The combined risks of reduced or increased function variants in cell death pathway genes differentially influence cervical cancer risk and herpes simplex virus type 2 infection among black Africans and the Mixed Ancestry population of South Africa

Cervical cancer is the second most common cancer and the highest cause of cancer related deaths across the globe. Approximately 500, 000 new cases of cervical cancer and nearly 300, 000 deaths (52 %) caused by cervical cancer are reported each year making it the third most common cancer in women of all ages worldwide. Cervical cancer is the second most common cancer in southern Africa with approximately 6,500 new incidents and the second highest cause of cancer mortality with around 3000 cervical cancer related deaths (53 %) annually in women of all ages [1]. Persistent infection with oncogenic types of human papillomavirus (HPV) is the main cause of cervical cancer. Many sexually active women become infected with HPV during their life time but 70–90 % of the individuals positive for HPV DNA are able to clear the infection. Only a small percentage of the women with HPV progress to develop cervical cancer over their time [2]. This suggests a possible role of additional risk factors such as, host and viral genetic factors as well as environmental and lifestyle factors in the development of the cancer of the cervix [3].

Herpes simplex virus type 2 (HSV-2) is a common sexually transmitted virus that causes genital infections. HSV-2 has a high prevalence rate throughout the world, more so in the developing nations. Sub-Saharan Africa particularly harbours a high incidence rate with more women being affected than men [4]. HSV-2 in conjunction with HPV has been implicated as a co-factor for cervical cancer [5]. Once inside a human cell, HSV-2 evades the immune system by overcoming the apoptosis of the cell which is triggered by Fas-FasL-Caspase pathway. Fas mediated cytolysis plays an important role in clearing the HSV-2 from genital epithelium [6].

Apoptosis or programmed cell death is one of the most crucial mechanisms of the human immune system to control viral infection in human body [7]. The interaction between cell surface death receptor Fas (FasR), together with Fas ligand (FasL) activates a caspase-cascade leading to activation-induced cell death (AICD) that eventually leads to apoptosis to eliminate the virus-infected cells [8, 9]. Among various caspases, caspase-8 is central to activating the caspase-cascade [10] and plays one of the most important roles in generating apoptotic signals [9]. Therefore functional polymorphisms in the cell death pathway genes encoding FasR, FasL and caspase-8 might interfere with the apoptotic process associated with the spread and clearance of virus-infections inside human body.

The FasR and FasL genes are mapped on chromosome 10q23 and 1q23 respectively. Three functional single nucleotide polymorphisms (SNPs) in these two genes (FasR-1377G > A, FasR-670A > G and FasL-844 T > C) have been well studied. FasR-1377G > A and FasR-670A > G polymorphisms disrupt the SP1 and STAT1 binding sites respectively leading to reduced Fas gene expression and decreased apoptosis [11, 12]. FasL-844 T > C is observed to show higher basal expression that inhibits the apoptotic activity of the FasR-FasL pathway [13]. These three SNPs have been associated with different diseases [11, 13–24] including cervical cancer in different populations [14, 25–30]. The other important factor in the apoptotic pathway, caspase-8 is encoded by CASP8 gene which is mapped on chromosome 2q33. A six nucleotide deletion of AGTAAG at the position 652 in the promoter region of CASP8 gene (CASP8 -652 6 N ins/del) have been well studied. This six nucleotide deletion destroys the SP1-binding site which reduces the caspase-8 expression and thereby decreasing the apoptotic activity of the caspase-cascade [31]. This polymorphism has been associated with different types of diseases [31–41] including cervical cancer in different populations [31, 41].

No studies to date have attempted to find out the combined risks of all the four polymorphisms from FasR (−1377G > A, −670A > G), FasL (−844 T > C) and CASP8 (−652 6 N ins/del) genes on cervical cancer, HPV infection or HSV-2 infection in any population. We therefore re-analysed our data evaluating the combined risks of CASP8 polymorphism in the presence of both FasR and FasL polymorphisms, on cancer of the cervix susceptibility, HPV infection and HSV-2 infection in South African women of black African and mixed-ancestry origin.

The observed genotype frequencies for all the polymorphisms were found to be in Hardy-Weinberg equilibrium (HWE) except for CASP8 -652 6 N ins/del which was slightly out of HWE (P = 0.045) in mixed-ancestry controls [30, 41]. FasR polymorphisms (−1377 and −670) were in strong linkage disequilibrium (LD) (P < 0.05) except in black African controls (P = 0.078) [30]. We have previously reported that the allele and genotype frequency distributions for FasR/FasL differ significantly between the ethnic groups (black African and mixed-ancestry) investigated [30]. Among all the baseline characteristics and secondary outcomes only smoking was found to be a significant confounding factor for cancer of the cervix as mentioned above. Therefore, all analyses were subsequently adjusted for both ethnicity and smoking by including in logistic regression models as covariates.

