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The polymorphisms of MSH6 gene are associated with AIDS progression in a northern Chinese population
Infection, Genetics and Evolution 42 (2016) 9–13
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Research paper
The polymorphisms of MSH6 gene are associated with AIDS progression in a northern Chinese population Chuntao Wang a,b,1, Chunyan Zhao c,1, Xuelong Zhang a, Lidan Xu a, Xueyuan Jia a, Haiming Sun a, Jingcui Yu d, Guangfa Zhang a, Ning He e, Qiuyan Li a, Yuandong Qiao a,⁎, Songbin Fu a,⁎ a
Laboratory of Medical Genetics, Harbin Medical University, Harbin 150081, China Department of Biology, Mudanjiang Medical University, Mudanjiang 157011, China Department of Medical Biology and Genetics, Harbin Medical University-Daqing, Daqing 163319, China d The Second Affiliated Hospital, Harbin Medical University, Harbin 150081, China e Department of Clinical Laboratory, Qiqihar Traditional Chinese Medicine Hospital, Qiqihar 161000, China b c
a r t i c l e
i n f o
Article history: Received 19 February 2016 Received in revised form 12 April 2016 Accepted 14 April 2016 Available online 16 April 2016 Keywords: MSH6 MSH2 SNPs HIV-1 AIDS Progression
a b s t r a c t It has been reported that DNA repair genes play an important role in HIV-1 infection and AIDS progression. One DNA repair pathway, the mismatch repair (MMR) is associated with a wide variety of tumors. However, the role of single nucleotide polymorphisms (SNPs) in the MMR genes and their importance in HIV-1 infection and AIDS progression remain unclear. In the present study, 479 HIV-1-infected and 487 healthy individuals from northern China were genotyped for nine SNPs in the MSH2 gene (rs13019654, rs4608577, rs4952887, rs6726691, rs10191478, rs12999145, rs1981929, rs2042649, rs2303428) and five SNPs in the MSH6 gene (rs2348244, rs3136245, rs3136329, rs2072447, rs7562048). Our results showed that the rs7562048 G allele frequency was significantly higher in the cases with the CD4+ T-lymphocyte count b 200 cells/μl than those with N 200 cells/μl (P = 0.001, OR = 1.811, 95% CI 1.255–2.614), which is in agreement with the result of the Bonferroni correction. The frequencies of the rs2348244 C allele and rs3136245 T allele were higher in the cases at clinical phase IV than those at clinical phase I + II + III (P = 0.026, OR = 1.591, 95% CI 1.056–2.398 and P = 0.019, OR = 1.749, 95% CI 1.096–2.791, respectively); however, this difference is not supported by the Bonferroni correction. There were no significant differences in the frequency of allele, genotype and haplotype of the 14 SNPs between HIV-1-infected individuals and healthy controls (P N 0.05). These results suggest that the rs7562048 is associated with the clinical features and that the MSH6 gene polymorphisms likely play an important role in the progression of AIDS in the northern Chinese population. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Acquired immune deficiency syndrome (AIDS) is a significant and challenging chronic infectious disease resulting from human immunodeficiency virus (HIV) infection (Bao et al., 2012; Weiss, 1993). HIV infection leads to a progressive decline in the functionality and the number of CD4+ T-lymphocyte, causing AIDS development (McCune, 2001). Studies have shown that there are a large quantity of apoptotic cells in the patients with HIV-1 infection, indicating that cell apoptosis is another mechanism responsible for the T-lymphocyte depletion in AIDS patients (Ameisen and Capron, 1991; Hel et al., 2006; LaurentCrawford et al., 1991; Terai et al., 1991). One route of cell apoptosis is induced by DNA damage (De Flora et al., 1996), and thus the DNA repair system may play a role in HIV-1 infection and AIDS progression. It has ⁎ Corresponding authors at: 157 Baojian Road, Nangang District, Harbin 150081, China. E-mail addresses: [email protected] (Y. Qiao), [email protected] (S. Fu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.meegid.2016.04.016 1567-1348/© 2016 Elsevier B.V. All rights reserved.
been reported that DNA repair genes are associated with HIV-1 infection and AIDS progression. Specifically, ERCC2 and ERCC3 genes can promote viral cDNA replication and reduce the CD4 cell count by preventing degradation of retroviral cDNA (Yoder et al., 2006). The DNA-PK protein encoded by the XRCC7 gene interacts with HIV-1 Tat to increase HIV-1 replication and transcription (Tyagi et al., 2011; Zhang et al., 2014). In addition, the polymorphisms of XRCC1, XPG and ERCC2 genes may influence individual variations in the DNA repair capacity and play an important role in the progression of HIV diseases (Sobti et al., 2011; Sobti et al., 2010; Sobti et al., 2009). The mismatch repair (MMR) is one of the DNA repair pathways, and its main function is to rectify base-pair mismatches to prevent gene mutation and maintain genomic stability and integrity (Burdett et al., 2001; Jiricny, 2006). The MMR pathway includes nine genes, of which, MSH2 and MSH6 are the two key components. The MSH2 gene is located on chromosome 2p21–22, and required for recognizing nucleotide mismatches that occur during DNA replication (Lo et al., 2011). The MSH6 gene is located on chromosome 2p16 and involved in the DNA base mismatch repair, reducing the spontaneous mutation rate (Suchy et al.,
