Lack of Association between TP73 G4C14-A4T14 Polymorphism and Cervical Cancer Risk in Overall and Asian Women: A Meta-Analysis

Background: Growing studies revealed the association between polymorphisms in Tumor Protein TP73 (TP73) and susceptibility to cancer, especially with gynecological cancers. but, the results remained inconsistent. This meta-analysis was carried out to examine the relationship of the TP73 G4C14-to-A4T14 polymorphism (hereafter, G4C14-to-A4T14) with susceptibility to cervical cancer globally and by ethnicity. Methods: Eligible studies were collected by retrieving PubMed, Scopus, Web of Science, Embase, Wan Fang, and CNKI published before 25 October, 2023. The pooled odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of such association. Results: A total of 10 case-control studies with 1804 cervical cancer cases and 2433 healthy controls were included to this study. The pooled results showed that TP73 G4C14-to-A4T14 polymorphism was not associated with cervical cancer risk in overall. in terms of stratified analyses by ethnicity, this polymorphism was not associated with risk of cervical cancer among East-Asian women. however, there was a significant association based source of control among hospital-based studies. Conclusions: Inconsistent with previous meta-analyses, our pooled results revealed that TP73 G4C14-to-A4T14 polymorphism might not be a risk factor for development of cervical cancer globally and among East-Asian women. Moreover, further studies examining the effect of gene-gene and gene-environment interactions may eventually provide a better knowledge.


Introduction
Cervical cancer is a major public health issue and the third most frequent cancer and the fourth leading cause of cancer death among women globally [1][2][3].It is estimated 57000 new cases and 311000 deaths in 2018 and will cause 474,000 women per year by 2030 [4,5].About 90% of the new cases and deaths worldwide in 2020 occurred in 662 2014 showed that the mean age of the cervical cases was 51.91 years and their 5-and 10-year survival rates were 58% and 50%, respectively [11,12].
The primary and main causative factor of cervical cancer is the high-risk oncogenic human papillomavirus (HR-HPV) [13][14][15].More than 90% of patients with cervical cancer show a positive long-lasting infection with certain types of HPV especially in western countries [16][17][18].Family history is beyond control when assessing the risks for cancer, but if the mother or sister of a patient has had cervical cancer, the likelihood of developing cancer increases by two to three times [10,19,20].Genetic factors contributing to the development of cervical cancer are largely unknown.There were reported a few familial clustering of cervical cancer cases indicating that highpenetrance germline variants are rare in this cancer and heritable risk of cervical cancer may be accounted for by low-and intermediate-penetrant genetic factors [21,22].However, some evidence from epidemiological studies have been shown that a genetic background could predispose to cervical cancer, and that some of the genes likely to be involved are IRF3, TLR2, EXO1, CYBA, XRCC1 and FANCA, OAS3, SULF1, IFNG, DUT, DMC1, GTF2H4, EVER1/2, ERAP1, LMP7, TAP2, TP53, TERT and IL-17 [23,24].Several studies showed that TP73 expression is up-regulated in cervical cancer tissues in comparison in normal cervical squamous epithelium, and negatively associated with clinical progression in cervical cancer patients [25,19].
Tumor Protein TP73 (TP73) is an essential member of a gene family that comprises TP63 (p63) and the wellcharacterized tumor suppressor TP53 (p53) [26][27][28].TP73 is plays important roles in embryonic development and differentiation, and located on chromosome 1p36-33, mapping on a region often deleted in different cancers [25,27].two single polymorphisms G4A (rs2273953) and C14T (rs1801173) in the 5′-UTR of exon 2 of the TP73 gene is reported.These polymorphisms are in complete disequilibrium with each other and are jointly referred to as G4C14-A4T14 [29,30].This set of polymorphisms are located above the translation initiation site and has been shown to affect TP73 gene expression levels by forming a stem-loop-like structure and therefore have functional outcomes [31,3].
In recent years, TP73 G4C14-A4T14 polymorphism have been reported implicated in the development of cervical cancer [32,25].Nevertheless, data arising from these published case-control studies were not consistent.With the rapid growth of literatures, there is increasing need to make meaningful inferences from a comprehensive and complex body of evidence, a thorough meta-analysis of the literature helps to explore more evidence of association between TP73 G4C14-A4T14 polymorphism and cervical cancer risk.thus, we performed this metaanalysis to examine the relationship of the TP73 G4C14to-A4T14 polymorphism with susceptibility to cervical cancer globally.To the best of our knowledge, this is the most comprehensive meta-analysis regarding the TP73 G4C14-A4T14 polymorphism and its association with cervical cancer risk.

