyperglycemia seen inT2DM leads to free radical production via glucose oxidation or other complexmechanisms and creates DNA damage. Polymorphisms of the two DNA repairgenes; XRCC1 and XRCC3 were investigated for their impact on disease riskand clinical parameters of T2DM patients. Materials and Methods: The patientgroup was comprised of 34 women and 40 men, a total of 74 patients, diagnosed with type 2 diabetes. The control group was randomly selected from thepopulation as 52 women and 50 men, a total of 102 individuals, whom did nothave diabetes. In order to determine the XRCC1 Arg194Trp (C/T) and XRCC3Thr241Met (C/T) polymorphisms, the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques were used. Results:Statistical analysis showed that the XRCC1 Arg194Trp and XRCC3 Thr241Metpolymorphisms have no association with type 2 diabetes and its clinical parameters compared to the control group. Conclusions: We believe that to clarifythe relationship between the XRCC1 Arg194Trp and XRCC3 Thr241Met polymorphisms and T2DM, the study should be carried on with an expanded studygroup
INTRODUCTION
Diabetes mellitus (DM) is a chronic and metabolicdisease described by elevated blood glucose levels.As a result of the pathological events occurring inthe genetic and immune structure, the absolute orrelative absence or inactivity of the insulin hormone secreted from pancreas β- cells, causes disorders in carbohydrate, protein and fat metabolismsand various events in all systems (1). One of every30 adults is diabetic and the prevalence is expected to double by 2030. Although both types of DMare increasing, type 2 diabetes mellitus (T2DM) isthe most prevalent form seen in 5-10% of the population in developed countries (2). T2DM is characterized by the disorders in insulin secretion oractivity and also the body becomes insensitive tothe metabolic effects of insulin (3).The molecular mechanisms that form the basisof T2DM are still unknown, yet many studies arefocused on enlightening the underlying geneticabnormalities. The hyperglycemia seen in T2DMleads to free radical formation through glucose oxidation, non-enzymatic protein glycation and complex mechanisms including the polyol pathway (4,5). The β-cells are more susceptible to oxidativestress since they contain lower levels of antioxidants compared to other tissues. Therefore oxidative stress plays an important role in diabetesdevelopment and the following complications (6).Organisms have developed several DNA repairmechanisms against certain agents to conserve theintegrity of their genetic material (7). These harmful agents that cannot be removed from DNA doplay a role in diabetes development. Also, the increased oxidative stress due to diabetes both worsens the prognosis and breaks the balance betweenoxidative stress and antioxidant mechanisms thushelping other genetic diseases to manifest (8,9). Inconditions of severe DNA damage, a cell selects theapoptotic path, but in mild damage it tries to solveit using the repair mechanisms. Thus, cells vital tothe immune system, such as the pancreatic β-cells,will be spared from destruction (10,11).In Base Excision Repair (BER), where minor pointdamages are repaired, there is a process of recognition and removal of the single strand oxidation,alkylation, hydrolysis and deamination relateddamages. One of the genes coding the proteins ofthe BER pathway is the X-ray repair cross-complementing group 1 (XRCC1) (12). The XRCC1 geneis located on chromosome 19 region q13.2, wheremore than 60 single nucleotide polymorphisms(SNPs) have been identified. Due to the functional importance and high allelic frequency the mostwidely studied polymorphism is the Cytosine/ Thymine (C/T) transition on codon 194 leading to a arginine to tryptophane substitution (13,14).Damages do not always occur on only a singlestrand of the genetic material. Oxidative stress,ionized radiation and even a normal physiologicprocess, somatic recombination, can damage boththe strands. Double strand damages are repairedwith two mechanisms, which are homologous recombination and non-homologous recombinationend joining. The X-ray repair complementinggroup 3 (XRCC3) protein plays a role in the homolog recombination (15). The XRCC3 gene is located on chromosome 14 region q32.3 (16), wheremore than 100 SNPs have been reported. The mostbroadly studied one is the C/T transition on exon7, codon 241 that is presented as a Thr/Met substitution (17).Until now, the effects of different polymorphismsof several DNA repair genes have been investigated on type 2 diabetes. However, the effects ofthe XRCC1 Arg194Trp and XRCC3 Thr241Metgene polymorphisms have not been studied.Therefore, the effect of DNA damage repair genepolymorphisms, XRCC1 Arg194Trp and XRCC3Thr241Met, have been investigated on type 2 diabetes mellitus patients for their relation with disease risk and clinical parameters in this study