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2002). The MSH6 encodes protein hetero-dimerization and functions as a bidirectional molecular switch to repair DNA replication errors (Umar et al., 1998). In fact, MSH2, MSH6 and other MMR genes are associated with an increased risk of cancers such as prostate cancer (Chen et al., 2003), pancreatic cancer (Liu et al., 2014), gastric cancer (Yamamoto et al., 1999), lung cancer (Jung et al., 2006; Slovakova et al., 2015), non-small cell lung cancer (Hsu et al., 2007), breast cancer (Feng and Radivoyevitch, 2009), ovarian cancer (Quaye et al., 2009), endometrial cancer (EC) (Beiner et al., 2006; Imai and Yamamoto, 2008; Lacey et al., 2011; Poplawski et al., 2015), and hereditary nonpolyposis colorectal cancer (HNPCC) (Lynch and Lynch, 2000; Papadopoulos et al., 1994; Park et al., 1999; Peltomaki and Vasen, 1997; Watson et al., 1994). However, there are no functional and association studies that have systematically assessed the association between the MMR genes, HIV-1 infection and AIDS progression. To further investigate the role of the polymorphisms in the MSH2 and the MSH6 genes in the development of HIV-1 infection and AIDS, we performed an association study of 14 single nucleotide polymorphisms (SNPs) in the MSH2 and the MSH6 genes in 966 northern Chinese individuals. Participants were genotyped to investigate whether the two genes polymorphisms were associated with the susceptibility to HIV-1 infection and the progression of AIDS. 2. Material and methods
Inc., and was based on a double ligation and multiplex fluorescence PCR. For quality control, a 5% random sample of cases and controls was genotyped twice to verify the genotyping accuracy, the reproducibility was 100%. 2.3. Statistical analysis After the genotypes of all participants were determined, the genotype and allele frequency were calculated by directly counting the number. The Chi-square test was used for examining the deviation from Hardy–Weinberg equilibrium (HWE) for all SNPs of the healthy group, the association between the allele, genotype frequency and susceptibility to HIV-1 infection, and the SNPs and the clinical features of cases (such as the CD4+ T-lymphocyte count and clinical stage). The haploView 4.2 software was used to evaluate the LD and the frequency of the haplotypes between HIV-1-infected individuals and health controls. The relative risk associated with rare alleles was estimated as an odds ratio (OR) and 95% confidence interval (CI). SPSS 17.0 (SPSS, Chicago, IL, USA) was used for all statistical analyses. The differences with a P value less than 0.05 were considered statistically significant, which was further subjected to a Bonferroni correction for multiple testing. Statistical power was assessed using the Genetic Power Calculator (Purcell et al., 2003). Considering 0.06% prevalence of the disease, a risk allele frequency of 17%, and an additive genetic model, we had at least 95% power to detect an OR of 1.5 at the 0.05 level.
2.1. Subjects 3. Results In this study, 479 HIV-1-infected male individuals who have sex with men (MSM) were recruited from Heilongjiang Center for Disease Control and Prevention (CDC). The age of the HIV-1-seropositive individuals ranged from 16 to 75 years old (mean age ± SD, 35.3 ± 11.55) and the average CD4+ T-lymphocyte count at that time point was 335 cells/μl (range, 3–1038 cells/μl). We categorized these patients as Category 1 (T-lymphocytes N200 cells/μl) or Category 2 (T-lymphocytes b 200 cells/μl) by the CD4+ T-lymphocyte count, and as Category A (Clinical phase I + II + III) or Category B (Clinical phase IV) by the clinical stage. The age and sex frequency-matched unrelated healthy controls (n = 487) were recruited from individuals who were HIV-1seronegative individuals diagnosed through a comprehensive medical test at the clinical laboratory of the second affiliated hospital of Harbin Medical University. The parents of each participant were individuals of the same nationality (for at least three generations) in nonsanguineous marriages. The age of the uninfected controls ranged from 16 to 75 years (mean age ± SD, 35.3 ± 11.59). All participants provided informed consent approved by the appropriate local authority. 2.2. SNPs selection and genotyping We included candidate SNPs based on the published literature and chose tagging SNPs based on linkage disequilibrium (LD) in the HapMap (HapMap Data Rel 24/phase II Nov08, on NCBI B36 assembly, dbSNP b126). The tagging SNPs were screened for minor allele frequency (MAF) ≥10% among Han Chinese in Beijing, China (CHB), and no representation by other tagging SNPs at a LD of r2 ≥ 0.80. We selected nine SNPs in MSH2 including rs13019654, rs4608577, rs4952887, rs6726691, rs10191478, rs12999145, rs1981929, rs2042649, and rs2303428 and five SNPs in MSH6 gene including rs2348244, rs3136245, rs3136329, rs2072447, and rs7562048. Of the 14 SNPs, rs6726691, rs12999145, rs3136245, rs3136329, and rs7562048 were selected from the literature and the others were tagging SNPs. Peripheral blood of all participants was collected and genomic DNA was extracted using the QIAamp blood kit (Qiagen, Germany). The 14 SNPs were genotyped using a custom-design 48-Plex SNPscan™ Kit (Genesky Bio-technologies Inc., Shanghai, China). This kit was developed according to patented SNP genotyping technology by Genesky Biotechnologies
