Genetic association of G2/M checkpoint genes with susceptibility to HIV-1 infection and AIDS progression in northern Chinese MSM population

MSM has a high risk of HIV infection. Previous studies showed that the cell cycle regulation played an important role in HIV-1 infection, especially in G2/M checkpoint. The ATR, Chk1, Cdc25C and CDK1 are key genes in G2/M checkpoint. However, the association between the SNPs of these genes and susceptibility to HIV-1 infection and AIDS progression remains unknown. Results 42 tSNPs of G2/M checkpoint genes were genotyped to analyze the association between these tSNPs and susceptibility to HIV-1 infection and AIDS progression among MSM (529 HIV − 1 seropositive men and 529 HIV-1 seronegative men) in northern China. The results showed that rs34660854-A and rs75368165-A in ATR gene and rs3756766-A in Cdc25C gene could increase the risk of HIV-1 infection (P = 0.049, P = 0.020 and P = 0.010, respectively), and ATR rs75069062 and Chk1 rs10893405 were signicantly associated with AIDS progression (P = 0.026 and P = 0.029, respectively). Besides, rs34660854 and rs75368165 in ATR gene, rs12576279 and rs540436 in Chk1 gene, rs3756766 in Cdc25C gene and rs139245206 in CDK1 gene were signicantly associated with HIV-1 infection under different models (P < 0.05). genetic variants genes northern AIDS: Acquired immune deciency syndrome; ATR:Ataxia Telangiectasia and Rad3-related kinase; Cdc25C:Cell division control protein 25 homolog C; CDK1:Cyclin-dependent kinase 1; Chk1:Checkpoint kinase 1; DSB:Double strand break; GMDR:Generalized multifactor dimensionality reduction; HIV:Human immunodeciency virus; HWE:Hardy-Weinberg equilibrium; MSM:Men who have sex with men; OR:Odd ratio; SNP:Single nucleotide polymorphism.

reports also showed that the genetic variants of G2/M checkpoint genes were associated with many kinds of diseases, i.e., the polymorphisms of ATR, Chk1 and CDK1 gene were signi cantly associated with breast cancer, while the polymorphisms of Cdc25C gene with hepatocellular carcinoma and so on. [15][16][17] However, still there is no clear report about the association between the genetic polymorphisms of ATR, Chk1, Cdc25C and CDK1 gene in G2/M checkpoint and susceptibility to HIV-1 infection and AIDS progression in northern Chinese MSM population.

Characteristics of the study subjects
The basic characteristics and clinical parameters of cases and controls were shown in the Table 1. There was no signi cant difference between cases and controls for age (P = 0.458). The detailed information of HWE test was listed in Table 2. All these tSNPs in controls were in HWE (P > 0.05) except Chk1 rs537046 (P = 0.049) and CDK1 rs16915503 (P = 0.029), which were excluded from further analysis.

Haplotype analysis
Strong linkage disequilibrium among tSNPs were observed and thirteen haplotypes in block 1 and block 2 of Chk1 gene and four haplotypes in block 1 of Cdc25C gene were identi ed (Additional le 2). A signi cant difference in haplotype distributions of Chk1 and Cdc25C gene between cases and controls were found ( Table 3). The frequencies of H7 (haplotype CCTCGACGC) of Chk1 gene and H4 (haplotype GGT) of Cdc25C gene in block 1 were higher in cases than in controls (P = 0.0054 and P = 0.0009, respectively). The association remained signi cant after correcting for multiple testing using 10,000 permutations with the Haploview program (P = 0.0452 and P = 0.0017). There was no signi cant difference in frequencies of other haplotypes (P > 0.05).

Gene-gene interaction analysis by GMDR
GMDR was applied to screen the possible gene-gene interaction combinations among 40 tSNPs in 4 genes and the results obtained from GMDR analysis were showed in Table 5. There was only one signi cant model involving ATR rs68065420, Chk1 rs1057733 and Cdc25C rs6861656 whose cross-validation consistency was 10/10 and test accuracy was 0.5578. Different combinations of high risk and low risk genotypes of three SNPs in the best model was summarized and an interaction analysis also conducted to explore the association between these combinations and susceptibility  (Table 6). Additionally, comparing the OR value of these 5 combinations, a trend of risk for HIV-1 infection progressively increased along with the high risk alleles observed in carrier participants (Fig. 2). The values in bold indicate the best gene-gene interaction model.

