Impact of CD40 gene polymorphisms on the risk of cervical squamous cell carcinoma: a case-control study

Background Cervical cancer is the fourth most common cancer among women worldwide. Genome-wide association studies have revealed multiple susceptible genes and their polymorphisms for cervical cancer risk. Therefore, we aimed to investigate the correlation between single nucleotide polymorphisms (SNPs) of the CD40 gene and susceptibility to cervical squamous cell carcinoma (CSCC) in a population from the northeastern Han Chinese population. Methods The three SNPs (rs1800686, rs3765459, and rs4810485) of the CD40 gene were analyzed by multiplex polymerase chain reaction (PCR) combined with next-generation sequencing methods in 421 patients with CSCC, 594 patients with high-grade squamous intraepithelial lesions (HSIL), and 504 healthy females. Multivariate logistic regression analysis was used to analyze the potential relationship between CD40 gene polymorphisms and CSCC, or HSIL. Results Our research results showed the AA genotype of rs1800686 had a protective effect on CSCC in comparison to the GG genotype and AG+GG genotypes (AA vs. GG: p = 0.0389 and AA vs. AG+GG: p = 0.0280, respectively). After FDR correction, the results were still statistically significant (p = 0.0389 and p = 0.0389, respectively). Similarly, rs3765459 showed a reduced risk association for CSCC in the codominant and recessive models (AA vs. GG: p = 0.0286 and AA vs. AG+GG: p = 0.0222, respectively). Significant differences remained after FDR correction (p = 0.0286 and p = 0.0286, respectively). However, these differences were no longer significant after the Bonferroni correction. In addition, the genotypes for the rs4810485 polymorphisms were associated with parity of the patients with CSCC. The genotypes for the rs3765459 polymorphisms were significantly correlated with the D-dimer of the patients with CSCC. The 3 SNPs genotypes of the CD40 gene were closely related to the squamous cell carcinoma antigen (SCC) of the patients with HSIL. Conclusions The CD40 gene may play a role in the occurrence and development of CSCC. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-023-11367-3.


Introduction
Cervical cancer is one of the leading causes of cancerrelated deaths in women.In 2020, it is estimated that 604,127 new cervical cancer cases and 341,831 deaths occurred worldwide [1,2].However, in China, there are about 109,741 new cases of cervical cancer and 59,060 deaths.China is still one of the countries with the highest incidence and mortality of cervical cancer [3].There are studies to indicate that risk factors including various sexual partners, early pregnancy, multiple pregnancies within a limited period of time, oral contraceptive pills, smoking habits, and limited access to health care play a role in cervical cancer development [4,5].Persistent human papillomavirus (HPV) infection is the most important risk factor for cervical cancer and squamous intraepithelial lesions (SIL) [6,7].However, not all patients infected with HPV will develop cervical cancer, and nearly 70-90% of patients can recover from this infection [8].More importantly, studies revealed that genetic heritability is one of the most common intrinsic factors that increase the probability of developing cervical cancer by almost 27% [9].According to an Iranian study, cervical cancer susceptibility is closely related to the interaction of HPV infection and host genetic factors.[10].
CD40 is composed of 277 amino acids and has a molecular weight of 40-50 kDa, which belongs to the tumor necrosis factor receptor (TNFR) superfamily.It is encoded by a gene located on chromosome 20q11-13 and comprises 8 introns and 9 exons [22][23][24].CD40 is expressed in a variety of cell types, including normal B lymphocytes, macrophages, endothelial cells, and dendritic cells [25].In addition to being expressed in normal lymphocytes, CD40 is also present in malignant hematopoietic cells, including leukemias, lymphomas, and solid cancers [26].Some studies have pointed out that CD40 expression is related to the increased risk of angiogenesis in some tumors [27].
Hill SC et al. [28] found that the expression of CD40 on HPV-infected lesions and advanced cervical squamous cell carcinoma (CSCC) is significantly higher than that of normal cervical tissues.These findings were further confirmed by Huang Q et al. [29].In vivo studies have shown that the expression of CD40 is correlated with HPV positivity, vascular endothelial growth factor (VEGF) expression, and microvessel density [27,30].In recent years, various studies have shown that CD40 gene polymorphisms are related to some malignant tumors, such as non-Hodgkin's lymphoma [31], breast cancer [32], and lung cancer [33,34].However, there were few reports on the relationship between the CD40 gene and cervical cancer [35,36].Therefore, this study aimed to explore the possible impact of CD40 gene polymorphisms on the occurrence of CSCC and high-grade squamous intraepithelial lesions (HSIL) in the northeastern Han Chinese population.