The information of the 14 SNPs and the allele frequency in the MSH2 and the MSH6 genes are shown in Table 1. All tested SNPs did not deviate from the Hardy–Weinberg equilibrium in the healthy group (P N 0.05). There were no significant differences in the allele frequency of the 14 SNPs between cases and controls (P N 0.05). Similarly, we found no associations between genotype frequency of the 14 SNPs and HIV-1 infection under different genetic models (P N 0.05) (Supplementary Table S1). In analyzing the associations between the 14 SNPs and clinical features of AIDS (Table 2), we found that there was a significant association between the rs7562048 and the CD4+ T-lymphocyte count in patients with the AIDS (P b 0.05). The frequency of the rs7562048 G allele was significantly higher in the cases with Category 2(T-lymphocytes b 200 cells/μl) compared with those with Category 1 (T-lymphocytes N 200 cells/μl) (P = 0.001, OR = 1.811, 95% CI 1.255–2.614). The difference between Category 1 and Category 2 was still statistically significance after the Bonferroni correction. There were significant associations of the rs2348244 and rs3136245 with the clinical stage of AIDS. The frequencies of the rs2348244 C allele and the rs3136245 T allele were higher in the cases with Category B (Clinical phase IV) than those with Category A (Clinical phase I + II + III) (P = 0.026, OR = 1.591, 95% CI 1.056–2.398 and P = 0.019, OR = 1.749, 95% CI 1.096– 2.791, respectively). However, the significance between Category A and Category B disappeared after the Bonferroni correction. There was strong LD between the nine SNPs in the MSH2 gene as well as the five SNPs in the MSH6 gene. Nine haplotypes in block 1 and eight haplotypes in blocks 2 and 3 were identified and the frequencies of these haplotypes are listed in Table 3. The frequency of the haplotype TCCAAGCTC in cases was higher than that in healthy controls (P = 0.0498). However, there were no differences in the frequencies of the other haplotypes between HIV-1-infected individuals and healthy cohorts and there was no significant association with the susceptibility to HIV-1 infection (P N 0.05). 4. Discussion To our knowledge, this is the first comprehensive study to systematically evaluate the association of the MSH2 and the MSH6 genes
C. Wang et al. / Infection, Genetics and Evolution 42 (2016) 9–13
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Table 1 The information and allele frequency of the 14 SNPs in in case and control cohorts. SNP
Gene
Chra
Risk allele
Allele frequency
rs13019654 rs4608577 rs4952887 rs6726691 rs10191478 rs12999145 rs1981929 rs2042649 rs2303428 rs2348244 rs3136245 rs3136329 rs2072447 rs7562048
MSH2 MSH2 MSH2 MSH2 MSH2 MSH2 MSH2 MSH2 MSH2 MSH6 MSH6 MSH6 MSH6 MSH6
2 2 2 2 2 2 2 2 2 2 2 2 2 2
G G T G T A A T C C C C G G
HCBc 0.756 0.167 0.162 0.389 0.812 0.556 0.860 0.500 0.302 0.442 0.326 0.189 0.182 0.186
a b c
cases 0.762 0.154 0.157 0.371 0.812 0.539 0.844 0.520 0.319 0.446 0.350 0.124 0.116 0.159
controls 0.751 0.140 0.156 0.364 0.789 0.523 0.838 0.509 0.317 0.437 0.345 0.114 0.086 0.134
P value
OR
95% CI
P for HWETb
0.580 0.391 0.956 0.775 0.194 0.480 0.688 0.641 0.919 0.712 0.842 0.487 0.159 0.133
1.061 1.117 1.007 1.028 1.159 1.067 1.051 1.043 1.010 1.034 1.019 1.103 1.235 1.214
0.860–1.310 0.868–1.437 0.786–1.291 0.852–1.239 0.927–1.449 0.892–1.275 0.824–1.342 0.873–1.247 0.834–1.223 0.864–1.238 0.845–1.229 0.837–1.452 0.921–1.656 0.943–1.562
0.963 0.090 0.407 0.361 0.741 0.856 0.108 0.757 0.211 0.282 0.527 0.452 0.050 0.103
Chr: chromosome. HWET: Hardy–Weinberg equilibrium test. HCB: Han Chinese in Beijing, China.
polymorphisms with the susceptibility to HIV-1 infection and progression toward AIDS. The DNA repair genes are involved in HIV-1 integration because this process will lead to the host genomic DNA damage. Therefore, analyzing the role of the DNA repair genes in HIV-1 infection and the progression of this disease likely provides a new effective target for AIDS therapy. In our study, we found that there is a significant association between the rs7562048 and the CD4+ T-lymphocyte count and that G allele can significantly promote the disease progression of AIDS. As the CD4+ Tlymphocyte count declines, the immunity of AIDS patients is seriously compromised, leading to tumors and other complications. Studies have shown that high grade malignant lymphomas are the part of the
AIDS-defining condition (Levine, 1993) and that colorectal malignancies are likely associated with the grade of an immunosuppression induced during HIV infection (Yeguez et al., 2003). In fact, the MSH6 is one of the important MMR genes that are known to play a key role in the carcinogenesis caused by a virus infection, such as the human papillomavirus-associated cervical carcinoma (Bahnassy et al., 2006) and the hepatitis C virus-associated hepatocellular carcinoma (Zekri et al., 2005), although direct association between the MSH6 gene and the HIV-1-associated cancers has not been reported yet. The rs7562048 is located in the MSH6 introns, which together with our results suggest that the MSH6 gene may modulate the disease severity and the ensuing AIDS progression. We propose that the rs7562048
Table 2 Association between the 14 SNPs and the clinical features of AIDS. SNPs
rs13019654 rs4608577 rs4952887 rs6726691 rs10191478 rs12999145 rs1981929 rs2042649 rs2303428 rs2348244 rs3136245 rs3136329 rs2072447 rs7562048
Allele
G T G T T C G C T G A G A G T C C T C T C T C T G C G A
CD4+ T-lymphocyte counta N200 cells/μl
b200 cells/μl
n
n
545 171 110 606 110 606 259 457 585 131 379 337 607 109 366 350 230 486 322 394 243 473 83 633 77 639 98 618
183 57 37 205 40 202 96 146 193 49 137 105 202 40 132 110 76 166 105 137 92 150 36 206 34 208 54 188
Clinical stageb P value
OR
95% CI
0.967
0.993
0.704–1.399
0.978
1.006
0.671–1.507
0.666
1.091
0.735–1.620
0.330
1.160
0.860–1.565
0.502
1.134
0.786–1.635
0.321
0.862
0.643–1.156
0.628
1.103
0.742–1.638
0.356
1.148
0.857–1.537
0.836
1.034
0.755–1.414
0.668
1.066
0.795–1.430
0.250
1.194
0.883–1.615
0.181
1.333
0.875–2.029
0.166
1.357
0.881–2.089
0.001
1.811
1.255–2.614
Clinical phase I + II + III
Clinical phase IV
n
n
653 203 129 727 134 722 310 538 699 157 454 402 725 131 440 416 278 578 371 485 310 546 106 750 99 757 138 718
75 25 18 84 16 86 43 59 79 23 62 40 84 18 58 44 28 74 56 46 25 77 13 89 12 90 14 88
The values in bold indicate that the differences are significant. a The CD4+ T-lymphocyte counts were divided into two groups: Category 1, N200 cells/μl; Category 2, b200 cells/μl. b Clinical stage: Category A, Clinical phase I + II + III; Category B, Clinical phase IV.
