Association Between Vitamin D Receptor Polymorphism and the Response to Helicobacter Pylori Treatment

Introduction

The spiral-shaped Gram-negative bacteria Helicobacter pylori (H. Pylori) exhibits a variety of processes and distinct virulence factors that impact the characteristics of the gastric mucosa and determine its adhesion and survival in this environment.¹ The main cause of persistent gastritis is infection with the H. Pylori bacteria. Nearly half of the world’s population is impacted.² Additionally, a study conducted on Egyptian patients by Abo-Amer et al discovered that 538 patients (83.3%) of Egyptian patients had H. pylori infection.³

Other gastrointestinal conditions such peptic ulcer disease, gastric adenocarcinoma, and gastric lymphoma are influenced by H. Pylori in their aetiology. H. pylori infection is linked to most duodenal ulcers and stomach malignancies.² The strains that reside in the stomach mucosa and how they interact with the host’s immune system determine the long-term colonisation of H. pylori and the production of chronic gastritis.² Additionally, the susceptibility and clinical development of the infection are also in part influenced by human genetic variations. They can create various levels of immune system mediators that are connected to infection rates, the eradication of H. pylori and colonisation by more virulent strains, the augmentation of stomach inflammation, and cell damage.⁴

The expression of the vitamin D receptor (VDR) in the stomach epithelia may improve as a result of H. pylori infection, according to a recent theory. Through the stimulation of cathelicidin antimicrobial peptides and the reduction of inflammatory cytokine and chemokine levels, it has been found in vitro that the active form of vitamin D, 1a,25-dihydroxy vitamin D3, exhibits immune modulators capabilities against this pathogen.⁵ In metanalysis done by Yang et al⁶ found that Vitamin D has a protective role in H pylori infection. Also, El Shahway et al,⁷ found that low level of Vitamin D associated with failure of treatment while Shafrir et al⁸ reported that increased vitamin D levels are associated with successful H. pylori eradication. The VDR gene, which spans a length of DNA larger than 60 kb, is found on the 12q13.11 chromosome.⁹ The restriction fragment length polymorphism (RFLP) for the FokI, BsmI, ApaI, and TaqI restriction enzymes is being utilised in investigations of genetic vulnerability to infectious diseases as well as response to treatment.^10–12

The FokI polymorphism (rs2228570; C>T), characterized by the change of thymine to cytosine (ATG to ACG)¹³ and ApaI polymorphism (rs7975232, A>C) where cytosine is changed to adenine.¹⁴ The FokI and Apal VDR polymorphism and H. pylori infection may go hand in hand.¹⁵ This research aimed to determine how variations in the vitamin D receptor gene affected how H. pylori infections responded to eradication therapy.

Subject and Methods

Subject

From 176 adult H. Pylori-positive patients, only 105 H. Pylori-positive patients who meet the eligibility criteria, a prospective cohort study was carried out at Mahala Hepatology Teaching Hospital during the period from May 2021 to Jan 2022. Research was approved by Mahala Hepatology Teaching Hospital research ethical committee. No animals were used for studies that are the basis of this research. All human procedures followed were in accordance with the guidelines of Helsinki Declaration of 1975. Informed consent was obtained from all participants of this study.

Methods

All of the enrolled patients had their H. pylori status confirmed as positive using the Eliza procedure in accordance with the manufacturer’s instructions.

The following is administered to all patients;

1) PCR was used to genotype all patients’ VDR gene polymorphisms.

2) The widely used clarithromycin-based triple therapy, which includes omeprazole 20 mg, amoxicillin 1000 mg, and clarithromycin 500 mg twice daily for 14 days.

3) Four weeks following the completion of treatment, a stool sample was tested for H. pylori Ag.

Laboratory Analysis

Genotyping and DNA extraction: The DNA was extracted using the QIAamp^® DNA Blood Mini Kit. Using a Nano Drop^® (ND-1000) Spectrophotometer (Nano Drop Technologies Inc., Washington, USA), the concentration of the isolated DNA was determined.

Genotyping of VDR SNPs: Applied Biosystems (Applied Biosystems-Life Technologies, Carlsbad, California, USA) genotyped the VDR FokI (rs2228570) and ApaI (rs7975232) SNPs.

All patients provided written, informed consent.

Exclusion Criteria

A proton pump inhibitor (PPI) was taken within the previous month by subjects or those who reported receiving H. pylori medication within the previous six months, being pregnant, abusing alcohol, or using illegal drugs were excluded from the study. Utilisation of corticosteroids, immunosuppressive therapy, anti-inflammatory medications, or antibiotics in the two months prior. Gastric surgery, renal failure, liver cirrhosis, autoimmune diseases with a history, liver cirrhosis, and cancers were disqualified.

Statistical Analysis

Statistical tests were performed using SPSS 21.0. Qualitative data were expressed as number and percentage and quantitative variables were expressed as the mean ± standard deviation (mean ±SD) for parametric data. Chi-square test was used to evaluate qualitative data. Regression analysis was done to determine the independent risk factors for the H. pylori eradication. A level for statistical significance was significant at p < 0.05.

Results

The study involved 105 patients, whose average age was 49.44 + 10.94. Male patients made up 56 (53.3%) and female patients were 49 (46.7%). Only 26 patients (24.8%) smoked, and their average BMI was 29.43 5.75. The average lab results for CBC, liver enzyme, and creatinine were displayed in (Table 1).