MATERIALS
Study Groups
This study was conducted with the approval of theIstanbul Medical Faculty Ethical Committee, Istanbul University. Two study groups have been included in this study. The patient group comprisedof 34 women and 40 male, a total of 74 patients,diagnosed with type 2 diabetes mellitus and whowere on follow up by the Division of Endocrinologyand Metabolic Diseases, Department of InternalMedicine, Istanbul Medical Faculty, Istanbul University. The control group included non-diabetic 52women and 50 men, a total of 102, non-diabetic,healthy individuals of the Turkish population
DNA Isolation and SNP Detection
In EDTA containing tubes, 10 ml of venous bloodsamples were obtained from the participants.Samples were stored at -20 °C until the genomicDNA isolation was performed using the salting outmethod (18). The primers used for the polymerasechain reaction (PCR) amplifications of the regionsof the XRCC1 Arg194Trp and XRCC3 Thr241Metpolymorphisms are given in Table 1. The reactionvolumes were set for a total of 25 μl as 16.2 μl apyrogenik water, 2.5 μl MgCl2 free (10X) buffer, 2.5 μlMgCl2 (25 mM) buffer, 1.5 μl dNTP (10 mM), 1 μlmix of forward (10 pmol) and reverse primers (10pmol), 0.3 μl Taq polimerase (5 U/μl) ve 1 μl 200ng/μl genomic DNA sample. The PCR mixes wereprepared on ice and in a sterile cabin.For the XRCC1 Arg194Trp polymorphism, thePCR reaction conditions were set as following theinitial denaturation of 95 ºC for 5 minutes, 95 ºCfor 30 sec, 58 ºC for 45 sec and 75 ºC 45 sec for 35cycles and a final elongation duration of 10 min at72 ºC. Then, for the XRCC3 Thr241Met, followingthe initial denaturation of 95 ºC for 5 minutes, 94ºC for 1 min, 58 ºC for 1 min and 75 ºC 1 min for30 cycles and a final elongation at 72 ºC for 5 min.The PCR yields were controlled on 2% agarose gelelectrophoresis.In order to determine the XRCC1 Arg194Trp andXRCC3 Thr241Met polymorphisms, obtainedPCR yields were digested with PvuII and Hin1II(Hsp92II) restriction enzymes, respectively. Thedigested yields were separated on 2% agarose gelelectrophoresis and genotyped after being viewedunder UV light. The obtained PCR and restrictionyields and genotyping of the polymorphisms areshown in Table 1.
Statistical Analysis
The statistical analysis was performed using SPSSversion 11.0 (SPSS inc. Chicago, USA). The statistical significance cutoff was taken as p<0.05. Thedistributions of the genotype and allele frequencies between study groups were evaluated usingthe Chi-square and Fisher’s exact test. The demographic data were compared between the studygroups using the Student’s T and Anova tests. Allele frequencies were calculated according to thegene counting method.
RESULTS
The demographic data of the study groups is givenin Table 2. There were no significant difference bymeans of age, gender, body mass index and smoking (p>0.05). Also, biochemical parameters of thestudy group given in Table 3 and no statisticallysignificance was found according to lipid profilesbetween the patient and control groups (p>0.05).The genotype and allele distributions were similar between the patient and control groups for the XRCC1 Arg194Trp polymorphism even that therewere no individuals were detected to carry the TTallele in both groups (p>0.05) (Table 4). likewise,there was no statistically significant difference forthe XRCC3 Thr241Met genotype and allele distributions (Table 5) The quantitative examinations of the demographic data for the XRCC1 Arg194Trp and XRCC3Thr241Met genotypes in the patient group areshown in Tables 6 and 7, respectively. According tothese findings no statistical significant differenceswere observed for either polymorphism.