P value
OR
95% CI
0.775
1.072
0.664–1.731
0.495
1.208
0.703–2.076
0.993
1.002
0.570–1.763
0.269
1.265
0.834–1.918
0.304
1.296
0.791–2.125
0.138
1.372
0.903–2.085
0.537
1.186
0.690–2.038
0.297
1.246
0.824–1.885
0.303
1.271
0.805–2.007
0.026
1.591
1.056–2.398
0.019
1.749
1.096–2.791
0.917
1.033
0.558–1.914
0.953
1.020
0.539–1.929
0.531
1.208
0.668–2.184
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C. Wang et al. / Infection, Genetics and Evolution 42 (2016) 9–13
Table 3 The frequency of haplotypes in the MSH2 and the MSH6 genes in case and control cohorts. Haplotype Block 1 TCCAGGCTC TCCAGTTTC TTGAAGTTT GCGGAGTTT TCCAAGTGT TCCAGTCTC GCGAAGTTT TCGAAGTTT TCCAAGCTC Block 2 TC CT TT CC Block 3 TCA CGG TCG CCG
Frequency
Case, control ratios
P value
0.212 0.158 0.149 0.145 0.133 0.073 0.032 0.025 0.017
197.6:749.8, 209.7:745.6 149.7:797.7, 153.9:801.4 143.7:803.7, 143.9:811.5 138.8:808.5, 139.6:815.7 135.2:812.2, 121.1:834.2 72.5:874.9, 67.8:887.5 28.9:918.4, 33.3:922.0 28.2:919.2, 19.8:935.6 21.9:925.5, 10.9:944.4
0.560 0.852 0.946 0.981 0.309 0.640 0.596 0.206 0.0498
0.428 0.334 0.225 0.014
413.9:544.1, 412.2:561.8 321.9:636.1, 323.0:651.0 209.1:748.9, 255.0:749.0 13.1:944.9, 13.8:960.2
0.693 0.838 0.502 0.921
0.850 0.105 0.031 0.010
800.9:155.1, 839.8:134.2 107.9:848.1, 94.4:879.6 37.1:918.9, 23.2:950.8 6.0:950.0, 13.4:960.6
0.132 0.253 0.058 0.098
The values in bold indicate that the differences are significant.
may regulate the transcription and the expression of the MSH6 gene, which interacts with HIV-1 to promote its replication and integration, leading to the decrease in the CD4+ T-lymphocyte count and the acceleration of this disease. Up to now, the mechanism underlying the interaction between the MSH6 and HIV-1 remains unclear, and our study opens a new field for further studying on the functional significance and the underlying mechanism of the association between the rs7562048 and AIDS progression. The differences of allele and genotype frequency of the 14 SNPs were not seen between the case and the control cohorts, which imply a null association of the MSH2 and the MSH6 genes polymorphisms with the susceptibility to HIV-1 infection in the northern Chinese population. In previous reports, the role of the MSH2 and the MSH6 genes polymorphisms was inconsistent in the cancer association studies. One report showed that the rs2072447 increased the colorectal cancer risk under the recessive model, which suggests that the MSH6 gene polymorphism is associated with the susceptibility to colorectal cancer (Tulupova et al., 2008). Some other studies showed that the MSH2 and the MSH6 genes polymorphisms were not associated with the susceptibility to the lung cancer in a Chinese population and the ovarian cancer in European and American populations (Lo et al., 2011; Mann et al., 2008). In our study, the difference of TCCAAGCTC haplotype in block 1 was found between case and control cohorts and the association was significant (P = 0.0498); however, the P value was closed to 0.05 and the frequency of the haplotype was too lower in cases and controls. Through further comprehensive analysis of the frequency distribution of alleles, genotypes and haplotypes, we verified that there is no association between the MSH2 and the MSH6 genes polymorphisms and HIV-1 infection. Here, we propose that the null association of the MSH2 and the MSH6 genes polymorphisms with the susceptibility to HIV-1 infection is because of the differences of disease types or genetic background. In conclusion, we found that the rs7562048 is associated with the clinical features and MSH6 gene polymorphisms play an important role in the progression of AIDS in a northern Chinese population. Nevertheless, larger scale association and functional studies as well as using more SNPs in the MMR pathway are needed to expand our initial findings, which would help us to fully understand the contribution of the MMR genes to HIV-1 infection and AIDS progression. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2016.04.016.
