IL4, IL13, GSTM1 and T1 variants and susceptibility to Schistosomiasis and associated bladder pathologies in Eggua, Nigeria

Failure of the human host to elicit adequate immune responses to the adult Schistosoma haematobium worm and continuous strong inflammatory responses to the eggs have been the main causes of bladder pathology in chronic Schistosomiasis. Identification of susceptibility biomarkers for schistosomiasisassociated bladder pathology is necessary in order to detect genetic factors responsible for the infection and spread of the disease. The aim of this study was to identify candidate-biomarkers for susceptibility to schistosomiasis and its associated pathologies. A total of 371 adult participants, comprising 130 males and 241 females from Eggua community, Ogun State, Nigeria, were randomly recruited into a cross sectional study from August 2012 to May 2014. They were screened for S. haematobium ova and bladder pathologies by microscopy and ultrasonography, respectively. Human host susceptibility to schistosomiasis and its associated bladder pathologies were determined by PCR genotyping of Interleukin (IL4 and IL13) genes, and glutathione-S-transferase (GSTT1 and GSTM1) genes. The overall prevalence of S. haematobium in the population was 29.3% (108/369). Bladder pathologies were observed in 32.3% (117/362) of the population. Polymorphisms in IL 4-590 and IL 13-1055 were observed in 24.1% and 9.3% schistosomiasis cases, respectively. The IL 13-1055 polymorphism did not indicate susceptibility to schistosomiasis in males (OR 0.7, 95% CI 0.3-2.1) but a slight risk was found in females (OR 1.1, 95% CI 0.7-1.7). Participants with GSTM1 and GSTT1 polymorphisms expressed elevated risks of bladder pathologies (OR = 4.3, 95% CI 2.0 9.2 and OR = 4.2, 95% CI 1.5 – 12.0, respectively), with the pathology and schistosomiasis group having more GST polymorphisms than bladder pathologies.


Introduction
Not less than 700 million persons are globally estimated to be infected with schistosomiasis and at least 206.4 million people required preventive treatment in 2016 (Yassir et. al., 2017;WHO, 2018). Nigeria is one of the most severely affected countries in Africa, with 29 million cases as at 2010 (Adenowo et. al., 2015). Indeed, a recent meta-analysis of schistosomiasis prevalence studies in Nigeria shows that the prevalence in Nigeria varied from 2-82.5% with a pooled prevalence of 34.7% (Abdulkadir et. al., 2017). Chronic morbidity during urinary schistosomiasis develops as a result of schistosome eggs that lodge in the bladder causing extensive tissue damage (Wilson et. al., 2007). The response to egg deposition could lead to calcification of the urinary bladder, infection, stone formation and mucosal proliferation (Zaghloul, 2012). Chronic infection with Schistosoma haematobium has been reported as a possible risk factor in the aetiology of bladder cancer (European Association of Urology, 2016;Onile et. al., 2016).
The intensity of schistosomiasis is reportedly influenced by a Schistosoma mansoni 1 (SM1) gene that is mapped to a region of chromosome 5 in the 5q31-q33 that codes for proteins that are associated with regulation of the Th2 response such as cytokines like Interleukin (IL)-3, IL-4, IL-5, IL-9, and IL-13 and Immunoglobulin (Ig) E (Dessein et. al., 2001;Gatlin et. al., 2009;Mbanefo et. al., 2014). Gatlin et. al., (2009) in a univariate analysis reported a significant correlation between resistance to reinfection with Schistosoma mansoni and the heterozygous (C/T) IL-13 -1055 genotype, any T allele in the Interferon (IFN)-c +874 genotype, and the heterozygous (C/T) in the IL-4 -590 genotype. Analysis of S. haematobium infection in Mali revealed that polymorphisms in the IL13 gene promoter at −1055 and −591 were associated with the rate of Schistosoma infection. The IL-13 alleles −1055C and −591A were preferentially transmitted to children with 10% highest infection rate, whereas −1055T was associated with the lowest infection levels (Kouriba et. al., 2005).
It is thought that schistosomes redirect the early cell-mediated immunity in infection to cytokines that would favour establishment of the infection. S. haematobium infectionassociated bladder damage is closely linked to immune reaction to the parasite deposited egg in the bladder which eventually induces chronic inflammation-related granulomatous injury. We propose that infected and re-infected persons are genetically directed to produce inappropriate cytokine responses that lead to the establishment of chronic infections.
Decrease in the activity of the carcinogenmetabolizing enzyme glutathione-S-transferase (GST) in human bladder cancer tissues have been associated with S. haematobium infection (Sheweita et. al., 2004). Somali et. al., (2003 and Yajie et. al., (2016) found that deficiencies in the GSTT1 gene confer an increased risk of bladder cancer. The inhibition of GST activity may enhance the effect of many environmental carcinogens such as N-nitrosamines, thereby reducing the capacity of detoxifying many endogenous compounds in the bladder (Yajie et. al., 2016).
In Nigeria, most studies have focused on the epidemiology of S. haematobium infection (Adenowo et. al., 2015 andAbdulkadir et. al., 2017), with limited information about the morbidity resulting from urinary schistosomiasis in adults (Nmorsi et. al., 2007;Ekwunife et. al., 2009;Onile et. al., 2016) while information on schistosomiasis susceptibility and resistance factors are lacking. This study was aimed at genotyping polymorphisms in Interleukin (IL-4 and IL-13) and GST (M1 and T1) genes and determining their association with genetic susceptibility to schistosomiasis and its pathologies.

