| Cervical cancer is the second most common malignancies in both of incidence and mortality rates in worldwide. Etiology of cervical cancer remains a major concern, and the most important finding is the close relationship between high-risk human papillomavirus (HR-HPV) and cervical cancer. Compelling evidences from both epidemiological and experimental studies have indicated that infection of oncogenic HR-HPV, predominantly types 16 and 18, are the major risk factors for the development of cervical cancer, and HR-HPV DNA is found in over 95% of cervical cancers. However, HR-HPV infection is frequent among sexually active women, with incidence ranging from 15% to 40%, most HR-HPV infections are transient and only a small proportion of infected women develop malignant cervical lesions. Therefore HR-HPV is considered to be a necessary but not sufficient cause for cervical cancer and other factors contribute to the carcinogenic process. Although age of first intercourse, number of sexual partners, more parities, cigarette smoking, race and low socioeconomic status consistently have been shown as risk factors for cervical cancer, none has been shown to be as a significant independent risk factor.Consequently, other factors maybe play a crucial role in those processes, especially genetics. Since Storey et al. reported that the Arg allele of p53 codon 72 increased cervical cancer compared Pro allele; many studies on the susceptibility of cervical cancer and SNP were carried on. However, the gene involved in almost were oncogene or antioncogene, metabolic gene. Seldom study focused on DNA repair gene. Regularly, DNA damage consequently initiates DNA repair process in the cells. Now known, DNA repair pathways maintain genetic integrity, and defects of them are associated with many different types of diseases, including cancer. More than 130 human DNA-repair genes have been described. It has been identified there are a number of single-nucleotidepolymorphisms (SNPs) in DNA-repair genes. Those SNPs may result in subtle alteration in structure and function of the repair enzymes, even in susceptibility of the host to the development of cancer.XRCC1 is involved in base excision repair (BER) and the repair of single-strand breaks. The XRCC1 acts as a scaffold for other DNA repair proteins, such as human AP endonuclease (APE1), DNA polymerase β, DNA ligase III, polynucleotide kinase, and PARP. At least 21 SNPs with the amino acid substitutions were identified within XRCC1 gene (dbSNP of NIH http://www.ncbi.nlm.nih.gov/SNP/index.html ). Especially three nonsynonymous SNPs, Argl94Trp, Arg280His, and Arg399Gln, reveal relatively high frequency. The variant genotype results in change of protein and has recently been focused on the relationship between these polymorphisms and susceptibility to cancers. Arg194Trp and Arg280His reside in a linker region connecting the domains that interact with PARP and DNA polymerase β, whereas Arg399Gln resides in the functionally important PARP-binding domain. To date, only one study evaluated the role of the Arg399Gln polymorphisms in XRCC1 gene in relation to cervical carcinoma in Japanese. To our knowledge, no study was reported so far with regard to the association of the Argl94Trp and Arg280His polymorphisms with risk of cervical carcinoma. The O~6-methylguanine DNA methyltransferase (MGMT) protein has a wide range of activities in normal tissues, and its evolutionary conservation indicates that it plays a fundamental role in cell physiology and genome maintenance. This is the only protein known to be involved in the cellular defense against alkylating agents within the mammalian DNA direct reversal repair pathway. Methyltransferase activity between human fibroblast cultures from different individuals varies as much as 20-fold, suggesting that genetic polymorphisms in MGMT are risk factors for the development of cancer. Several SNPs have been reported in MGMT, such as Leu84Phe, Ile143Val, and Lysl78Arg. The significance of these SNPs in cervical cancer has not been investigated.Therefore, in the case-control study, we investigated the genotype distributions of the XRCC1 Arg194Trp, Arg280His, Arg399Gln and MGMT Leu84Phe, Ile143Val, and Lys178Arg polymorphisms in the patients with cervical carcinoma, CIN and control, to explore the role of SNPs in DNA repair genes in carcinogenesis of the cervix.Part IComparison between two Methods for SNP GenotypingObjective: To select a suitable method for great scale single nucleotide polymorphism (SNP) genotyping by comparing the two methods: RFLP and MAMA-PCR. Methods: Recruited 200 healthy women seen for gynecologic examinations at the Women's Hospital School of Medicine at Zhejiang University during October 2004 -March 2005, we randomly selected 50 among them for XRCC1 Argl94Trp and XRCC1 Arg399Gln genotyping by MAMA-PCR and by RFLP, then compared the results with sequencing's. The whole 200 healthy women were genotyped by MAMA-PCR. Results: 1. For MAMA-PCR, the genotypes were distinguished for the heterozygote presenting two bands and the wild type or variant type presenting single different band respectively. The results were all consistent with the results of sequencing. No discordant pairs were observed. 