Association between XRCC3 Thr241Met SNP and systemic lupus erythematosus in Han Chinese patients in Taiwan, and a meta-analysis of healthy populations
Yng-Tay Chen1, Shih-Yin Chen1, 2, Ying-Ju Lin1, 2, Chung-Ming Huang3, Yuan-Yen Chang,4 Fuu-Jen Tsai1, 2,5,*
1 Human Genetic Center, China Medical University Hospital, Taichung, Taiwan. 2 Graduate Institute of China Medical Science, China Medical University, Taichung, Taiwan.
3 Division of Immunology and Rheumatology, China Medical University Hospital, Taichung, Taiwan.
4 Department of Microbiology and Immunology, and Institute of Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan.
5College of Health Science, Asia University, Taichung, Taiwan.
Running Title: XRCC3 SNP and SLE
Y-T Chen and S-Y Chen contributed equally to this paper.
*Correspondence to: Fuu-Jen Tsai, Human Genetic Center, China Medical University Hospital, 2 Yuh-Der Road, Taichung 40447, Taiwan.
Email: [email protected] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Abstract
X-ray repair cross-complementing group 3 (XRCC3) plays a crucial role in mammalian DNA repair processes. The polymorphism of XRCC3, rs861539 (Thr > Met at codon 241), is common in populations worldwide. This study analyzed the relationship between this functional single nucleotide polymorphism (SNP) and systemic lupus erythematosus (SLE) in the Han Chinese population in Taiwan (HC-TW). Genotyping was performed using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) on 163 SLE patients and 191 healthy participants in the control group. The data showed that the genotype frequency at codon 241 did not differ significantly between the SLE patients and the healthy participants in the control group; however, the allele frequency analysis indicated a significant difference between these groups. In addition, we used the genotype and allele frequencies of 191 healthy HC-TW participants for comparison with HapMap populations. The results indicated a significant difference of XRCC3 Thr241Met allele and genotype frequencies between the HC-TW population and HapMap populations, except for the other Han Chinese populations. A prior study showed that Thr241 > Met substitution in XRCC3 protein was positive as damaging and functional consequences as well. This is the first study to demonstrate the difference of XRCC3 Thr241 > Met variant between the HC-TW population and HapMap population.
Keywords: systemic lupus erythematosus (SLE); X-ray repair cross-complementing group 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
3 (XRCC3); Single nucleotide polymorphisms (SNPs); Genotype; Haplotype.
Introduction
Systemic lupus erythematosus (SLE) is a complex multi-factorial auto-immune disease that is characterized by B-cell hyperactivity and auto-antigen production against nuclear constituents and other self-antigens (1, 2). SLE may be induced by genetic, hormonal, environmental factors, viral infection (3, 4). Exposure to ultraviolet (UV) light are considered crucial environmental factors (5). Defective immune regulatory mechanisms induced by genetic mutations can result in the dysregulation of inflammatory cytokine production and insufficient clearance of apoptotic cells or immune complexes, which activate anti-self-immune responses (6-9). Single nucleotide polymorphism (SNP) typing can be used to determine the potential molecular mechanisms in whole genomes (10, 11); the interactions between environmental factors and genetic variants in SLE remain unclear. Hypersensitivity to UV irradiation is frequently diagnosed in patients with SLE. Recently, deficiencies in the ability to repair DNA damage and the consequent abnormal levels of apoptotic bodies have been identified as causative factors for SLE. Clinical studies have further demonstrated that a number of SLE patients are highly deficient in DNA repair (12-14),suggesting that genetic variations in the genes encoding DNA repair proteins contribute to the susceptibility to SLE. Among the genes involved in the DNA 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
repair pathways, the X-ray repair cross-complementing (XRCC) gene family can protect mammalian cells from DNA damage caused by ionizing radiation (15, 16). Consistent with this observation, previous studies have indicated that the XRCC 4, 5, 7, and 9 proteins function at DNA break sites to trigger the development of autoimmune diseases (17-20). A prior study indicated that XRCC1 is the genetic factor that interacts with environmental factors, such as UV irradiation, to promote the pathogenesis of SLE in the HC-TW population (21). DNA polymorphisms in the XRCC1 and XRCC3 have been an area of
intense current investigation (22).