Authors' contribution All authors have contributed to the paper. C.T.W., C.Y.Z., Y.D.Q., and S.B.F. participated in the design of the study. X.Y.J., H.M.S., and J.C.Y assisted with experiment preparation. C.T.W., C.Y.Z., X.L.Z., L.D.X., G.F.Z., N.H., and Q.Y.L. performed the experiments. C.T.W., and C.Y.Z fulfilled the statistical analysis. C.T.W., C.Y.Z., Y.D.Y., and S.B.F. wrote the manuscript. All authors read and approved the final version. Acknowledgements This work was funded by the Program for Changjiang Scholars and the Innovative Research Team in University (IRT1230); the National Natural Science Foundation of China (81373220); Natural Science Foundation of Heilongjiang Province of China (D2007-04); the Post-doctoral Foundation of Heilongjiang Province (LRB08-340); Wu Lien-Teh Youth Science Foundation of Harbin Medical University (WLD-QU1405). We also gratefully acknowledge the numerous sample donors for making this work possible. References Ameisen, J.C., Capron, A., 1991. 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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Research paper
The polymorphisms of MSH6 gene are associated with AIDS progression in a northern Chinese population Chuntao Wang a,b,1, Chunyan Zhao c,1, Xuelong Zhang a, Lidan Xu a, Xueyuan Jia a, Haiming Sun a, Jingcui Yu d, Guangfa Zhang a, Ning He e, Qiuyan Li a, Yuandong Qiao a,⁎, Songbin Fu a,⁎ a
Laboratory of Medical Genetics, Harbin Medical University, Harbin 150081, China Department of Biology, Mudanjiang Medical University, Mudanjiang 157011, China Department of Medical Biology and Genetics, Harbin Medical University-Daqing, Daqing 163319, China d The Second Affiliated Hospital, Harbin Medical University, Harbin 150081, China e Department of Clinical Laboratory, Qiqihar Traditional Chinese Medicine Hospital, Qiqihar 161000, China b c
a r t i c l e
i n f o
Article history: Received 19 February 2016 Received in revised form 12 April 2016 Accepted 14 April 2016 Available online 16 April 2016 Keywords: MSH6 MSH2 SNPs HIV-1 AIDS Progression
a b s t r a c t It has been reported that DNA repair genes play an important role in HIV-1 infection and AIDS progression. One DNA repair pathway, the mismatch repair (MMR) is associated with a wide variety of tumors. However, the role of single nucleotide polymorphisms (SNPs) in the MMR genes and their importance in HIV-1 infection and AIDS progression remain unclear. In the present study, 479 HIV-1-infected and 487 healthy individuals from northern China were genotyped for nine SNPs in the MSH2 gene (rs13019654, rs4608577, rs4952887, rs6726691, rs10191478, rs12999145, rs1981929, rs2042649, rs2303428) and five SNPs in the MSH6 gene (rs2348244, rs3136245, rs3136329, rs2072447, rs7562048). Our results showed that the rs7562048 G allele frequency was significantly higher in the cases with the CD4+ T-lymphocyte count b 200 cells/μl than those with N 200 cells/μl (P = 0.001, OR = 1.811, 95% CI 1.255–2.614), which is in agreement with the result of the Bonferroni correction. The frequencies of the rs2348244 C allele and rs3136245 T allele were higher in the cases at clinical phase IV than those at clinical phase I + II + III (P = 0.026, OR = 1.591, 95% CI 1.056–2.398 and P = 0.019, OR = 1.749, 95% CI 1.096–2.791, respectively); however, this difference is not supported by the Bonferroni correction. There were no significant differences in the frequency of allele, genotype and haplotype of the 14 SNPs between HIV-1-infected individuals and healthy controls (P N 0.05). These results suggest that the rs7562048 is associated with the clinical features and that the MSH6 gene polymorphisms likely play an important role in the progression of AIDS in the northern Chinese population. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Acquired immune deficiency syndrome (AIDS) is a significant and challenging chronic infectious disease resulting from human immunodeficiency virus (HIV) infection (Bao et al., 2012; Weiss, 1993). HIV infection leads to a progressive decline in the functionality and the number of CD4+ T-lymphocyte, causing AIDS development (McCune, 2001). Studies have shown that there are a large quantity of apoptotic cells in the patients with HIV-1 infection, indicating that cell apoptosis is another mechanism responsible for the T-lymphocyte depletion in AIDS patients (Ameisen and Capron, 1991; Hel et al., 2006; LaurentCrawford et al., 1991; Terai et al., 1991). One route of cell apoptosis is induced by DNA damage (De Flora et al., 1996), and thus the DNA repair system may play a role in HIV-1 infection and AIDS progression. It has ⁎ Corresponding authors at: 157 Baojian Road, Nangang District, Harbin 150081, China. E-mail addresses: [email protected] (Y. Qiao), [email protected] (S. Fu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.meegid.2016.04.016 1567-1348/© 2016 Elsevier B.V. All rights reserved.
been reported that DNA repair genes are associated with HIV-1 infection and AIDS progression. Specifically, ERCC2 and ERCC3 genes can promote viral cDNA replication and reduce the CD4 cell count by preventing degradation of retroviral cDNA (Yoder et al., 2006). The DNA-PK protein encoded by the XRCC7 gene interacts with HIV-1 Tat to increase HIV-1 replication and transcription (Tyagi et al., 2011; Zhang et al., 2014). In addition, the polymorphisms of XRCC1, XPG and ERCC2 genes may influence individual variations in the DNA repair capacity and play an important role in the progression of HIV diseases (Sobti et al., 2011; Sobti et al., 2010; Sobti et al., 2009). The mismatch repair (MMR) is one of the DNA repair pathways, and its main function is to rectify base-pair mismatches to prevent gene mutation and maintain genomic stability and integrity (Burdett et al., 2001; Jiricny, 2006). The MMR pathway includes nine genes, of which, MSH2 and MSH6 are the two key components. The MSH2 gene is located on chromosome 2p21–22, and required for recognizing nucleotide mismatches that occur during DNA replication (Lo et al., 2011). The MSH6 gene is located on chromosome 2p16 and involved in the DNA base mismatch repair, reducing the spontaneous mutation rate (Suchy et al.,