2. For RFLP, the wild-type Arg194 allele produces a single band representing the entire 485 bp fragments, and the variant 194Trp allele has 396 and 89 bp fragments because it gains a RFLP site. The wild-type allele (Arg/Arg) at the Arg399Gln site generates 2 DNA bands (384 and 133 bp), the variant allele (Gln/Gln) has a single 517 bp fragment, and the heterozygote (Arg/Gln) displays all 3 bands (517, 384 and 133 bp). No discordant pairs were observed in three different methods for assaying the same polymorphisms. 3. The distributions of the genotypes of the SNPs among the 200 health women were all in accordance with the Hardy-Weinberg equilibrium. The allelic frequencies of two SNPs in XRCC1 were both consist with prior reports in a Chinese population.Conclusion: MAMA-PCR is a simple and rapid method for SNP genotyping.Part IIThe SNP of XRCC1/MGMT and the susceptibility to cervicalcarcinomaObjective: To evaluate contribution of SNPs of XRCC1 and MGMT gene to the risk of cervical carcinomaMethods: Patients at five hospitals in Zhejiang Province including Women's Hospital School of Medicine at Zhejiang University with histologically confirmed primary cervical carcinoma were recruited between March 2004 and May 2006. 1012 eligible patients (539 with cervical carcinoma and 473 with CIN) and 800 eligible controls agreed to provide blood samples for genotyping. Control selection criteria included no positive findings during the gynecological examination, no history of cancer, age matching to the patients. Phenol-chloroform was used to extract DNA. The SNPs of XRCC1/MGMT were determined by MAMA-PCR.Results: 1. There were no significant differences in ages between the three groups. 2. Genotype and allele frequencies of both XRCC1 Arg399Gln and Argl94Trp among carcinomas, CINs and controls were significantly different. However, genotype and allele frequencies of XRCC1 Arg280His and MGMT Leu84Phe, Ile143Val, Lys178Arg among carcinomas, CINs and controls were not significantly different. 3. Logistic regression analysis revealed that women carrying homozygous Gln399Gln (AA) genotype had a significantly 2.32-fold increased risk for cervical carcinoma (95% CI 1.47-3.65), heterozygous Arg399Gln (GA) genotype were also had a increased risk for cervical carcinoma, with OR 1.58 (95% CI 1.24-2.00), compared with women carrying XRCC1 Arg399Arg (GG) genotype. When we combined the Arg399Gln (GA) and Gln399Gln (AA) genotypes assuming a codominant allele effect, the combined GA/AA variant genotypes were associated with a 68% (OR = 1.68; 95% CI 1.34 -2.10) increased risk for cervical carcinoma. Similarly, compared with Argl94Arg (CC) wild-type genotype, elevated risks were associated with the Trpl94Trp (TT) or their combined genotypes CT/TT both for cervical carcinoma [ORs and 95%CIs being 2.09 (1.45-3.02) and 1.28 (1.03-1.59), respectively]. For MGMT gene, Logistic regression analysis revealed that, compared with the MGMT Lysl78Lys (AA) genotype, women homozygous for theArg178Arg (GG) genotype had a 2.48-fold increase in the risk for cervical carcinoma (95% CI = 1.02-6.03), and a combination of the Arg178Arg (GG) and Lys178Arg (GA) genotypes increased the risk by 60%. (OR = 1.63 [95% CI = 1.01-2.61]). Those heterozygous for the Lys178Arg (GA) genotype, however, did not have an increased risk for cervical carcinoma. Similarly, compared with the wild-type Ile143Ile (AA) genotype, those homozygous for Val143Val (GG) had an elevated risk for carcinoma (OR = 2.44 [95% CI = 1.01-5.95]), but those heterozygous for Ilel43Val (GA) did not. Neither the Phe84Phe (TT) nor the Leu84Phe (CT) genotype elevated the risk for cervical carcinoma compared to the Leu84Leu (CC) genotype. 4. It showed the very similar results of squamous cell carcinoma to the overall carcinoma. No significant differences among adenocarcinomas and other types and between CIN and CIN II-III were found. 