Table 1 Baseline Characteristic Features of the H. Pylori Infected Patients

As seen in Table 1, the distribution of TT, CT, and CC genotypes among the patients under study was 10.5%, 53.3%, and 36.2%, respectively. As shown in Table 2, the proportions of males and females with VDR rs 2228570 gene polymorphisms are 7.1%, 14.3%, 50.0%, and 57.1%, respectively, in TT, CT, and CC. The distribution of the genotypes AA, AC, and CC for the VDR rs 7975232 gene polymorphisms was 13.3%, 48.6%, and 38.1%, respectively, among the overall study population. According to Table 2, the percentages of males and females with VDR rs 7975232 gene polymorphisms are as follows: AA (12.5% and 14.3%), AC (42.9% and 55.1%), and CC (44.6% and 30.6%).

Table 2 Gene Polymorphism of VDR in Relation to Gender

In 79 of the individuals under study, H pylori was completely eradicated (75.2% success rate). The successful eradication of H. pylori and VDR rs 2228570 gene polymorphisms was more prevalent in CT gene polymorphism (64.6%) compared to non-responders (19.2%), while treatment failure was more prevalent in CC gene polymorphism (73.1% in non-responders compared to responders 24.1%), which is statistically significant (Table 3). In regards to the eradication of H. pylori and VDR rs7975232 gene polymorphisms, the success of eradication was more prevalent in AC gene polymorphism (54.4%) vs non-responders (30.4%), while all patients (14) with gene AA (17.7%) are responders to standard treatment, while the failure of treatment was more prevalent in CC gene polymorphism (69.2% in non-responder vs 27.8% in responders) which is statistically significant.

Table 3 Gene Polymorphism of VDR in Relation to the Treatment Outcome of H.pylori

The VDR rs 2228570 and VDR rs 7975232 gene polymorphisms were found to be independent risk factors for the elimination of H. pylori after regression analysis (Table 4). There were significant correlations between H.Pylori response to eradication therapy and VDR rs 2228570 and VDR rs 7975232 alleles (Table 5).

Table 4 Logistic Regression of H.Pylori Response to Eradication Therapy

Table 5 Logistic Regression Between H.Pylori Response to Eradication Therapy and VDR Rs 2228570 and VDR Rs 7975232 Alleles

Discussion

The virulent strains of H. pylori are linked to gastric ulcers and cancer because they cause extensive immune cell infiltration in the gastric mucosa and cause noticeable inflammatory reactions.¹⁶ Due to its ability to neutralise gastric acid through the potent action of its urease enzymes⁴ and the activity of glutamyl transpeptidase enzyme, which supports its proliferation and survival in the gastric mucosa,¹⁷ H. pylori can persist in the gastric mucosa for an extended period of time during infection. Treatment with vitamin D inhibits both mechanisms.¹⁸ Because the vitamin D metabolites are implicated in the induction of antimicrobial activity and have anti-inflammatory and immune-system modulating properties.^19,20 Failure to eradicate H. pylori may be associated with vitamin D deficiency, necessitating vitamin D supplementation prior to H. pylori eradication.²¹ VDR has a role in the immune-modulating abilities of active vitamin D.²² Vitamin D receptor gene polymorphisms (ApaI and TaqI) lead to decreased vitamin D level which associated with the response to treatment. In patients with H. pylori infection, Guo et al⁵ found a statistically significant positive connection between chronic inflammation and VDR mRNA expression.

105 patients who tested positive for H. pylori participated in the study. Our findings demonstrated a substantial correlation between patients’ responses to H. pylori treatment and polymorphisms in the VDR gene (ApaI and TaqI). Also, the eradication of H. pylori is at risk due to polymorphisms in the VDR gene (ApaI and TaqI). According to Mohamed et al’s findings, H. pylori infection and the FokI and Apal VDR polymorphism are related.¹⁵ Additionally, Qadir et al discovered a substantial correlation between the VDR Bsml SNP and increased risk of gastric cancer, particularly in obese individuals.²³ In contrast to Martins et al,²⁴ who claimed that there were no appreciable changes between patients with H. pylori infection and control groups in terms of the allelic and genotypic distribution of the FokI and ApaI polymorphisms of the VDR gene. Additionally, Eom et al²⁵ discovered no conclusive associations between vitamin D intake and the prevalence of gastric cancer, and genetic variation related to vitamin D had no impact on these associations. The rates of H. pylori eradication do, however, appear to be related to vitamin D levels, according to Huang et al.²⁶ The discrepancy in the findings could be attributed to the complex interactions between genetic, environmental, and societal factors that lead to H. pylori infections. Together with ethnic characteristics, these interactions—which are crucial for illness development and genetic makeup—were most likely responsible for the disparity in allelic frequencies observed in the research population. The results of Mohamed et al¹⁵ were consistent with the lack of a statistically significant variation in the sex distributions across the various genotypes. This is consistent with our findings.

In our study, the usual triple therapy had a 75.2% H. pylori eradication rate, and other studies have reported rates between 61 and 77%.^27–30 In contrast to other studies, certain investigations^31,32 showed greater rates (85–94%). Therefore, it makes sense to assume that variations in eradication rates are correlated with the degree of adherence and/or the local susceptibility pattern of H. pylori in the investigated areas. A meta-analysis has recommended that antibiotic selection be localised in light of this regional diversity.³³ The present study found that there was a significant difference in the response to eradication therapy of H.Pylori regarding VDR (ApaI and TaqI) gene polymorphism. Also, the independent risk factors for H. pylori eradication were VDR rs 2228570 and VDR rs7975232 gene polymorphisms. There is also, a significant correlation between H. Pylori response to eradication therapy and VDR rs 2228570 and VDR rs 7975232 alleles.

Conclusion

According to our knowledge, this is the first study to have discovered a connection between H pylori positivity and the FokI and Apal VDR polymorphism, which are separate risk factors for H pylori eradication. There is also, a significant correlations between H. Pylori response to eradication therapy and FokI and Apal VDR polymorphism alleles. To evaluate the findings, more research with numerous populations is required.