DISCUSSION
Diabetes mellitus is a metabolic disease characterized by high blood sugar (hyperglycemia) developed due to the partial or complete absence ofinsulin. T2DM is the most common type of diabetesand although almost all the patients have a family history, the disease has not yet been explained with a single genetic mechanism (19). Owing tothe gene-environment interactions some polymorphisms found in DNA repair genes cause a promotion in disease tendencies since they affect the capacity of DNA repair. Impairments in the XRCC1and XRCC3 increase genetic instability and susceptibility to DNA damaging agents (20). In this study, the prevalence of the XRCC1 Arg-194Trp (C/T) and the XRCC3 Thr241Met (C/T)polymorphisms and their impacts on T2DM development was investigated. This study, which wasconducted on a Turkish population, is the first tofocus on this hypothesis. According to the demographic data collected the study group, comprisedof 74 patients and 102 controls, no statistically significant difference was observed in terms of age,gender, smoking status, body mass index and lipidprofile distributions. Since there are no other studies directed to the XRCC1 Arg194Trp polymorphism and a T2DM patient group for comparisonto this study, the disease mechanism was attempted to be comprehended based on the other studiesrelated to the XRCC1 Arg194Trp polymorphismand other patient groups or another polymorphismassociated with T2DM.The effect of the XRCC1 Arg194Trp polymorphismon patients of glaucoma with open angle havebeen investigated and no connection was found.On the other hand, the XRCC1 399 Arg/Gln polymorphism was indicated as a risk factor for the development of glaucoma with open angle (21). Therole of the XRCC1 399 Arg/Gln polymorphism onT2DM and diabetic nephropathy was studied andno significant relation was observed (22, 23). However, Narne et al., suggested that the XRCC1 399Arg/Gln polymorphism increases the risk of diabetic nephropathy (24). According to the findingsof a study, where 207 prostate cancer patients and235 healthy controls were included, it was reported that the Arg194Trp polymorphism decreasedthe risk of the disease’s development (25). In thestudy that Deligezer et al., conducted on Turkishbreast cancer patients and controls to investigate the effects of the Arg194Trp and Arg399Glnpolymorphisms, because both the alleles showedsimilar frequency in both groups, they concluded that there were no associations between theseXRCC1 variants and breast cancer development(26). Moreover, in the study of Demokan et al., on XRCC1 Arg194Trp polymorphism of 95 patientsof head and neck cancer, no significant association was found (27). Again, in a study of Dumanet al., on 73 Turkish chronic lymphatic leukemiapatients, the allele frequency was found similarwith the control group (28). While the mutant allele frequency was found to be 4-5% by Deligezeret al., (26) in both study groups, it was 1.37% inthe patient group and there were no mutant allelecarriers in the control group in the Duman et al.study (28).In this study, the allele frequencies for the XRCC1Arg194Trp polymorphisms were detected to besimilar between the patient and control groups.Thus, compatible with the three other studiesconducted on the Turkish population by Deligezeret al., Demokan et al. and Duman et al., the protective effects observed in other populations havenot been seen and no statistical significances havebeen encountered. Moreover, the Trp/Trp genotypefrequency is quite low in the Turkish population.There were no homozygous mutant allele carriersin either study group of this study.So as to elucidate the function and molecularmechanisms of the polymorphisms of the XRCCand the other related BER components, the studygroup should be expanded and meticulous studiesshould be carried out.The other polymorphism of this study, the XRCC3Thr241Met, showed no statistical significance between the study groups in terms of genotype andallele distributions. Furthermore, the genotypedistributions did not show any associations withany of the demographic criteria.In a study on 160 acute myeloid leukemia (AML)patients and 161 controls, the XRCC3 Met/Met wasshown to increase the risk of AML 2 fold (29). Similarly, in a study on 140 colorectal cancer patientsand 280 controls, both heterozygous and homozygous mutant genotype increased the risk of colorectal cancer 3 fold (30). Sangrajrang et al., (31) investigated the association between the XRCC3Thr241Met polymorphism and breast cancer in aThai population and found an association with theMet allele. In addition, Narter et al., (32) evaluatedthe relation between the XRCC3 Thr241Met polymorphism and bladder cancer. They found that theXRCC3 T allele distribution had a significant difference between the study groups and the mutantallele created protection against the risk of bladdercancer development by 4.87 fold.There were no other studies in the literature thatinvestigated the relationship between the XRCC1Arg194Trp and XRCC3 Thr241Met polymorphismsand type 2 diabetes mellitus and/or lipid profiles in the Turkish population. Thus our study is the firstto focus on the mentioned polymorphisms in such agroup and we believe that it might be a data sourcefor the further studies.