5. Frequencies of the 4 common haplotypes of XRCC1 were significantly different in the carcinoma and control groups. Using 194Arg-280Arg-399Arg as the reference, which was the most common type in control and CIN group, subjects carrying the 194Trp-280Arg-399Arg had a significantly 1.98-fold increased risk of cervical carcinoma (95% CI=1.62-2.40). The OR for 194Arg-280His-399Arg and 194Arg-280Arg-399Gln were 1.85 (95% CI =1.41-2.41) and 2.30 (95% CI= 1.86-2.85), respectively. The frequencies of the five most common haplotypes of MGMT were also significantly different between the patients and control groups. Using the most common haplotypes 84Leu (C)-143Ile (A)-178Lys (A) as a reference, women carrying the 84Phe (T)-143Val (G)-178Arg (G) haplotypes had a significantly increased risk for cervical carcinoma (adjusted OR = 1.87 [95% CI = 1.07-3.27]). The risk for cervical carcinoma was not elevated for women with other haplotypes, including 84Leu (C)-143Val (G)-178Arg (G), 84Leu (C)-143Val (G)-178Lys (A), and 84Phe (T)-143Ile (A)-178Lys (A). It showed the very similar results of squamous cell carcinoma to the overall carcinoma. No significant differences among adenocarcinomas and other types and between CIN were found.Conclusion: XRCC1/MGMT polymorphisms including genotypes and haplotypes contribute to susceptibility to the development of cervical carcinoma.Part IIIMultivariant analysis on the correlation among the SNP of XRCC1/MGMT, HR-HPV infection, and behavior risk factors in susceptibility to cervical carcinomaObjective: To evaluate the correlation of the SNP of XRCC1/MGMT, HR-HPV, behavior risk factors and susceptibility to cervical carcinomaMethods: The subjects were recruited as Part II. After providing signed informed consent, participants responded to a standard questionnaire about their sexual and reproductive history, including the number of sexual partners, the age at first intercourse, the age at first full-term pregnancy and parities (including full-term pregnancy and abortion at or after 28 weeks). 1012 eligible patients (539 with cervical carcinoma and 473 with CIN) and 800 eligible controls agreed to provide blood samples for genotyping and correctly completed the questionnaire. Of these, 675 patients (260 with cervical carcinoma and 415 with CIN) and 410 controls agreed to provide cervix brush-off samples for detection of HR-HPV. High risk HR-HPV-DNA was detected by hybrid-capture II assay.Results: 1. HR-HPV infection rates in carcinoma and CIN patients were 90.4% and 85.8%, respectively, whereas in control women, the infection rate was only 31.0%. Thus, the infection rate in both patient groups was significantly higher than in the control group (x~2= 366.310, P<0.001). The sexual and reproductive histories, including age at first intercourse, number of sexual partners, and age at first full-term pregnancy among the patients with carcinomas, patients with CIN, and controls were not significantly different, but the parities (including full-term pregnancy and abortion at or after 28 weeks) was significantly different among the three groups (x~2= 18.235; P<0.001) (Table 1). 2. All six variant genotypes did not confer higher risk of HR-HPV infection. 3. Risk factors for cervical carcinoma and potential confounders were kept in multivariable models at significantly level a=0.05, permission error =0.10, used Forward analyses, except HR-HPV infection, the multivariable logistic regression involved in number of sexual partners, age at first intercourse, parities (including full-term pregnancy and abortion at orafter 28 weeks), and age at first full-term pregnancy. 4. The results for XRCC1/MGMT genotypes in the HR-HPV positive group were very similar to the results in the overall carcinoma group. 5. The results for XRCC1/MGMT haplotypes in the HR-HPV positive group were very similar to the results in the overall carcinoma group. 6. In addition, the effect of XRCC1 codon 194 variant genotypes (CT/TT) and MGMT codon 178 variant genotypes (GG+GA) were only existed in women who had multiple sexual partner (>1), early onset of sexual activity (≤20 years), high parity (>3), and young age at first full-term pregnancy (≤22 years) (adjusted OR and 95% CI being [1.28 (1.00-1.63), 1.43 (1.04-1.76), 1.55 (1.74-3.81), 1.44 (1.26-2.53), 3.23 (1.14-5.82), 2.58(1.23-4.41), 2.52(1.75-3.45) and 3.22 (1.20-4.66)], respectively). While, XRCC1 codon 399 variant genotypes (GA/AA) increased the risk of cervical carcinoma in all the subgroup except the HR-HPV negative group.Conclusions:1. The genotypes and the haplotypes of XRCC1 Arg194Trp, MGMT Lys178Arg and Ile143Val increase the risk of cervical carcinoma only in the women with HR-HPV infection or with behavior risk factors, whereas XRCC1 Arg399Gln increases the risk of cervical carcinoma in all groups except HR-HPV negative, suggested that XRCC1 Arg399Gln is a SNP conferred more risk for cervical carcinoma.2. The SNPs of XRCC1/ MGMT do not confer higher risk of HR-HPV infection.
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