XRCC3 is an X-ray repair cross-complementing gene family that can protect mammalian cells from DNA damage induced by ionizing radiation (15, 16).Cells lacking XRCC3 are unable to form Rad51 foci after radiation damage, and demonstrate genetic instability and increased sensitivity to DNA damaging agents (23, 24). The SNP within XRCC3 have been identified at codon 241, which is located in exons 7 (25). Molecular epidemiological studies have linked XRCC3 polymorphism to increased risk of breast cancer (26), lung cancer (27), skin cancer (28), and colorectal cancer (29). The results have been inconsistent (30). The SNP at codon 241 is also linked to reduced DNA repair activity (31). Typically, the allele frequencies of genes often differ substantially between populations, and thus, ethnic-specific association studies are required to confirm genetic association in different populations. In this present
60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78
study, we investigated whether the XRCC3 rs861539 SNP contributes to SLE susceptibility in HC-TW populations and confirm genetic association in different populations using a
meta-analysis approach.
Materials and Methods
Patients and sample collection
A total of 163 SLE patients and 191 healthy controls were recruited from the China Medical University Hospital in Taiwan. All SLE patients met the criteria for SLE as defined by the American Rheumatism Association. People who underwent health examinations at the same hospital were recruited for the healthy control group. Informed consent was obtained from all participants, and was approved by the Institutional Review Board (IRB) at the China Medical University.
Genomic DNA extraction and genotyping
All blood samples were collected using venipuncture for genomic DNA isolation. Genomic DNA was extracted from peripheral blood leukocytes according to standard protocols (Genomic DNA kit; Qiagen, Valencia, CA, USA). We used the following primers: Forward:
5'-GCTCGCCTGGTGGTCATC-3' and reverse: 5'-CTTCCGCATCCTGGCTAAAAA-5'-GCTCGCCTGGTGGTCATC-3'. The PCR products were amplified with an initial denaturation at 95°C for 5 min, followed by 40 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98
cycles of 95°C for 10 s, 56°C for 10 s, 72°C for 20 s, and one additional cycle of 72°C for 5 min. DNA fragments containing the polymorphism were detected using PCR-RFLP, and CviA II digestion was used to detect codon 722 C/T.
Identification of eligible studies and data extraction
A literature search was conducted for studies that examined associations between the
XRCC3 Thr241Met polymorphism and SLE. We utilized the MEDLINE citation index to
identify articles in which the XRCC3 Thr241Met polymorphism was determined in SLE patients and controls (until December 2012). In addition, all references mentioned in identified articles were reviewed to identify additional studies not indexed by MEDLINE. The following information was extracted for each study: author, year of publication, ethnicity of the study population, demographics, numbers of cases and controls.
Meta-analysis
Population data were selected using a population data source (www.hapmap.org) for meta-analysis. The HapMap database was used to identify the allele and genotype frequencies of XRCC3 Thr241Met polymorphisms in various ethnic populations. The populations were as follows: Tunisian (TUN), Utah residents with Northern and Western European ancestry from the CEPH collection (CEU), Tuscan in Italy (TSI), Luhya in Webuye , Kenya (LWK), Maasai in Kinyawa, Kenya (MKK), Mexican ancestry in Los Angeles, California (MEX), 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117
Gujarati Indians in Houston (GIH), Yoruban in Ibadan, Nigeria (YRI), African ancestry in Southwest USA (ASW), Japanese in Tokyo, Japan (JPT), Han Chinese in Beijing China (HCB), Chinese in Metropolitan Denver, Colorado, Texas (CHD), and Han Chinese in Taiwan (HC-TW).