Acknowledgements: This study was supported byThe Research Support Unit of Istanbul University, project no: 6529. In this study, the experimentalwork was carried out by Muhammed Oguz Gokceas his Master’s Thesis in Istanbul University andthe study was proposed and guided by Prof. Dr.Umit Zeybek.
Conflict of Interest: The authors declare thatthey have no conflict of interest.
REFERENCES
1. King H, Aubert RE, Herman, WH. Global burden of diabetes,1995-2025: prevalence, numerical estimates, and projections. Diabetes care 1998; 21:1414-1431.
2. Whiting DR, Guariguata L, Wei C, et al. IDF Diabetes Atlas:Global estimates of the prevalence of diabetes for 2011 and2030. Diabetes Res Clin Pr 2011; 94:311-321.
3. Gillies CL, Abrams KR, Lambert PC, et al. Pharmacologicaland lifestyle interventions to prevent or delay type 2 diabetesin people with impaired glucose tolerance: systematic reviewand meta-analysis. Bmj 2007; 334:299.
4. Lappas M, Hiden U, Desoye G, et al. The role of oxidativestress in the pathophysiology of gestational diabetes mellitus.Antioxidants & redox signaling 2011; 15:3061-3100.
5. Lipinski B. Pathophysiology of oxidative stress in diabetes mellitus. Journal of diabetes and its complications 2001; 15:203-210.
6. Cheeseman KH, Slater TF. An introduction to free radical biochemistry. British medical bulletin 1993; 49:481-493.
7. Merecz A, Markiewicz L, Sliwinska A, et al. Analysis of oxidative DNA damage and its repair in Polish patients with diabetes mellitus type 2: Role in pathogenesis of diabetic neuropathy. Advances in medical sciences 2015; 60:220-230.
8. Paolisso G, Giugliano D. Oxidative stress and insulin action: isthere a relationship? Diabetologia 1996; 39:357-363.
9. Blasiak J, Arabski M, Krupa R, et al. DNA damage and repair intype 2 diabetes mellitus. Mutation research 2004; 554:297-304.
10. Fleck O, Nielsen O. DNA repair. Journal of cell science 2004;117:515-517.
11. Bertram C, Hass R. Cellular responses to reactive oxygenspecies-induced DNA damage and aging. Biological chemistry2008; 389:211-220.
12. Shen M, Hung RJ, Brennan P, et al. Polymorphisms of the DNArepair genes XRCC1, XRCC3, XPD, interaction with environmental exposures, and bladder cancer risk in a case-controlstudy in northern Italy. Cancer epidemiology, biomarkers &prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2003; 12:1234-1240.
13. Hu Z, Ma H, Chen F, et al. RCC1 polymorphisms and cancer risk: a meta-analysis of 38 case-control studies. Cancerepidemiology, biomarkers & prevention : a publication of theAmerican Association for Cancer Research, cosponsored bythe American Society of Preventive Oncology 2005; 14:1810-1818.
14. Hung RJ, Hall J, Brennan P, et al. Genetic polymorphisms inthe base excision repair pathway and cancer risk: a HuGE review. American journal of epidemiology 2005; 162:925-942.
15. Sonoda E, Hochegger H, Saberi A, et al. Differential usage ofnon-homologous end-joining and homologous recombination indouble strand break repair. DNA repair 2006; 5:1021-1029.