Study subjects
The case groups of this case-control study were continuously recruited from the same center (Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China) from September 2014 to October 2018.The case groups included 421 patients with CSCC and 594 patients with HSIL.101 patients with CSCC and 362 patients with HSIL agreed to obtain cervical brushing exfoliated cells to do HR-HPV detection.92 were found to be HPV positive and 9 were HPV negative in the CSCC patients, and 341 were found to be HPV positive and 21 were HPV negative in the HSIL patients.Further stratification by HPV sub-type shows that 51 samples were HPV 16 and/or 18 in the CSCC patients and 132 samples were HPV 16 and/or 18 in the HSIL patients.All patients were confirmed by pathology experts of the Second Affiliated Hospital of Harbin Medical University.All the women with benign cervical benign lesions, cervical benign tumors, other cervical malignant tumors, and cervical lesions who had a history of preoperative radiotherapy and chemotherapy and other cancers were excluded from the study.
The 504 cases in the control group were all those who went to the physical examination center of the hospital for routine health examinations at the same time.The control group had no history of cancer or family history of cancer, and the results of the Thinprep cytologic test (TCT) were normal.The results of the routine blood examination and other biochemical indicators were within the reference value range.Patients with any gynecological diseases, a history of gynecological diseases, gynecological surgery, hypoimmunity and immunological diseases, skin or genital condyloma, and other cancers were excluded.
All of the cases and controls agreed with the ethics of the study and signed informed consent, which was approved by the ethics committee of the Second Affiliated Hospital of Harbin Medical University.

Selection of SNPs
Based on the characteristics of the East Asian population in the dbSNP database (minor allele frequency [MAF] > 15%) and tracking the relevant literature [35,36], we selected four SNPs of the CD40 gene.They were rs1800686 (G > A), rs1883832 (T > C), rs3765459 (G > A), and rs4810485 (T > G).Rs1800686 was located in the 508-bp upstream region, rs1883832 was located in the Kozak consensus sequence of the 5´-UTR, rs3765459 was located in the intron 8 of the CD40 gene, and rs4810485 was located in the intron 1 of the CD40 gene.

Statistics
The SPSS software package, version 24.0 (SPSS, Institute Inc.), was used to analyze the clinical characteristics between cases and controls.The genotypic distribution of each SNP in control subjects was tested for departure from Hardy-Weinberg equilibrium (HWE) using an exact test.There were six inheritance models, including the allele model, the codominant model, the dominant model, the recessive model, the overdominant model, and the genotypic model.The 95% confidence interval (CI) and odds ratio (OR) were calculated through multiple logistic regression analysis to evaluate the potential association between CD40 gene polymorphisms and CSCC or HSIL.Empirical p values were calculated by 10,000 permutation tests using the Max (T) permutation procedure implemented in PLINK [39].We used the false discovery rate (FDR) (p < 0.05 was statistically significant) and the Bonferroni correction (p < 0.016 (0.05/3) was statistically significant) to control for multiple hypothesis testing.SHEsis software was used to implement the Haplotype analysis between groups [40].For all statistical analyses, p < 0.05 was considered statistically significant.

Quality control and SNP genotype
All of the tested SNPs were in agreement with the HWE in the control population of this study (p > 0.05) except for rs1883832 (p = 0.016), and therefore it was excluded from the analysis (Supplementary Table S1).In addition, quality control was set up for the genotypes of several samples.The genotype calling rate in 73 quality control samples was 98.50%, which fully improved the reliability of the follow-up research results.

Clinical characteristics of the study population
The clinical characteristics of the CSCC group, the HSIL group, and the control group were shown in Table 1.The ages of the patients in the CSCC group and the HSIL group were significantly higher than those in the control group.The ages, menarche ages, fibrinogen (FIB), D-dimer, and squamous cell carcinoma antigen (SCC) of the patients with CSCC were significantly higher than those with HSIL.While the prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) of the patients with CSCC were significantly lower than those with HSIL.The percentages of smoking and amenorrhea were higher in the CSCC group than those in the HSIL group.In addition, there was a significant difference in parity in the CSCC group compared with the HSIL group.