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C. Wang et al. / Infection, Genetics and Evolution 42 (2016) 9–13
2002). The MSH6 encodes protein hetero-dimerization and functions as a bidirectional molecular switch to repair DNA replication errors (Umar et al., 1998). In fact, MSH2, MSH6 and other MMR genes are associated with an increased risk of cancers such as prostate cancer (Chen et al., 2003), pancreatic cancer (Liu et al., 2014), gastric cancer (Yamamoto et al., 1999), lung cancer (Jung et al., 2006; Slovakova et al., 2015), non-small cell lung cancer (Hsu et al., 2007), breast cancer (Feng and Radivoyevitch, 2009), ovarian cancer (Quaye et al., 2009), endometrial cancer (EC) (Beiner et al., 2006; Imai and Yamamoto, 2008; Lacey et al., 2011; Poplawski et al., 2015), and hereditary nonpolyposis colorectal cancer (HNPCC) (Lynch and Lynch, 2000; Papadopoulos et al., 1994; Park et al., 1999; Peltomaki and Vasen, 1997; Watson et al., 1994). However, there are no functional and association studies that have systematically assessed the association between the MMR genes, HIV-1 infection and AIDS progression. To further investigate the role of the polymorphisms in the MSH2 and the MSH6 genes in the development of HIV-1 infection and AIDS, we performed an association study of 14 single nucleotide polymorphisms (SNPs) in the MSH2 and the MSH6 genes in 966 northern Chinese individuals. Participants were genotyped to investigate whether the two genes polymorphisms were associated with the susceptibility to HIV-1 infection and the progression of AIDS. 2. Material and methods
Inc., and was based on a double ligation and multiplex fluorescence PCR. For quality control, a 5% random sample of cases and controls was genotyped twice to verify the genotyping accuracy, the reproducibility was 100%. 2.3. Statistical analysis After the genotypes of all participants were determined, the genotype and allele frequency were calculated by directly counting the number. The Chi-square test was used for examining the deviation from Hardy–Weinberg equilibrium (HWE) for all SNPs of the healthy group, the association between the allele, genotype frequency and susceptibility to HIV-1 infection, and the SNPs and the clinical features of cases (such as the CD4+ T-lymphocyte count and clinical stage). The haploView 4.2 software was used to evaluate the LD and the frequency of the haplotypes between HIV-1-infected individuals and health controls. The relative risk associated with rare alleles was estimated as an odds ratio (OR) and 95% confidence interval (CI). SPSS 17.0 (SPSS, Chicago, IL, USA) was used for all statistical analyses. The differences with a P value less than 0.05 were considered statistically significant, which was further subjected to a Bonferroni correction for multiple testing. Statistical power was assessed using the Genetic Power Calculator (Purcell et al., 2003). Considering 0.06% prevalence of the disease, a risk allele frequency of 17%, and an additive genetic model, we had at least 95% power to detect an OR of 1.5 at the 0.05 level.
2.1. Subjects 3. Results In this study, 479 HIV-1-infected male individuals who have sex with men (MSM) were recruited from Heilongjiang Center for Disease Control and Prevention (CDC). The age of the HIV-1-seropositive individuals ranged from 16 to 75 years old (mean age ± SD, 35.3 ± 11.55) and the average CD4+ T-lymphocyte count at that time point was 335 cells/μl (range, 3–1038 cells/μl). We categorized these patients as Category 1 (T-lymphocytes N200 cells/μl) or Category 2 (T-lymphocytes b 200 cells/μl) by the CD4+ T-lymphocyte count, and as Category A (Clinical phase I + II + III) or Category B (Clinical phase IV) by the clinical stage. The age and sex frequency-matched unrelated healthy controls (n = 487) were recruited from individuals who were HIV-1seronegative individuals diagnosed through a comprehensive medical test at the clinical laboratory of the second affiliated hospital of Harbin Medical University. The parents of each participant were individuals of the same nationality (for at least three generations) in nonsanguineous marriages. The age of the uninfected controls ranged from 16 to 75 years (mean age ± SD, 35.3 ± 11.59). All participants provided informed consent approved by the appropriate local authority. 2.2. SNPs selection and genotyping We included candidate SNPs based on the published literature and chose tagging SNPs based on linkage disequilibrium (LD) in the HapMap (HapMap Data Rel 24/phase II Nov08, on NCBI B36 assembly, dbSNP b126). The tagging SNPs were screened for minor allele frequency (MAF) ≥10% among Han Chinese in Beijing, China (CHB), and no representation by other tagging SNPs at a LD of r2 ≥ 0.80. We selected nine SNPs in MSH2 including rs13019654, rs4608577, rs4952887, rs6726691, rs10191478, rs12999145, rs1981929, rs2042649, and rs2303428 and five SNPs in MSH6 gene including rs2348244, rs3136245, rs3136329, rs2072447, and rs7562048. Of the 14 SNPs, rs6726691, rs12999145, rs3136245, rs3136329, and rs7562048 were selected from the literature and the others were tagging SNPs. Peripheral blood of all participants was collected and genomic DNA was extracted using the QIAamp blood kit (Qiagen, Germany). The 14 SNPs were genotyped using a custom-design 48-Plex SNPscan™ Kit (Genesky Bio-technologies Inc., Shanghai, China). This kit was developed according to patented SNP genotyping technology by Genesky Biotechnologies