Discussion
It is known that many viruses including HIV-1 induce cell cycle arrest in G2 phase via G2/M checkpoint activation through a variety of mechanisms. [18] HIV-1 is a subtype of HIV, that functions like retrovirus and the integration after reverse transcription is important for its proliferation. [19] The integration of HIV-1 can cause DSB of CD4 + T cell and activate G2/M checkpoint that leads to cell cycle arrest. [20,21] This mechanism will also affect the proliferation of HIV-1. [22] Therefore, it is critical to investigate the association between G2/M checkpoint and HIV-1. In this study, 42 tSNPs in ATR, Chk1, Cdc25C and CDK1 gene were genotyped to analysis the association with susceptibility to HIV-1 infection and AIDS progress among MSM population in the northern China.
ATR, as a sensor of DNA damage, contributes to cell cycle arrest, DNA damage repair and stable replication of cells after being activated, which is an important kinase of avoiding apoptosis for cells. [23,24] A study also found that Vpr-induced structural alteration of DNA can trigger ATR-mediated DNA damage response and contributed to HIV-1 infection. [13] Previous reports showed that rs13091637, which also located in the ATR intronic region and was in strong linkage disequilibrium with rs34660854 in Chinese population of and, was signi cantly associated with melanoma and breast cancer. [25][26][27] The results in our study showed that rs34660854-A and rs75368165-A in ATR gene were signi cantly associated with increased susceptibility to HIV-1 infection. These two SNPs also showed signi cant differences under codominant and dominant model. It indicated that the rs34660854-A and rs75368165-A in ATR gene were the pathogenic factors for HIV-1 infection. The genotypes carrying risk alleles of these tSNPs were more likely to infect HIV-1 among MSM population in the northern China. By analyzing the effect of the SNPs on AIDS progression, rs75069062 showed a difference between clinical phase I/II/III and clinical phase IV. Although rs34660854, rs75368165 and rs75069062 were all located in the intronic regions, they might be responsible for affecting gene function at transcription level, splicing enhancer or silencer and other mechanisms.
Chk1 was phosphorylated and activated by ATR after DNA damage had been sensed by ATR at G2/M checkpoint. The genetic mutations of Chk1 gene can cause many kinds of disease such as breast cancers, colorectal cancers, human lymphoid neoplasms and so on. [28][29][30] However, there are few reports which clearly explain the association between Chk1 and HIV-1 infection. Our results showed that rs12576279 and rs540436 in Chk1 gene were signi cantly associated with HIV-1 infection risk under codominant and dominant model. The results found that the individuals carrying rs12576279-T were at lower risk for HIV-1infection. It also indicated that the genotypes including rs540436-T were pathogenic factors for HIV-1 infection. In addition, rs10893405 also showed signi cant differences between clinical phase I/II/III and IV by analyzing the association between the polymorphisms of Chk1 gene and AIDS progression. It indicated that the rs10893405-G was the risk factor of AIDS progression. Furthermore, a haplotype (H7) and haploid allele of Chk1 gene was signi cantly associated with HIV-1 infection susceptibility. These three SNPs were all located in the intronic regions and we didn't nd any publications about them in NCBI database (https://www.ncbi.nlm.nih.gov). Moreover, the functional studies about these SNPs with HIV-1 infection needed to be carried out.
The protein Cdc25 is a key inducer for the entry of M phase and controls the timing of mitosis. It includes three homologues i.e. Cdc25A, Cdc5B and Cdc25C. [31] Cdc25C, which are phosphorylated and inactivated by Chk1, play important roles in the process of G2/M checkpoint. [32] Many reports showed that Cdc25A and Cdc25B were associated with breast cancers, colorectal cancers, non-small cell lung cancers and so on. But there were few reports about the association between Cdc25C and carcinogenesis. [33] Vpr, which is an important protein of HIV-1, can trigger G2 arrest by inhibiting the Cdc25C phosphatase activities. However, no study reported the association between the polymorphisms of Cdc25C and HIV-1 infection and AIDS progression. [12] Our results showed that rs3756766-A in Cdc25C gene was signi cantly associated with increased susceptibility to HIV-1 infection. Moreover, this SNP also showed a signi cant association under codominant and dominant model. It indicated that the rs3456766-A was the pathogenic factor of HIV-1 infection. Hence the genotypes including allele A could increase the cumulative risk of HIV-1 infection.
CDK1-Cyclin B1 complex plays an important role in the process of G2/M transition. The activation and nuclear accumulation of this complex are key events for G2/M transition. [34,35] In the process of G2/M checkpoint, Cdc25C phosphatase activates CDK1 by removing two inhibitory phosphates from Thr14 and Tyr15. [36] A study showed that the Vif of HIV-1 could impair the mitotic entry by interfering with CDK1-Cyclin B1 complex activation causing cell cycle arrest. [10] However, the association of CDK1 polymorphisms and HIV-1infection and AIDS progression remains unclear. The results in our study showed that CDK1 rs139245206 was signi cantly associated with HIV-1 infection under codominant and recessive model. Although this SNP located in intron regions, it might regulate gene transcription level by binding with transcription factors.
Gene-gene interaction is extremely important, because many genes involve the complex process of G2/M checkpoint regulating cell cycle and the role of a single gene may be nite. Therefore, GMDR software was used to investigate the impact of interaction between ATR, Chk1, Cdc25C and CDK1 gene polymorphisms on HIV-1 infection susceptibility.
Our results showed a three-locus model including ATR rs68065420, Chk1 rs1057733 and Cdc25C rs6861656, and participants with rs68065420-AA and rs1057733-TT and rs6861656-TT genotype had the highest HIV-1 infection risk.
We also observed a drift of progressively increased risk of HIV-1 infection along with the high risk alleles observed in carrier participants.