Including and Excluding Criteria
We set these inclusive criteria for recruited publications: a) studies with case-control or cohort design; 2) published studies in English, Chinese and Farsi; 3) studies evaluated the association of TP73 G4C14-A4T14 polymorphism with risk of cervical cancer; and 4) enough and available data to figure out odds ratios (ORs) and 95% confidence intervals (CIs).In addition, we also restricted these exclusive criteria: 1) studies without control group (case only studies); 2) insufficient data offered to analyze or unavailable data; 3) studies were carried out based on animals and in vitro studies; 4) studies evaluated the association of other polymorphisms at TP73 genes with cervical cancer; 5) case reports, case series, letters, comments, reviews, and previous meta-analyses 6) overlapped data or duplication.

Data extraction
Eligible studies containing the required data were selected and the data were organized for further analysis by comprehensive screening.All recruited studies had to be seriously scanned by two individual researchers separately.If there was a dispute between the two researchers, they would reach a consensus by discussing or a third researcher.We extracted the following information from eligible studies: name of the first author, year of publication, country of origin, ethnicity of participants, genotyping methods, source of controls, total numbers of cases and controls, genotyping method, genotypes frequencies of cases and controls, minor allele frequencies (MAFs) and Hardy-Weinberg equilibrium test in control subjects.

Statistical Analysis
An ethical approval was not necessary as this study was a meta-analysis based on previous studies.The strength of association of TP73 G4C14-A4T14 polymorphism with risk of cervical cancer was measured by odds ratios (ORs) with 95% confidence intervals (CIs).The statistical significance of the pooled OR was determined using the Z-test.Pooled estimates of the OR were obtained by calculating a weighted average of OR from each study.The pooled ORs was calculated under all five genetic models, i.e., allele (AT vs. GC), homozygote (AT/AT vs. GC/GC), heterozygote (GC/AT vs. GC/GC), dominant (AT/AT+ GC/AT vs. GC/GC), and recessive (AT/AT vs. GC/AT+GC/GC).A χ2-based Q test was calculated for assessing the heterogeneity among recruited investigations and if the P-value of Q test exceeded 0.05 that meant there was no obvious heterogeneity [33,34].In addition, I 2 -value was used to quantify the proportion of the between study heterogeneity (range of 0 to 100%: I 2 =0-25%, no heterogeneity; I 2 =25-50%, moderate heterogeneity; I 2 = 50-75%, large heterogeneity; I 2 =75-100%, extreme heterogeneity).Random-effect models (DerSimonian-Laird method) would be adopted for analyses if I 2 was >50%.Otherwise, analyses would be conducted with fixed-effect models (Mantel-Haenszel method) [35][36][37].Genotype frequencies of controls for each study using goodness-of-fit test (chi-square) and a p-value less than 0.05 was considered as significant disequilibrium (HWE-violating) [38,39].Sensitivity analysis was conducted by excluding one study at a time to examine the stability of the pooled results [38,40,41].Begg's funnel plot and Egger's linear regression test were applied to assess potential publication bias, in which P<0.05 was considered statistically significant.All of the statistical calculations were performed using Comprehensive Meta-Analysis (CMA) software version 2.0 (Biostat, USA).Two-sided P-values < 0.05 were considered statistically significant.

Results
The selection process of eligible studies is presented in Figure 1.Initially, 316 papers were obtained through publication search in electronic databases and other sources.Then, de-duplicate all the documents we have retrieved, and then remove the documents irrelevant to TP73 G4C14-A4T14 polymorphism or cervical cancer by reading the title and abstract of the article.Therefore, 76 publications were deleted for obvious irrelevance.Finally, a total of 10 case-control studies [42][43][44][45][46][47][48][49]25].with 1804 cervical cancer cases and 2433 healthy controls were included in this meta-analysis.The studies were published between 2004 and 2022, and nine studies were published in English and one in Chinese.These studies were published among Japanese, Hong Kong, Indian, Chinese and Portuguese women.Among these studies, nine studies were conducted among Asians and one study among Caucasian women.Six studies were populationbased (PB) studies and remaining were hospital-based (HB) studies, and used a case-control study design.High resolution melt analysis (HRM) and TaqMan.The genotype, allele and minor allele frequency (MAF) in each study are shown in Table 1.Moreover, the distribution of genotypes in the controls was in agreement with Hardy-Weinberg equilibrium (HWE) for all selected studies, except for one study.