16. Yamada NA, Hinz JM, Kopf VL, et al. XRCC3 ATPase activity isrequired for normal XRCC3-Rad51C complex dynamics and homologous recombination. The Journal of biological chemistry2004; 279:23250-23254.
17. Xu ZY, Loignon M, Han FY, et al. Xrcc3 induces cisplatin resistance by stimulation of Rad51-related recombinational repair, S-phase checkpoint activation, and reduced apoptosis.The Journal of pharmacology and experimental therapeutics2005; 314:495-505.
18. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleicacids research 1988; 16:1215.
19. Wilder RP. Education and mortality in type 2 diabetes. Diabetes care 2003; 26:1650.
20. Ramadan RA, Desouky LM, Elnaggar MA, et al. Associationof DNA repair genes XRCC1 (Arg399Gln), (Arg194Trp) andXRCC3 (Thr241Met) polymorphisms with the risk of breastcancer: a case-control study in Egypt. Genetic testing and molecular biomarkers 2014; 18:754-760.
21. Szaflk JP, Cuchra M, Przybylowska-Sygut K, et al. Association of the 399Arg/Gln XRCC1, the 194 Arg/Trp XRCC1, the326Ser/Cys OGG1, and the 324Gln/His MUTYH gene polymorphisms with clinical parameters and the risk for development of primary open-angle glaucoma. Mutation research2013; 753:12-22.
22. Kasznicki J, Krupa R, Blasiak J, et al. Association betweenpolymorphisms of the DNA repair genes XRCC1 and hOGG1and type 2 diabetes mellitus in the Polish population. PolskieArchiwum Medycyny Wewnetrznej 2009; 119:122-128.
23. Narne P, Ponnaluri KC, Siraj M, et al. Polymorphisms in oxidative stress pathway genes and risk of diabetic nephropathyin South Indian type 2 diabetic patients. Nephrology 2014;19:623-629.
24. Narne P, Ponnaluri KC, Siraj M, et al. Association Analysis ofPolymorphisms in Genes Related to Oxidative Stress in SouthIndian Type 2 Diabetic Patients with Retinopathy. Ophthalmicgenetics 2016; 37:1-8.
25. Xu Z, Hua LX, Qian LX, et al. Relationship between XRCC1polymorphisms and susceptibility to prostate cancer in menfrom Han, Southern China. Asian journal of andrology 2007;9:331-338.
26. Deligezer U, Dalay N. Association of the XRCC1 gene polymorphisms with cancer risk in Turkish breast cancer patients. Experimental & molecular medicine 2004; 36:572-575.
27. Demokan S, Demir D, Suoglu Y, et al. Polymorphisms of theXRCC1 DNA repair gene in head and neck cancer. Pathologyoncology research 2005; 11:22-25.
28. Duman N, Aktan M, Ozturk S, et al. Investigation of Arg399Glnand Arg194Trp polymorphisms of the XRCC1 (x-ray cross-complementing group 1) gene and its correlation to sister chromatid exchange frequency in patients with chronic lymphocyticleukemia. Genetic testing and molecular biomarkers 2012;16:287-291.
29. Voso MT, Fabiani E, D’Alo F, et al. Increased risk of acutemyeloid leukaemia due to polymorphisms in detoxifiation andDNA repair enzymes. Ann Oncol 2007; 18:1523-1528.
30. Jin MJ, Chen K, Song L, et al. The association of the DNA repair gene XRCC3 Thr241Met polymorphism with susceptibilityto colorectal cancer in a Chinese population. Cancer geneticsand cytogenetics 2005; 163:38-43.
31. Sangrajrang S, Schmezer P, Burkholder I, et al. The XRCC3Thr241Met polymorphism and breast cancer risk: a case-control study in a Thai population. Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals2007; 12:523-532.
32. Narter KF, Ergen A, Agachan B, et al. Bladder cancer and polymorphisms of DNA repair genes (XRCC1, XRCC3, XPD, XPG,APE1, hOGG1). Anticancer research 2009; 29:1389-1393.