Conclusions
In our study, we investigated the association between genetic polymorphisms of G2/M checkpoint genes and HIV-1 infection and AIDS progression. In conclusion, rs34660854 and rs37568165 in ATR gene, rs12576279 and rs540436 in Chk1 gene, rs3756766 in Cdc25C and rs139245206 in CDK1 gene were associated with susceptibility to HIV-1 infection and rs75069062 in ATR gene and rs10893405 in Chk1 gene were associated with AIDS progression among MSM population in northern Chinese. It revealed that G2/M checkpoint played a crucial role in HIV-1 infection and AIDS progression. Although, the positive results showed no differences after Bonferroni correction because there were so many SNPs included in this study. However, the results we got were still positive for HIV prevention and should not be ignored due to the false negatives. This nding will provide the theoretical basis and basic data for the prevention of HIV infection and the treatment of AIDS. Moreover, in our further studies, more related researches are needed using larger sample size and different populations to verify our ndings.

DNA extraction and genotyping
The DNA of every participant was extracted from peripheral blood of every individual using the QIAamp blood kit (Qiagen, Hilden, Germany). And the genotyping of these 42 tSNPs was carried out utilizing the SNPscan Kit (Genesky Biotechnologies Inc., Shanghai, China). 53 participants (5% of all participants) were selected randomly whose 42 tSNPs were double-genotyped in order to ensure the accuracy of the results and the accuracy rate was 100%.

Statistical analysis
The difference of age distribution between case and control groups was compared by Student's t-test. Hardy-Weinberg equilibrium (HWE) in the controls was checked by Chi-square test. The associations of these 42 tSNPs with susceptibility to HIV-1 infection and AIDS progression were determined using Chi-square test, and the effect size was expressed as odds ratio (OR) with 95% con dence interval (95% CI). The statistical analyses were performed by SPSS v. 22.0 statistical software (IBM-SPSS, Inc., Chicago, USA). Haploview v4.2 was used to analyze the linkage disequilibrium (LD) and haplotype, and 10,000 permutations were run to compute the p-values. P < 0.05 was considered statistically signi cant. Generalized Multifactor Dimensionality Reduction (GMDR) v 0.9 (http://www.ssg.uab.edu/gmdr/) was utilized to explore the possible gene-gene interaction.

Figure 1
Distrubution of genotypes of the tSNPs with signi cant difference in cases and controls. Condomiant 1, homozygous including risk allele versus homozyrous including non-risk-allele; Codominant 2, heterozygous versus homozygous including risk allele. The distribution of genotypes of the other tSNPs were showed in Additional le 1.