Statistical analysis
The allelic and genotype frequency distributions for the polymorphism of SLE were determined through χ2 analysis using SPSS software (version 10.0, SPSS Inc. Chicago, Illinois, US). A P value of less than 0.05 was considered statistically significant. Allelic and genotype frequencies were expressed as percentages of the total number of alleles and genotypes. Odds ratios (OR) were calculated from allelic and genotype frequencies with a 95% confidence interval (95% CI). Haplotypes were determined using the Bayesian statistical method, which was available in the program Phase 2.1.32. Adherence to the
Hardy-Weinberg equilibrium constant was tested using a χ2 test with one degree of freedom. Pair-wise Chi square (χ2) tests were also performed between HC-TW and
HapMap populations using the allele frequencies in a 22 contingency table to determine whether the HC-TW population significantly differed from other populations.
Result
The SNP rs861539: C > T at exon 7 causes Threonine to Methionine substitution at codon 241 of XRCC3 protein (Thr241>Met). PCR-RFLP product for rs861539 showed in Fig.1. 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136
Table I shows the genotype distribution values of XRCC3 Thr241Met polymorphism in SLE patients and the healthy control. No statistically significant difference was observed in the genotype frequency of XRCC3 Thr241Met polymorphisms between the SLE patients and the healthy control. However, a statistically significant difference was observed in allele frequency of XRCC3 Thr241Met polymorphism between the SLE patients and the healthy control (P<0.05).
Furthermore, we determined the association among XRCC3 SNP and clinical and biochemical manifestations in SLE patients. The results showed that no significant difference occurred between the clinical and biochemical manifestations of SLE patients with XRCC3 genotype and allele distribution (Table 2).
We compared the XRCC3 Thr241Met polymorphism of healthy females and males in the HC-TW population, and the results indicated that no significant difference occurred between females and males in this population (Table 3). The XRCC3 Thr241Met T/T
genotype was not observed in the healthy HC-TW population.
We compared the XRCC3 Thr241Met between the healthy HC-TW population and the healthy HapMap population. The meta-analysis results, based on 13 studies, indicated that the genotype of XRCC3 Thr241Met polymorphism differed significantly between the HC-TW population and HapMap populations worldwide (Table 4). The Thr/Thr (CC), Thr/Met (CT), and Met/Met (TT) genotype frequencies in the HC-TW population were 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155
0.922, 0.078, and 0, respectively (Table 4). The wild-type (C) and variant (T) alleles frequencies were 0.961 and 0.039, respectively. For HapMap populations, the variant allele frequency was 0.041 among the CHD population, and 0.474 among the TUN population (Table 4). The HC-TW population differed significantly from all HapMap populations
(P<0.01), except for Chinese populations, such as the HCB and CHD (P>0.05).
Discussion
UV irradiation is a powerful inducer of SLE, and genetic variation determines the susceptibility of a person to the development of SLE. We previously reported that SLE patients were associated with DNA repair gene XRCC1 polymorphism in the HC-TW population (21). Patients with SLE are commonly observed in RA patients. Previously report found that genetic polymorphisms of the DNA repair gene uracil-DNA glycosylase (UNG), 8-oxoguanine glycosylase 1 (OGG1), and N-methylpurine-DNA glycosylase (MPG) are associated with the susceptibility of RA in Taiwan population (32-34). We attempted to determine whether the functional SNP of XRCC3 Thr241Met is the genetic factor that reduces protection from DNA damage induced by UV exposure in SLE patients. The genetic polymorphism of DNA repair gene XRCC3 was significantly associated with SLE in the HC-TW population. The results suggest that the HC-TW population with genotype T/C at codon 241 of XRCC3 have a higher risk of developing SLE. No 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174
association between XRCC1 Arg399Gln, XRCC3 Thr241Met polymorphism and SLE patients was observed in Brazilian populations (35). XRCC3 is one of the DNA repair genes, and encodes for a protein participating in the homologous recombination repair (HRR) of DNA double-strand breaks and cross-links (31, 36, 37). It is a member of an emerging family of Rad-51-related proteins that may participate in HRR to maintain chromosome stability and repair DNA damage (24). These phenotypes are a result of failure to initiate HRR and aberrant processing of HRR intermediates (31). Our results indicated that XRCC3 Thr241Met SNP associated with SLE disease in the HC-TW population and the distribution profile of the SNP is significant different compared with other non-Han-Chinese populations.