The information of the 14 SNPs and the allele frequency in the MSH2 and the MSH6 genes are shown in Table 1. All tested SNPs did not deviate from the Hardy–Weinberg equilibrium in the healthy group (P N 0.05). There were no significant differences in the allele frequency of the 14 SNPs between cases and controls (P N 0.05). Similarly, we found no associations between genotype frequency of the 14 SNPs and HIV-1 infection under different genetic models (P N 0.05) (Supplementary Table S1). In analyzing the associations between the 14 SNPs and clinical features of AIDS (Table 2), we found that there was a significant association between the rs7562048 and the CD4+ T-lymphocyte count in patients with the AIDS (P b 0.05). The frequency of the rs7562048 G allele was significantly higher in the cases with Category 2(T-lymphocytes b 200 cells/μl) compared with those with Category 1 (T-lymphocytes N 200 cells/μl) (P = 0.001, OR = 1.811, 95% CI 1.255–2.614). The difference between Category 1 and Category 2 was still statistically significance after the Bonferroni correction. There were significant associations of the rs2348244 and rs3136245 with the clinical stage of AIDS. The frequencies of the rs2348244 C allele and the rs3136245 T allele were higher in the cases with Category B (Clinical phase IV) than those with Category A (Clinical phase I + II + III) (P = 0.026, OR = 1.591, 95% CI 1.056–2.398 and P = 0.019, OR = 1.749, 95% CI 1.096– 2.791, respectively). However, the significance between Category A and Category B disappeared after the Bonferroni correction. There was strong LD between the nine SNPs in the MSH2 gene as well as the five SNPs in the MSH6 gene. Nine haplotypes in block 1 and eight haplotypes in blocks 2 and 3 were identified and the frequencies of these haplotypes are listed in Table 3. The frequency of the haplotype TCCAAGCTC in cases was higher than that in healthy controls (P = 0.0498). However, there were no differences in the frequencies of the other haplotypes between HIV-1-infected individuals and healthy cohorts and there was no significant association with the susceptibility to HIV-1 infection (P N 0.05). 4. Discussion To our knowledge, this is the first comprehensive study to systematically evaluate the association of the MSH2 and the MSH6 genes
C. Wang et al. / Infection, Genetics and Evolution 42 (2016) 9–13
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Table 1 The information and allele frequency of the 14 SNPs in in case and control cohorts. SNP
Gene
Chra
Risk allele
Allele frequency
rs13019654 rs4608577 rs4952887 rs6726691 rs10191478 rs12999145 rs1981929 rs2042649 rs2303428 rs2348244 rs3136245 rs3136329 rs2072447 rs7562048
MSH2 MSH2 MSH2 MSH2 MSH2 MSH2 MSH2 MSH2 MSH2 MSH6 MSH6 MSH6 MSH6 MSH6
2 2 2 2 2 2 2 2 2 2 2 2 2 2
G G T G T A A T C C C C G G
HCBc 0.756 0.167 0.162 0.389 0.812 0.556 0.860 0.500 0.302 0.442 0.326 0.189 0.182 0.186
a b c
cases 0.762 0.154 0.157 0.371 0.812 0.539 0.844 0.520 0.319 0.446 0.350 0.124 0.116 0.159
controls 0.751 0.140 0.156 0.364 0.789 0.523 0.838 0.509 0.317 0.437 0.345 0.114 0.086 0.134
P value
OR
95% CI
P for HWETb
0.580 0.391 0.956 0.775 0.194 0.480 0.688 0.641 0.919 0.712 0.842 0.487 0.159 0.133
1.061 1.117 1.007 1.028 1.159 1.067 1.051 1.043 1.010 1.034 1.019 1.103 1.235 1.214
0.860–1.310 0.868–1.437 0.786–1.291 0.852–1.239 0.927–1.449 0.892–1.275 0.824–1.342 0.873–1.247 0.834–1.223 0.864–1.238 0.845–1.229 0.837–1.452 0.921–1.656 0.943–1.562
0.963 0.090 0.407 0.361 0.741 0.856 0.108 0.757 0.211 0.282 0.527 0.452 0.050 0.103
Chr: chromosome. HWET: Hardy–Weinberg equilibrium test. HCB: Han Chinese in Beijing, China.
polymorphisms with the susceptibility to HIV-1 infection and progression toward AIDS. The DNA repair genes are involved in HIV-1 integration because this process will lead to the host genomic DNA damage. Therefore, analyzing the role of the DNA repair genes in HIV-1 infection and the progression of this disease likely provides a new effective target for AIDS therapy. In our study, we found that there is a significant association between the rs7562048 and the CD4+ T-lymphocyte count and that G allele can significantly promote the disease progression of AIDS. As the CD4+ Tlymphocyte count declines, the immunity of AIDS patients is seriously compromised, leading to tumors and other complications. Studies have shown that high grade malignant lymphomas are the part of the
AIDS-defining condition (Levine, 1993) and that colorectal malignancies are likely associated with the grade of an immunosuppression induced during HIV infection (Yeguez et al., 2003). In fact, the MSH6 is one of the important MMR genes that are known to play a key role in the carcinogenesis caused by a virus infection, such as the human papillomavirus-associated cervical carcinoma (Bahnassy et al., 2006) and the hepatitis C virus-associated hepatocellular carcinoma (Zekri et al., 2005), although direct association between the MSH6 gene and the HIV-1-associated cancers has not been reported yet. The rs7562048 is located in the MSH6 introns, which together with our results suggest that the MSH6 gene may modulate the disease severity and the ensuing AIDS progression. We propose that the rs7562048
Table 2 Association between the 14 SNPs and the clinical features of AIDS. SNPs
rs13019654 rs4608577 rs4952887 rs6726691 rs10191478 rs12999145 rs1981929 rs2042649 rs2303428 rs2348244 rs3136245 rs3136329 rs2072447 rs7562048
Allele
G T G T T C G C T G A G A G T C C T C T C T C T G C G A
CD4+ T-lymphocyte counta N200 cells/μl
b200 cells/μl
n
n
545 171 110 606 110 606 259 457 585 131 379 337 607 109 366 350 230 486 322 394 243 473 83 633 77 639 98 618
183 57 37 205 40 202 96 146 193 49 137 105 202 40 132 110 76 166 105 137 92 150 36 206 34 208 54 188
Clinical stageb P value
OR
95% CI
0.967
0.993
0.704–1.399
0.978
1.006
0.671–1.507
0.666
1.091
0.735–1.620
0.330
1.160
0.860–1.565
0.502
1.134
0.786–1.635
0.321
0.862
0.643–1.156
0.628
1.103
0.742–1.638
0.356
1.148
0.857–1.537
0.836
1.034
0.755–1.414
0.668
1.066
0.795–1.430
0.250
1.194
0.883–1.615
0.181
1.333
0.875–2.029
0.166
1.357
0.881–2.089
0.001
1.811
1.255–2.614
Clinical phase I + II + III
Clinical phase IV
n
n
653 203 129 727 134 722 310 538 699 157 454 402 725 131 440 416 278 578 371 485 310 546 106 750 99 757 138 718
75 25 18 84 16 86 43 59 79 23 62 40 84 18 58 44 28 74 56 46 25 77 13 89 12 90 14 88
The values in bold indicate that the differences are significant. a The CD4+ T-lymphocyte counts were divided into two groups: Category 1, N200 cells/μl; Category 2, b200 cells/μl. b Clinical stage: Category A, Clinical phase I + II + III; Category B, Clinical phase IV.