Materials and Methods
The study design, area, methods and participants have been described previously (Onile et. al., 2016;Anumudu et. al., 2019). Briefly, a cross-sectional study was carried out from August 2012 to May 2014 in Eggua ( Figure   1), a rural agrarian community where S. haematobium infections are prevalent. Children and participants with human immunodeficiency virus (HIV) were excluded from the study because the main objective was to determine the effect of chronic urinary schistosomiasis on the health of the adults within the community. A total of 371 participants between the ages of 30 and >60 years were drawn from the village as they convened at the community health Centre following the initial announcement by the community leader. This study was a continuation of previously published work by Onile et. al., (2016  3.

Parasitology
Study volunteers provided blood (2 mL) by venipuncture and urine (for egg count) specimens. The urine samples were collected between 10:00am and 2:00pm for maximum egg yield and were processed for parasitological examination and egg count (as previously described (Onile et. al., 2017;Adebayo et. al., 2017;Olayinka et. al., 2020). The urine sediment (obtained by centrifuging 10ml of sample at 5000 rpm for 5 minutes) was examined microscopically to identify Schistosoma haematobium ova characterised by the presence of a terminal spine. The eggs were counted and the intensity of infection classified as light if ≤50 eggs/10 mL of urine and heavy if >50 egg/10 mL urine were present (Nmorsi et. al., 2007;Onile et. al., 2016). In addition to microscopy, detection of macro and microhaematuria (urinalysis) for schistosomiasis was also done as described in Onile et. al., (2017) 4.

Ultrasound and Pathology
A blind ultrasound examination was carried out by a radiologist for each participant in the study and classification of bladder pathologies was as previously described (Onile et. al., 2016).

5.
DNA Extraction and Purification DNA was purified from the blood samples using Thermos Scientific GeneJET Whole Blood Genomic DNA purification kit (Lithuania), following the manufacturer's instructions. DNA concentration was measured by spectrophotometry. Aliquots (10 µL) of all samples was taken and subsequently adjusted to provide standard stock solutions of 20ng/µL. The A280/A260 ratio was estimated to provide an indication of the quality of the sample. Only samples that provided a yield of >20 ng/µL and A280/A260 ratio >1.8 and <1.95 were included for genotyping analysis.