Genotype and allele association analysis
The genotype and allele frequencies of each SNP in the control group and the CSCC group were summarized in Table 2.We found that compared with the control group, rs1800686 was significantly associated with CSCC in the codominant model and the recessive model (AA vs. GG: p = 0.0389 and AA vs. AG + GG: p = 0.0280, respectively).After FDR correction, the results were still statistically significant (p = 0.0389 and p = 0.0389, respectively).There were obvious differences in the genotype frequencies of rs3765459 between the CSCC group and the control group in the codominant model and the recessive model (AA vs. GG: p = 0.0286 and AA vs. AG + GG: p = 0.0222, respectively).Significant differences remained after FDR correction (p = 0.0286 and p = 0.0286, respectively).After 10,000 permutations, the results were still statistically significant (p < 0.05, all).The consistency of 10,000 permutations further supports the reliability of our results.However, these associations did not survive the Bonferroni correction.
Further stratification by HPV status showed the CD40 gene SNPs (rs1800686, rs3765459, and rs4810485) were not associated with the risk of HPV infection status (HPV positive, HPV 16 and/or 18, and HPV negative) in the patients with CSCC when compared to controls.Similarly, there were also no significant correlations between the CD40 gene SNPs and the risk of HPV infection status in the patients with HSIL, as shown in Table 3 (p > 0.05, all).
The genotype and allele frequencies of each SNP in the control group and the HSIL group were summarized in Supplementary Table S2.The results showed that the genotype and allele frequencies of rs1800686, rs3765459, and rs4810485 were not significantly different in the HSIL group compared with the control group (p > 0.05, all).After 10,000 permutations, the results were still not statistically significant (p > 0.05, all).
The genotype and allele frequencies of each SNP in the HSIL group and the CSCC group were analyzed, as shown in Supplementary Table S3.No differences for alleles and genotypes were identified for the comparison of three SNPs (rs1800686, rs3765459, and rs4810485) between the CSCC group and the HSIL group.(p > 0.05, all).After 10,000 permutations, there were still no obvious correlation (p > 0.05, all).
The 3 SNPs genotypes of the CD40 gene in patients with CSCC and HSIL by clinical features were summarized in Table 4.We found that in the CSCC group, the genotypes for the rs4810485 polymorphisms were associated with parity (p = 0.0378), and the genotypes for the rs3765459 polymorphisms were significantly correlated with D-dimer (p = 0.0346).The genotypes for the rs1800686, rs3765459, and rs4810485 polymorphisms were closely related to SCC in the HSIL group (p = 0.0280, p = 0.0199, and p = 0.0042, respectively).
Tables 5 and 6 summarized the genotypic associations of three SNPs with risk factors related to CSCC and HSIL, respectively.For rs4810485 in the CSCC group, the parity between patients with the T allele and the G allele was different (p = 0.0138), and the parity between patients with the TT + TG genotype and the GG genotype was different (p = 0.0338).For rs3765459 in the CSCC group, the D-dimer between patients with the AA genotype and the AG + GG genotype was different (p = 0.0326).In the HSIL group, rs4810485 was significantly associated with parity in the dominant model and the genotypic model (p = 0.0237 and p = 0.0417, respectively).The 3 SNPs genotypes of the CD40 gene were significantly correlated with SCC in the HSIL group, and the allele, codominant, dominant, and genotypic models confirmed the credibility of that conclusion, respectively (p < 0.05, all).

Haplotype analysis
Considering the linkage disequilibrium of genes, haplotype analysis for CD40 gene polymorphisms was performed (Supplementary Table S4).Results showed that there were no significant differences between the CSCC group and the control group, the HSIL group and the control group, and the CSCC group and the HSIL group, respectively (p > 0.05, all).