P value
OR
95% CI
0.775
1.072
0.664–1.731
0.495
1.208
0.703–2.076
0.993
1.002
0.570–1.763
0.269
1.265
0.834–1.918
0.304
1.296
0.791–2.125
0.138
1.372
0.903–2.085
0.537
1.186
0.690–2.038
0.297
1.246
0.824–1.885
0.303
1.271
0.805–2.007
0.026
1.591
1.056–2.398
0.019
1.749
1.096–2.791
0.917
1.033
0.558–1.914
0.953
1.020
0.539–1.929
0.531
1.208
0.668–2.184
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C. Wang et al. / Infection, Genetics and Evolution 42 (2016) 9–13
Table 3 The frequency of haplotypes in the MSH2 and the MSH6 genes in case and control cohorts. Haplotype Block 1 TCCAGGCTC TCCAGTTTC TTGAAGTTT GCGGAGTTT TCCAAGTGT TCCAGTCTC GCGAAGTTT TCGAAGTTT TCCAAGCTC Block 2 TC CT TT CC Block 3 TCA CGG TCG CCG
Frequency
Case, control ratios
P value
0.212 0.158 0.149 0.145 0.133 0.073 0.032 0.025 0.017
197.6:749.8, 209.7:745.6 149.7:797.7, 153.9:801.4 143.7:803.7, 143.9:811.5 138.8:808.5, 139.6:815.7 135.2:812.2, 121.1:834.2 72.5:874.9, 67.8:887.5 28.9:918.4, 33.3:922.0 28.2:919.2, 19.8:935.6 21.9:925.5, 10.9:944.4
0.560 0.852 0.946 0.981 0.309 0.640 0.596 0.206 0.0498
0.428 0.334 0.225 0.014
413.9:544.1, 412.2:561.8 321.9:636.1, 323.0:651.0 209.1:748.9, 255.0:749.0 13.1:944.9, 13.8:960.2
0.693 0.838 0.502 0.921
0.850 0.105 0.031 0.010
800.9:155.1, 839.8:134.2 107.9:848.1, 94.4:879.6 37.1:918.9, 23.2:950.8 6.0:950.0, 13.4:960.6
0.132 0.253 0.058 0.098
The values in bold indicate that the differences are significant.
may regulate the transcription and the expression of the MSH6 gene, which interacts with HIV-1 to promote its replication and integration, leading to the decrease in the CD4+ T-lymphocyte count and the acceleration of this disease. Up to now, the mechanism underlying the interaction between the MSH6 and HIV-1 remains unclear, and our study opens a new field for further studying on the functional significance and the underlying mechanism of the association between the rs7562048 and AIDS progression. The differences of allele and genotype frequency of the 14 SNPs were not seen between the case and the control cohorts, which imply a null association of the MSH2 and the MSH6 genes polymorphisms with the susceptibility to HIV-1 infection in the northern Chinese population. In previous reports, the role of the MSH2 and the MSH6 genes polymorphisms was inconsistent in the cancer association studies. One report showed that the rs2072447 increased the colorectal cancer risk under the recessive model, which suggests that the MSH6 gene polymorphism is associated with the susceptibility to colorectal cancer (Tulupova et al., 2008). Some other studies showed that the MSH2 and the MSH6 genes polymorphisms were not associated with the susceptibility to the lung cancer in a Chinese population and the ovarian cancer in European and American populations (Lo et al., 2011; Mann et al., 2008). In our study, the difference of TCCAAGCTC haplotype in block 1 was found between case and control cohorts and the association was significant (P = 0.0498); however, the P value was closed to 0.05 and the frequency of the haplotype was too lower in cases and controls. Through further comprehensive analysis of the frequency distribution of alleles, genotypes and haplotypes, we verified that there is no association between the MSH2 and the MSH6 genes polymorphisms and HIV-1 infection. Here, we propose that the null association of the MSH2 and the MSH6 genes polymorphisms with the susceptibility to HIV-1 infection is because of the differences of disease types or genetic background. In conclusion, we found that the rs7562048 is associated with the clinical features and MSH6 gene polymorphisms play an important role in the progression of AIDS in a northern Chinese population. Nevertheless, larger scale association and functional studies as well as using more SNPs in the MMR pathway are needed to expand our initial findings, which would help us to fully understand the contribution of the MMR genes to HIV-1 infection and AIDS progression. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2016.04.016.
Authors' contribution All authors have contributed to the paper. C.T.W., C.Y.Z., Y.D.Q., and S.B.F. participated in the design of the study. X.Y.J., H.M.S., and J.C.Y assisted with experiment preparation. C.T.W., C.Y.Z., X.L.Z., L.D.X., G.F.Z., N.H., and Q.Y.L. performed the experiments. C.T.W., and C.Y.Z fulfilled the statistical analysis. C.T.W., C.Y.Z., Y.D.Y., and S.B.F. wrote the manuscript. All authors read and approved the final version. Acknowledgements This work was funded by the Program for Changjiang Scholars and the Innovative Research Team in University (IRT1230); the National Natural Science Foundation of China (81373220); Natural Science Foundation of Heilongjiang Province of China (D2007-04); the Post-doctoral Foundation of Heilongjiang Province (LRB08-340); Wu Lien-Teh Youth Science Foundation of Harbin Medical University (WLD-QU1405). We also gratefully acknowledge the numerous sample donors for making this work possible. References Ameisen, J.C., Capron, A., 1991. 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