The variant T allele frequencies were 0.039 in HC-TW, 0.041 in CHD, and 0.062 in HCB populations. However, the variant T allele frequencies were 0.116 in JPT, 0.429 in CEU, 0.461 in TSI, and 0.474 in TUN populations. The results of meta-analysis, based on 13 studies, indicated that the genotype and allele frequencies of the XRCC3 Thr241Met polymorphism differed significantly between the HC-TW population and HapMap populations, except for the Han Chinese populations. The XRCC3 systematic function remains unclear. Walker Boxes A and B are two potential ATP-binding domains (38), and suggest that the transition from hydrophilic Threonine to a hydrophobic Methionine can affect ATP-binding and DNA repair efficiency (31, 39). A recent study indicated that the 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193
variant allele of TUN differed significantly from all HapMap populations, except the CEU and TSI populations (38). We found that the variant allele of the HC-TW population differed significantly from all HapMap non-Han-Chinese populations. These results indicate that XRCC3 Thr241Met SNP differs among populations worldwide.
This study identified the polymorphism in the DNA repair gene XRCC3 Thr241Met and its associations with SLE risk in the HC-TW population. The meta-analysis of XRCC3 Thr241Met polymorphism showed a significant difference between the HC-TW population and the HapMap populations.
Acknowledgements
This work is supported in part by Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH101-TD-B-111-004), China Medical University (CMU100-S-06) and China Medical University Hospital (DMR-101-041) in Taiwan. The first author fellowship (YT Chen) is supported by China Medical University (CMU101-AWARD-01), Taiwan. 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208
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1 2 3
(CC) (CT) (TT)
Figure.1. Electrophoresis of PCR-RFLP product for rs861539. The sizes of PCR amplified products was 214 base pair (bp), after CviA II digestion, line 1 is CC genotype (214 bp). Line 2 was CT genotype, the fragments were 214 bp, 112 bp, and 102 bp. Line 3 was TT genotype, fragments were 112 bp and 102 bp.
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Table 1. Allelic and genotype frequencies of XRCC3 polymorphisms among the Han Chinese SLE patients in Taiwan and the healthy control group.
Polymorphism s SLE N (%) Control N (%) p-valuea OR (95% CI) N=163 N=191 XRCC3 241 T T 1 (0.6) 0 (0) 0.06 -(rs861539) T C 24 (14.7) 15 (7.9) 2.04 (1.03-4.04) C C 138 (84.7) 176 (92.1) 1 T 26 (8.0) 15 (3.9) 0.02* 2.12 (1.10-4.08) C 300 (92.0) 367 (96.1) 1
aGenotype and allelic frequencies were compared between SLE patients and normal controls by x2 tests.
*Indicates statistical significance. 318
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Table 2. Comparison of clinical and biochemical manifestations of SLE patients with XRCC3 genotype and allele distribution.
Clinical features Genotype Allele C C C T T T p-valuea C T p-valuea ANA 11 0 2 0 1 1.000 2 4 0 2 2 1.000 Immunologic 91 1 5 1 0.645 1 9 7 1 7 0.782 Hemato-logic 58 1 0 0 1.000 1 2 6 1 0 0.824 CNS 14 1 0 0.728 2 9 1 0.486 Renal 49 7 0 0.885 1 0 5 7 0.495 Serositis 25 2 0 0.490 5 2 2 0.267 Arthritis 60 1 3 1 0.265 1 3 3 1 5 0.116 Mucosal ulcer 30 7 1 0.184 6 7 9 0.133 Photo-sensitivity 52 8 1 0.792 1 1 2 1 0 0.824 Discoid lupus 16 2 0 1.000 2 4 2 1.000 Malar rash 61 8 1 0.459 1 3 0 1 0 0.821
a When compared with normal controls by x2 tests.