1.
Genotyping for IL 4 and IL 13 Genes PCR for IL-13 and IL-4 Single nucleotide polymorhisms (SNPs) was performed for 108 samples from the participants that tested positive for urinary schistosomiasis by microscopy, using modifications from the original methods (references in Table 1), which enhanced optimal amplification. PCR for IL-13 -1055 C/T was conducted in a 25 µl reaction mixture containing 100 ng DNA.
Initial denaturation was performed at 95 o C for 3 min followed by 30 cycles of PCR with the following conditions: 95 o C for 30 sec, 62 o C for 30 sec for annealing, 72 o C for 1min for amplification, and a final extension at 72 o C for 3 min. This was done using the Hot Start Taq 2X Master Mix (M0496L, BioLabs, New England).
PCR for IL-13 -591 A/G was conducted in a 25 µL reaction mixture containing 100 ng DNA and was done using the Hot Start Taq 2X Master Mix (M0496L, BioLabs, New England). Initial denaturation was performed at 94 o C for 5 min followed by 30 cycles of PCR with the following conditions: 94 o C for 1 min, 61 o C for 45 sec for annealing, 72 o C for 45 sec for amplification, and a final extension at 72 o C for 3 min.
PCR for IL-13 +130 G/A was conducted in a 25 µL reaction mixture containing 100 ng DNA and the PCR reaction was done using the Hot Start Taq 2X Master Mix (M0496L, BioLabs, New England). Initial denaturation was performed at 94 o C for 5 min followed by 34 cycles of PCR with the following conditions: 94 o C for 1 min, 60 o C for 45 sec for annealing, 72 o C for 45 sec for amplification, and a final extension at 72 o C for 3 min.
PCR for IL-4 -590 C/T was conducted in a 25µL reaction mixture containing 100 ng DNA, using the Hot Start Taq 2X PCR Master Mix (M0496L, BioLabs, New England). Initial denaturation was performed at 95 o C for 5 min followed by 30 cycles of PCR with the following conditions: 94 o C for 30 sec, 59 o C for 30 sec for annealing, 72 o C for 30 sec for amplification, and a final extension at 72 o C for 3 min. 21 Purified PCR amplicons were further sent for sequencing (Fig. 3).

GSTM1 and GSTT1 Genotyping
A total of 219 samples were genotyped for GST polymorphisms; these included 118 urinary tract pathology and 101 control cases. The control cases were randomly selected from among participants. The genotypes GSTM1-null and GSTT1-null, produced no GSTM1 and GSTT1 protein and consequently completely lack GSTM1 and GSTT1 enzymatic activity (Matic et. al., 2016).

Genotyping for GSTM1 Polymorphism
This reaction was used to distinguish between GSTM1-active and GSTM1-null individuals. Two primers (G1 and G2) were used to amplify GSTM1 complementary DNA sequences (Brockmoller et. al., 2000). The G1 and G2 amplified a 500-base pair (bp) product specific for the GSTM1 gene. The presence of a GSTM1-null polymorphism was concluded from the absence of the specific 500-base pair fragment. 5μL DNA was amplified in a final volume of 25µL amplification reaction of 30 cycles, using the Hot Start Taq 2X Master Mix (M0496L, BioLabs, New England) according to the manufacturer's instructions.

2.
Genotyping for GSTT1 Polymorphism The 25 µL volume amplification reaction for GSTT1 gene was done in a duplex PCR assay with a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers used as internal control, for determination of GSTT1 and GSTT1-null genotype. A 5 μL DNA template was amplified in a final volume of 25 µL amplification reaction of 30 cycles, using the Hot Start Taq 2X Master Mix (M0496L, BioLabs, New England) according to the manufacturer's instructions.

Statistical Analysis
Statistical analysis was done using SPSS version 20.0 to determine the χ 2 and Odds ratio with P value set at <0.05.

1.
Prevalence of urinary schistosomiasis and bladder pathology A total of 371 participants (130 males and 241 females) aged 30-90 years were recruited for the study. While 369 were eventually screened for S. haematobium infection and 362 for bladder pathologies, some of the volunteers were excluded from the study using the exclusion criteria ( Table 2). The mean age of the participants was 48.6 ±0.6 years. The overall prevalence of S. haematobium in the sampled population was 29.3% (108/369), 42 (11.4%) in males and 66 (17.9%) in females ( Table 2). The Eggua community had the highest prevalence of infection 58 (16.0%), while Ibeku 9 (2.5%) had the least prevalence of infection (Table 2). Bladder pathologies were observed in 32.3% (117/362) of sampled population (Table 2).  (Table 3).  GST gene polymorphisms in susceptibility to schistosomiasis-associated bladder pathologies Amplification of GSTM1 and GSTT1 genes yielded 500bp and 400bp fragments, respectively, among the sampled population. The GSTMI amplicons were found in 36 (30.8%) pathology cases and 7 (7.1%) control samples while GSTT1 was found in 20 (17.1%) pathology cases and 4 (4.1%) control samples.