Comparing the different polymorphisms in smoking positive and HSV-2 positive cases and controls did not show any significant association (results not shown here) either.

The cell death pathway is one of the most essential pathways in human immune system. This pathway is central to and regulates cellular apoptosis which is one of the mechanisms by which the immune system controls infections caused by pathogenic viruses [7]. Disruptions in cell death pathway can lead to impaired apoptotic mechanism which may affect the ability of the immune system to control the spread of virus-infections in cells. Three functional polymorphisms in FasR (FasR-1377, FasR-670) and FasL genes (FasL-844) and one in CASP8 gene (CASP8 -652 6 N ins/del), all in cell death pathway are known to disrupt the apoptotic mechanism by altering the level of expression of Fas and caspase-8 proteins, respectively [11–13, 31]. All the four polymorphisms have been investigated in different types of cancers such as breast [32, 33, 37], bladder [19, 40], lung [20, 23], squamous cell carcinoma [15, 22], colorectal [35], pancreatic [36], different leukaemia [11, 16], cervical [14, 25–31, 41] and several other pathological conditions [11, 13–23, 31–41], all presenting with conflicting results. Previously we have investigated these four polymorphisms in cervical cancer in South African women of black African and mixed-ancestry origin [24, 30, 41]. Comparing women with invasive cervical cancer with age, ethnicity and domicile (urban/rural) matched controls did not show any significant association for FasR and FasL polymorphisms with cervical cancer [30]. However, a secondary analysis only in the control group found a statistically significant association with FasR-1377A (P = 0.008) and FasR-1377/FasR-670 AG haplotype (P = 0.0001) with reduced risk of HSV-2 infection [24]. When CASP8 -652 6 N ins/del polymorphism was compared between cervical cancer cases and controls no significant association with cervical cancer was observed [41]. However, further analysis within only controls showed an association (positive) of CASP8 -652 6 N del/del (P = 0.03) polymorphism with high-risk HPV infection (only among black Africans) and a weak association (positive) with abnormal cytology (P = 0.048) suggesting a susceptible role for this genotype in the development of pre-cancers but not in cervical cancer [41]. The combined risks of all the four polymorphisms of FasR, FasL and CASP8 on cervical cancer, HPV infection, pre-cancerous lesions and HSV-2 infection have not been investigated in any population yet.

Since all the four polymorphisms play vital roles in the cell death pathway leading to altered mechanism of cellular apoptosis it is valuable to investigate the combined risks of CASP8 polymorphism with any (and all) of the FasR/FasL polymorphisms in cervical cancer, pre-cancerous lesions, HPV infection and HSV-2 infection. None of the combinations of the polymorphisms had any significant association with cancer of the cervix in both populations combined and also when stratified for mixed-ancestry population, which is in accordance with our previous individual results with FasR/FasL [30] and CASP8 polymorphisms [41]. However, when stratified among only black Africans a new reduced risk for cervical cancer was observed with CASP8 -652 6 N del combined with FasR-670 A allele (P = 0.019, CPS = −2.34). In the same population, a marginal susceptible risk to cervical cancer was also observed when CASP8 -652 6 N ins was combined with FasR-1377 G allele (P = 0.047, CPS = 1.99). This could be due to different factors. The black Africans are a homogenous population with well defined genetic conformation unlike the mixed-ancestry population that carries a highly mixed genetic makeup. It is also well known that disease susceptibility varies from population to population due to varying level of genetic markers present in various population.

When the combined risks of the polymorphisms were investigated only in the controls, a reduced risk for HSV-2 infection was observed with CASP8 -652 6 N ins + FasR-1377A. This is in consistence with our previous results [24] that showed a reduced risk for HSV-2 infection with FasR-1377A allele alone in the control group as mentioned above. CASP8 promoter polymorphism individually have also been associated with reduced risk of cutaneous melanoma, lung, esophageal, gastric, breast, colorectal and cervical cancer [31, 34] in different population even though in our own population we found an increased risk of this polymorphism with cervical cancer [41].

None of the combinations of the polymorphisms had any significant association with any other parameters in the control group suggesting the combined risks of the polymorphisms are not associated with smoking, high-risk HPV infection and abnormal cytology status in the larger South-African female population.

The present study showed a reduced risk to cervical cancer combining CASP8 -652 6 N del with FasR-670 A allele (P = 0.019, CPS = −2.34) and a marginal susceptible risk to cervical cancer with CASP8 -652 6 N ins + FasR-1377G (P = 0.047, CPS = 1.99), only in black Africans. This study also showed a reduced risk to HSV-2 infection with CASP8 -652 6 N ins + FasR-1377A in the control group. The results must be interpreted cautiously. This was a sub-group analysis of our original study and the results could be influenced due to several possible population or biological confounding factors that are hitherto unrecognized. Future studies on larger cohorts are warranted to further confirm these findings and to elucidate the mechanisms involved.