Evidence synthesis
Table 2 listed the main results of the meta-analysis of TP73 G4C14-A4T14 polymorphism and cervical cancer risk.We pooled all the 10 case-control studies together to assess the overall association between this polymorphism and risk of cervical cancer.Pooled analysis did not show a significant association between TP73 G4C14-A4T14 polymorphism and cervical cancer risk under all the five genetic models (Figure 2A-2E).The studies were further stratified based on the ethnicity or country.When subgroup analysis by ethnicity was performed, a significant association between TP73 G4C14-A4T14 polymorphism and cervical cancer risk not found among Asian and Chinese women.The meta-analysis results for the Asian and Chinese women are listed in Table 2.Moreover, in the stratified analysis by source of controls, results revealed that the IL TP73 G4C14-A4T14 polymorphism was associated with cervical cancer risk in HB group under four genetic models, i.e., allele (AT vs. GC: OR= 1.280, 95% CI 1.082-1.516,p=0.004), homozygote (AT/AT vs. GC/GC: OR= 1.652, 95% CI 1.079-2.529,p=0.021), dominant (AT/AT+ GC/AT vs. GC/GC: OR= 1.292, 95% CI 1.050-1.591,p=0.016), and recessive (AT/AT vs. GC/ AT+GC/GC: OR= 1.574, 95% CI 1.037-2.391,p=0.033), but not among PB group.

Heterogeneity test
Based on the results, there was a moderate level of heterogeneity was found between the included studies under two genetic models, i.e., heterozygote (GC/AT vs. GC/GC: I 2 =62.11 and PH=0.005) and dominant (AT/AT+ GC/AT vs. GC/GC: I 2 =55.56 and PH=0.016).Thus, a subgroup analysis was conducted to explore the predefined possible source of heterogeneity.Subgroup analyses showed that ethnicity was not significant source of heterogeneity in this meta-analysis (Table 2).

Sensitivity Analysis
Sensitivity analyses were performed after sequential  this study, results of which indicate that results remain unchanged.

Publication Bias
The Begg's funnel plot and Egger's test were performed to assess the publication bias.The shapes of the Begger's funnel plots did not show any evidence of publication bias, except under dominant (AT/AT+ GC/ AT vs. GC/GC: P Beggs =55.56 and P Eggers =0.016).Thus, we applied the Duval and Tweedie non-parametric ''trim and fill'' method to the publication bias (Figure 3).The results showed that the current meta-analysis with and without ''trim and fill'' did not draw different results, indicating that our results were statistically reliable.Overall, the results suggest this meta-analysis is not affected by publication biases.

Discussion
It is well known that single nucleotide polymorphisms (SNPs) are the most common sources of human genetic variation, which may contribute to an individual's susceptibility to cancer [50,11,28].Here, we have carried out a meta-analysis based on 10 case-control studies with 1804 cases and 2433 controls to obtain a more conclusive result on relationship between TP73 G4C14-A4T14 polymorphism and cervical cancer.To the best of our knowledge, this is so far the most comprehensive meta-analysis on assocition between TP73 G4C14-A4T14 polymorphism and cervical cancer.Pooled analyses indicated that TP73 G4C14-A4T14 polymorphism was not correlated with cervical cancer in overall and ethnicity.Moreover, when we stratified data by source of controls, we noticed that significant associations between G4C14-A4T14 polymorphism and cervical cancer were only existed in controls with hospital-based.[29].In 2017, Liang et al., elucidated the role of TP73 G4C14-A4T14 polymorphism on cervical cancer development by performing a meta-analysis.Their pooled data included a total of 635 cases and 998 control subjects, and showed that TP73 G4C14-A4T14 polymorphism was associated with susceptibility to cervical cancer [51].In the same year, Feng et al., in a meta-analysis based on three studies indicated that this variant at TP73 gene might be associated with an increased risk of cervical cancer [25].
Our meta-analysis has some advantages which these advantages strongly guaranteed a more accurate and reliable conclusion.First, we attempted to find as many published studies by means of various searching approaches, which may enhance the authenticity and reliability of the analysis.Second, the well-designed search and selection method significantly increased the statistical power of this meta-analysis and the number of included studies and sample sizes were greatly enlarged than previous meta-analyses.Third, sensitivity analysis also revealed that the our results were not influenced by any individual study.Finally, the subgroup analysis is 668 sufficient and performed under different subgroups.
In summary, our pooled results revealed that TP73 G4C14-to-A4T14 polymorphism was not associated with an increased risk of cervical cancer globally and among Asian and chinese women.Future studies with large sample size are encouraged to validate our results and to prove the clinical relevance of TP73 G4C14-to-A4T14 polymorphism in the development of cervical cancer.Moreover, further studies examining the effect of genegene and gene-environment interactions may eventually provide a better knowledge.

Figure 3 .
Figure 3. Begg's Funnel Plots of IL TP73 G4C14-A4T14 Polymorphism with Cervical Cancer Risk for Publication bias Test under the Dominant model.Before (blue) and after (red) ''Trim-and-Fill'' method.

Table 1 .
Characteristics of the Studies Included in Meta-Analysis

Table 2 .
Summary Risk Estimates for Association between TP73 Polymorphism and Risk of Cervical Cancer SOC, Source of Controls; HB, Hospital Based; PB, Population Based.