Discussion
The incidence rate of cervical cancer has been kept high in China.The morbidity age has shown a younger trend in recent years.Cervical cancer susceptibility is closely linked to host genetic factors.As a host genetic factor, genetic variation at susceptibility loci may have a significant impact on the risk of cervical cancer [41].Previous studies on CD40 gene polymorphisms and cervical cancer have been very limited.The association of CD40 gene polymorphisms with the risk of CSCC development in the northeastern Han Chinese population was first investigated in our study.With further study of the CD40 gene, it may be a useful biomarker for evaluating the risk of developing cervical cancer and may also be used as a target for therapy.These two roles of CD40-promoting immune responses and angiogenesis-have opposing effects on the development of a tumor.Immune responses that are tailored to the tumor prevent tumor growth, whereas enhanced angiogenesis promotes tumor growth by giving the tumor cells nutrition and a path to distant organs [42].Cervical cancer is more dependent on angiogenesis than other cancers [43].Angiogenesis, the development of new blood vessels, is essential for the initiation of tumors, their growth, and metastasis [44].The process of tumor-related angiogenesis is regulated by various pro-angiogenic factors, such as VEGF and interleukin 6 (IL-6), which are the dominant regulators of the proliferation of endothelial cells and the formation of new blood vessels [45,46].The activation of CD40 can upregulate VEGF, IL-6, and other factors through MAPK, PI3K/Akt, and other signal transduction pathways and then promote tumor angiogenesis by inhibiting endothelial cell apoptosis and promoting the growth of vascular endothelial cells, so as to promote the occurrence and development of cervical cancer [44,47,48].Zhang et al. [49] reported that some SNPs in the coding region can change the amino acid sequence of proteins, while some SNPs in the non-coding region may still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA.CD40 gene SNPs can affect the transcription and translation efficiency of CD40, which can determine the effective expression of CD40.
In this study, we investigated the potential effects of rs1800686, rs3765459, and rs4810485 polymorphisms on the occurrence of CSCC and HSIL.Our results showed that rs1800686 and rs3765459 were associated with the risk of CSCC.To date, no study has discussed the association of the rs1800686, rs3765459, and rs4810485 polymorphisms with CSCC risk.Rs1800686 and rs3765459 may be closely related to the pathological classification of cervical cancer.The AA genotype of rs1800686 had a protective effect on CSCC in comparison to the AG + GG genotypes in the recessive model, and these data were statistically significant (CSCC vs. control: OR = 0.552, 95% CI = 0.325-0.938,p = 0.0280).Similarly, rs3765459 showed a reduced risk association for CSCC in the recessive model (CSCC vs. control: OR = 0.527, 95% CI = 0.304-0.912,p = 0.022).The same results appeared in the codominant model (AA vs. GG).Even after 10,000 permutations and FDR correction, the associations held (p < 0.05).Although the interaction effect was not significant after Bonferroni correction, it also suggested a high potential interaction effect as Bonferroni correction was a conservative and stringent correction test.Studies in larger patient cohorts would contribute to elucidating this interaction.Rs1800686 is located in the 5' near-gene region of the CD40 gene, where mutations can modulate CD40 promoter activity.Rs3765459 is located in intron 8 of the CD40 gene, where mutations can lead to abnormal splicing.Therefore, the AA genotypes of rs1800686 and rs3765459 may inhibit CD40 expression.The decrease in CD40 expression levels can down-regulate VEGF, IL-6, and other factors, reducing the risk of CSCC by promoting endothelial cell apoptosis, inhibiting the growth of vascular endothelial cells, and inhibiting tumor angiogenesis.Shuang et al. [32] demonstrated that the rs1800686 and rs3765459 AA genotypes may increase the risk of breast cancer.They observed that the rs1800686 and rs3765459 AA genotypes were higher in patients  with breast cancer compared with the control group.A possible explanation for the inconsistencies between the rs1800686 and rs3765459 polymorphisms and different types of cancer is that these polymorphisms might have different genetic effects on different diseases.Similar results can be seen in the studies of Chen et al. [50] and Yi et al. [51], in which they found that the impacts of genetic polymorphisms may be different according to the types of cancer.Yan et al. [36] reported the association between CD40 gene polymorphisms and cervical cancer in the Yunnan Han population, pointing out that there was no significant association between rs4810485 and cervical cancer.Purushotham Krishnappa et al. [35] pointed out that rs1800686 and rs4810485 were not related to the occurrence of cervical cancer in the Malaysian population; however, for rs3765459, the G allele increased the risk of cervical cancer (p = 0.010).In our study, we discovered no significant associations between the three SNPs of the CD40 gene and the risk of HSIL.The same results appeared in the comparison of CSCC and HSIL women.The reasons for these negative results remain unknown, but two possibilities should be considered.First, it may be because of genetic trait differences; as we know, genetic polymorphisms in human genes are distinct in different ethnicities, populations, and geographic regions.In addition, even though we might find a potential link between the disease-causing gene and the disease itself, cervical cancer is a multi-factorial disease, and individual exposure to diverse environmental factors and genetic backgrounds may cause different results.
Rs4810485 had no significant correlation with the genetic susceptibility of CSCC (p > 0.05).However, in the CSCC group, the genotypes for the rs4810485 polymorphisms were associated with parity.The genotypes for the rs3765459 polymorphisms were significantly correlated with the D-dimer of the patients with CSCC.The 3 SNPs genotypes of the CD40 gene were significantly correlated with the SCC of the patients with HSIL, suggesting that the CD40 gene polymorphisms genotypes may affect the level of SCC in the patients with HSIL, and further researches are needed.
Studies conducted in vivo and in vitro found that, in contrast to healthy cervical epithelium and non-cancerous keratinocyte cell lines transfected with HPV-DNA, oncogenic HPV positive cervical cancer and HPV-positive cervical cancer cell lines expressed CD40 at high levels [28,52].This disparity may explain why HPV infection persists by evading the immune system.The reason cervical cancer has a bad prognosis when the HPV-18 genotype is present may be due to this immune evasion [53].Unfortunately, we did not find an interaction between the CD40 gene SNPs (rs1800686, rs3765459, and rs4810485) and the risk of HPV infection status (HPV positive, HPV 16 and/or 18, and HPV negative) in CSCC patients; more samples are required in future studies.
Since CD40 gene polymorphisms may correlate with CSCC susceptibility, it is convinced that clarification of how polymorphisms of the CD40 gene have affected mRNA expression and protein expression and how the protein performs in the pathogenesis of the disease is meaningful and helpful.It is widely accepted that mRNA may be regulated by SNPs [54].Therefore, the mutation of the CD40 gene may affect the expression of CD40 and play an anti-tumor role.In our study, we did not discuss the association between these polymorphisms and CD40 expression in CSCC and HSIL.Functional studies in the future are necessary to elucidate how polymorphisms regulate CD40 expression, especially their role in cervical cancer pathogenesis.
There were some limitations in the present study.First of all, we found the CD40 gene SNPs (rs1800686, rs3765459, and rs4810485) with specific HPV genotypes did not influence the patients' susceptibility towards the developments of HSIL or CSCC; however, the conclusion is to proceed with caution because only 463 patients (45.6%) underwent HR-HPV detection.Second, the association between CD40 gene polymorphism and its expression level was not investigated, which many previous reports have confirmed.Third, cervical cancer is a complex disease affected by multiple genes, but we only studied the limited sites of the CD40 gene, and other functional genes and SNPs have not been explored.Therefore, the role of CD40 and other genes in the occurrence and development of cervical cancer remains to be further studied.