Confidence Interval, b odds ratio, ref-Reference
The risks associated with combined activities of the GSTM1 and GSTT1 genes were also examined and a higher risk of having bladder pathology was found in participants with one of either of the active genes (OR= 3.4, 95% CI 1.9-6.2) compared to those having both null genotypes (OR= 0.6, 95% CI 0.6-0.8). There was no distinctive risk found in having a high intensity of pathology with any of the GST genotypes when the combined variant and active genotype were considered, but there was a slight risk of mild bladder pathology in the participants in the presence of both inactive variants (both null genotype) when compared to those with either one alone (OR= 1.2, 95% CI 0.7-2.4).
There was a slight risk of bladder pathology among the smokers and S. haematobiuminfected participants with GSTT1 and GSTM1 null polymorphisms (Supplementary Table 1).  Table 2).

Infection and Pathology
The overall prevalence (29.3%) in this study was higher than several reported cases in Nigeria (Nmorsi et. al., 2007;Dawet et. al., 2012;Ugochukwu et. al., 2013;Olayinka et. al., 2020). This could further explain the continuous and long-time exposure to infection and possibly contribute to S. haematobium subtle morbidity.
The consistently higher frequency of light intensity of S. haematobium infection observed in this study as previously seen in Onile et. al., (2016) could be explained by some possible level of gradual development of acquired protected immunity by adults in this community due to chronic exposure to schistosomiasis (Barbosa et. al., 2006). Also, according to the WHO Expert Committee (WHO, 2002), prevalence and intensity of infection have been directly related to the patterns of variation with age with a reported decline in adults, an assertion supported by studies in Nigerian populations (Pukuma and Musa, 2007;Dawaki et. al., 2016). Pearce and MacDonald, (2002) also reported an obvious pattern of agedependent intensity of infection where those who are below the age of puberty carry the most parasites, and those in older age groups are generally less heavily infected.
The association between S. haematobium infection and the presence of urinary tract abnormalities was consistent in our studies and similar to other previous reports (Nmorsi et. al., 2007;Ekwunife et. al., 2009;Onile et. al., 2016;Serieye et. al., 1996). Also, S. haematobium infection has been associated with a two to tenfold increase in the risk of bladder squamous cell carcinoma, as well as being a potential cause of kidney damage (Driguez et. al., 2016).
In fact, in some of the regions where S. haematobium is endemic, bladder cancer has been marked as the most common cancer in men and the second in women, just behind breast cancer, and accounts for as much as 30% of all cancer cases (Botelho et. al., 2010). A meta-analysis of the estimated disease burden showed that morbidity and mortality attributed to schistosomiasis increases with DALYs (disability-adjusted life years) which had risen to about ~20% increase in the past 20 years (Driguez et. al., 2016;Murray et. al., 2013). A retrospective review of clinical records of bladder cancer cases in Sokoto, Nigeria between 1999 and 2004 showed a 4.7 fold rise in the number of bladder cancer cases, with squamous cell carcinoma composed of 65.1% of histologically verified cases and 50% of the squamous cell carcinoma showed evidence of chronic urinary schistosomiasis (Mungadi and Malami, 2007).