Abbreviations: ANA, antinuclear antibody; CNS, central nervous system; SLE, systemic lupus erythematosus.
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Table 3. Genotype distributions and allele frequencies of XRCC3 SNP among the HC-TW population.
Genotype Total (N=191) Woman (N=157) Man (N=34) Allele frequency HWE
p-valuea
N Freq N Freq N Freq Wild-type Variant
rs861539: C > T (p.Thr241 > Met)
C/C 176 0.922 143 0.911 33 0.971
C/T 15 0.078 14 0.089 1 0.029 (C)0.961 (T)0.039 0.571
T/T 0 0 0 0 0 0
a When compared with normal controls by x2 tests. 328
Table 4. Genotype and allele frequencies of XRCC3 p.Thr241>Met in HapMap and HC-TW population.
Genotype frequency Allele frequency Pair-wise p-value between HC-TW and other
populations Population
Geno. Freq. Geno. Freq. Geno . Freq.
Wild-type Variant allele allele
XRCC3 C > T (p.Thr241 > Met) (C) (T) genotype allele
TUN (n = 154) C/C 0.312 C/T 0.428 T/T 0.260 0.526 0.474 9.19E-32 4.45E-41 CEU (n = 226) C/C 0.310 C/T 0.522 T/T 0.168 0.571 0.429 7.94E-36 2.45E-38 TSI (n = 204) C/C 0.324 C/T 0.431 T/T 0.245 0.539 0.461 1.28E-33 7.98E-42 LWK (n = 220) C/C 0.582 C/T 0.364 T/T 0.055 0.764 0.236 2.94E-14 1.15E-15 MKK (n = 310) C/C 0.645 C/T 0.310 T/T 0.045 0.800 0.200 2.13E-11 8.73E-13 MEX (n = 114) C/C 0.719 C/T 0.246 T/T 0.035 0.842 0.158 5.32E-06 3.04E-07 GIH (n = 202) C/C 0.574 C/T 0.356 T/T 0.069 0.752 0.248 1.70E-14 1.49E-16 YRI (n = 288) C/C 0.660 C/T 0.319 T/T 0.021 0.819 0.181 2.64E-10 8.47E-11 ASW (n = 114) C/C 0.684 C/T 0.281 T/T 0.035 0.825 0.175 2.43E-07 1.34E-08 JPT (n = 224) C/C 0.777 C/T 0.214 T/T 0.009 0.884 0.116 2.00E-04 5.15E-05
HCB (n = 274) C/C 0.876 C/T 0.124 T/T 0.000 0.938 0.062 0.116 0.126
CHD (n = 218) C/C 0.917 C/T 0.083 T/T 0.000 0.959 0.041 0.881 0.884
HC-TW (n =
191) C/C 0.922 C/T 0.078 T/T 0.000 0.961 0.039
Population data source: (www.hapmap.org) n number of individuals
TUN Tunisian, CEU Utah residents with Northern and Western European ancestry from the CEPH collection, TSI Tuscan in Italy, LWK Luhya in Webuye, Kenya, MKK Maasai in Kinyawa, Kenya, MEX Mexican ancestry in Los Angeles, California, GIH Gujarati Indians in Houston, YRI Yoruban in Ibadan, Nigeria, ASW African ancestry in Southwest USA, JPT Japanese in Tokyo, 330
331 332 333 334
Japan, HCB Han Chinese in Beijing China, CHD Chinese in Metropolitan Denver, Colorado, Texas, HC-TW Han Chinese in Taiwan.
p < 0.05, a significant difference between HC-TW and other populations p > 0.05, no significant difference between HC-TW and other populations
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