Conclusion
In conclusion, the study presented is the first to analyze the CD40 gene polymorphisms in a northeastern Han Chinese population with CSCC and HSIL, which revealed that rs1800686 and rs3765459 were significantly associated with the decreased risk of CSCC.In addition, the genotypes for the rs4810485 polymorphisms were associated with parity of the patients with CSCC.The genotypes for the rs3765459 polymorphisms were significantly correlated with the D-dimer of the patients with CSCC.The 3 SNPs genotypes of the CD40 gene were closely related to the SCC of the patients with HSIL.Despite the fact that interactions became insignificant after Bonferroni correction, these findings provide novel evidence for risk assessment and personalized intervention in Chinese women with CSCC.

Table 4
1.173(0.265-5.196)CSCC, cervical squamous cell carcinoma; HSIL, high-grade squamous intraepithelial lesions; p, the p value was adjusted by age; OR, Odds Ratio for the recessive model with controls as reference group; CI, confidence interval p < 0.05 was considered as statistically significant (bold) Analysis of 3 SNPs genotypes of the CD40 gene in patients with CSCC and HSIL by clinical features squamous cell carcinoma; HSIL, high-grade squamous intraepithelial lesions; PT, prothrombin time; APTT, activated partial thromboplastin time; TT, thrombin time; FIB, fibrinogen; SCC, squamous cell carcinoma antigen; p, p value p < 0.05 was considered as statistically significant (bold) CSCC, cervical squamous cell carcinoma; PT, prothrombin time; APTT, activated partial thromboplastin time; TT, thrombin time; FIB, fibrinogen; SCC, squamous cell carcinoma antigen; p, p value p < 0.05 was considered as statistically significant (bold)

Table 1
Clinical characteristics of all subjects in this study CSCC, cervical squamous cell carcinoma; HSIL, high-grade squamous intraepithelial lesions; PT, prothrombin time; APTT, activated partial thromboplastin time; TT, thrombin time; FIB, fibrinogen; SCC, squamous cell carcinoma antigen; p, p value a Comparison between CSCC and controls b Comparison between HSIL and controls c Comparison between CSCC and HSIL.p < 0.05 was considered as statistically significant (bold)

Table 2
Distribution of 3 SNPs genotypes and alleles of the CD40 gene in patients with CSCC and controls CSCC, cervical squamous cell carcinoma; p, p value a Comparison between CSCC and controls b Calculated using multivariate logistic regression analysis c Adjusted by age d Adjusted by age and then calculated using 10,000 permutations for each model to correct the multiple test p < 0.05 was considered as statistically significant (bold)