Genetic Susceptibility to Schistosomiasis
The presence of polymorphisms in cytokine genes IL 4-590 C/T and IL13-1055 C/T among the S. haematobiuminfected participants in this study was similar to findings from some previous studies (Kouriba et. al., 2005;Gatlin et. al., 2009;He et. al., 2008). Gatlin et. al., (2009)  shown a marked increase in the plasma levels of IL-5 and IL-13 in individuals identified as being resistant to schistosome infection (Leenstra et. al., 2006). Therefore, to understand the role of this heterozygous cytokine as a susceptibility or resistance factor, further post-treatment follow-up study will be required among the infected participants to establish the role of these cytokines as has been done in other similar studies (Cameron et. al., 2006;Gatlin et. al., 2009). Gatlin et. al., (2009 had reported that individuals with polymorphisms at positions IL-13 -1055 and IL-4 -590 are more likely to require fewer reinfections and treatments to become resistant to reinfection than individuals who are homozygous at either position. Another study in Mali revealed an association between a singlenucleotide polymorphism in the STAT6 gene at 12q13.3 and intensity of infection by S. haematobium; this polymorphism had an additive effect with IL13 −1055 (He et. al., 2008).

Other analyses of S. haematobium infection in
Mali revealed that in chromosomal region 5q31-q33, polymorphisms in the IL13 gene promoter at position −1055 and −591 were associated with the infection rate: alleles −1055C and −591A were preferentially transmitted to children with 10% highest infection rate, whereas −1055T associated with the lowest infection levels (Kouriba et. al., 2005). IL4 −590T allele has been associated with high IgE production, thereby having increased resistance to infection (Russell et. al., 2015).
In this study, GSTM1 and GSTT1 null polymorphisms were shown to significantly increase the risk of structural bladder pathology. This agrees with Arnaldo et. al., (2000), Aktas et. al., (2001), Cengiz et. al., (2007, Yajie et. al., (2016) and Jobaida et. al., (2016) who also observed a risk of bladder cancer with GSTM1 and GSTT1 null polymorphisms. Okkels et. al., (1996) and Arnaldo et. al., (2000) reported that the association of GSTM1 null genotype with bladder tumour was more apparent in a group with less aggressive tumours, as we also observed in this study. This could further support the presence of GSTM1 null genotype among the urinary tract pathology cases (an indicator of early stage of possible progression to bladder cancer). Among the bladder pathology cases, the distribution of the polymorphisms was relatively similar to the control group with slightly higher GSTM1 null genotype in the control cases. This is similar but considerably higher than what was reported in several other studies (Okkels et. al., 1996;Arnaldo et. al.,2000;Jobaida et. al., 2016). The role of GSTT1 null genotype in bladder cancer risk remains unresolved. Several studies suggested an increased risk (Moore et. al., 2004;Yajie et. al., 2016;Jobaida et. al., 2016;Ying et. al., 2016), but others suggested no risks or low risks (Karagas et. al., 2005;McGrath et. al., 2006;Matic et. al., 2016). In the present study, an elevated risk of bladder cancer was found among the S. haematobium infected participants; and this was more in smokers with null GSTM1 and GSTT1 polymorphisms. This finding is similar to those observed by Moore et. al., (2004) and Yu et. al., (2017) where elevated risk to bladder cancer was only seen in smokers with the GSTT1 null polymorphism and seems to buttress the observation that smoking is the most important risk factor for susceptibility to bladder cancer.

Conclusion
The prevalence of urinary schistosomiasis among adults is relatively high in the study area when compared with other areas in Nigeria. Individuals with bladder pathologies could have heavy or light intensity of schistosomiasis or have no existing infection at all. However, long term exposure to schistosomiasis is necessary for the development of bladder pathology which may eventually advance to cancer. IL 13-1055 polymorphisms did not indicate susceptibility to schistosomiasis in males, but a slight risk was found in females. GSTM1 and GSTT1 polymorphisms were associated with elevated risk of bladder pathology with the pathology and schistosomiasis group having more GST polymorphisms than the group with only bladder pathology.

Limitation of the study
In order to elucidate properly the role of GST (GSTM1 and GSTT1) polymorphisms in susceptibility to urinary schistosomiasis associated bladder pathology, it is important to conduct a repeat of this study with a much larger sample size. Also, molecularly characterized schistosomiasis negative control samples will be required to further establishe the relationship (susceptibility/resistance) between the cytokines (IL-13 -1055 and IL-4 -590) and urinary schistosomiasis.
participated in the study. The CIA thanks Nuria Malats for discussions.

Funding
The CIA acknowledges The World Academy of Science associate fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.