Polymorphism in SNP rs972283 of the KLF14 gene and genetic disposition to peptic ulcer

Introduction and Aim: Kruppel Like Factor 14 (KLF14) gene plays an important role in metabolic illnesses and is also involved in the regulation of many other biological processes. This study's objective was to determine whether or not the KLF14 single-nucleotide-polymorphism (SNP) known as rs972283 was linked to an increased risk of peptic ulcer disease in the population that was being investigated. Materials and Methods: Participants in this study included 71 people who had been diagnosed with peptic ulcers and 50 people who were considered to be healthy controls. In order to genotype the KLF14 SNP rs972283, an amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was carried out, and the PCR results were then sequenced. Results: Genotypes (GG, AG, and AA) were significantly different in people who had peptic ulcers compared to those who were in the control group (chi-square=7.703, 5.317 and 4.294) respectively. AG and AA genotypes for KLF14 gene were correlated with a high risk of peptic ulcer (P≤0.05) (Odds Ratio (O.R.) =6.343 and 2.441) respectively. Patients with peptic ulcer had a significantly greater incidence of the allele A gene (30.3%), whereas healthy people had a much higher incidence of the G allele (86%). Patients who carried the AG genotype and had a chronic H. pylori infection were found to have a highly significant correlation with one another (P≤0.01, O.R. =1.218). Similarly, there was a higher frequency of the G allele (84.6%), in people who had peptic ulcers, but there was a higher frequency of the A allele (39%), in cases of chronic infection. Conclusion: According to the findings of this research, a variant in the KLF14 gene called rs972283 is linked to an increased risk of peptic ulcer illness.


INTRODUCTION
early four million people throughout the world are diagnosed with peptic ulcer disease every year.This condition is defined by the formation of ulcers in the lining of the stomach, small intestine, and esophagus (1).The prevalence and incidence of peptic ulcer disease have contributed to an increase in the global burden of illness.A number of factors, including geography, environment, age, gender, socioeconomic position, stress, nutrition, alcohol use, and drug use, among others, play a significant role in the development of this disease (2).Recent research has demonstrated that genetic factors are a significant contributor to the development of peptic ulcer disease and gastritis, as well as the factors that play a part in the disease's etiology (3).Multiple pieces of research have found a correlation between having an infection with the bacterium H. pylori and having peptic ulcer disease.Peptic ulcer disease can be identified by the presence of excruciating ulcers in the lining of the stomach.These investigations have also demonstrated that the development of peptic ulcers is affected by the genetic polymorphisms of the host as well as the virulence genes carried by H. pylori.Peptic ulcers can also be caused by a combination of genetic and environmental causes (4,5).It has been shown that some polymorphisms in the genes for interleukin and Toll-like receptors can influence a person's susceptibility to gastrointestinal conditions that are associated with H. pylori (6,7).In a manner parallel to this, it has been demonstrated that the genetic variations that are present among the several distinct SNPs of H. pylori are substantial risk factors that increase susceptibility to peptic ulcer disease (8,9).Differentiation, invasion, proliferation, and apoptosis are just some of the biological processes that Kruppellike transcription factors (KLF) play a role in.This family of transcription factors comprises a large group (KLF1 to KLF17) (2,3).KLF14 gene has a wide expression in different tissues and has a role in metabolic disorders and cancerous diseases (10).The genetic instability of the KLF14 gene has been shown to alter chromosomal expression and increase the risk of developing peptic ulcers and gastric cancer (10).In order to determine whether or not SNPs in the KLF14 N gene play a part in the progression of peptic ulcer disease, researchers have analyzed a significant number of these variants.Polymerase chain reaction (PCR) was applied in detection of pathogenic bacteria and parasites (11)(12)(13)(14)(15)(16), and genetic diseases (17,18).Consequently, the objective of this study was to utilize Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) in order to investigate whether or whether there is a link between the KLF14 SNPrs972283 (A>G) polymorphism and the development of peptic ulcers.

Study population
Mucosal biopsy samples were collected from seventyone patients (between February to June 2011) aged between 20 and over 60 years with peptic ulcers, and being treated at Medical City Hospital, Baghdad.The control group consisted of individuals without peptic ulcer disease who were between the ages of 25 and 46 and gave blood.The healthy samples were acquired from these individuals.This research was performed in the labs of the Institute of Genetic Engineering and Biotechnology for Graduate Studies at the University of Baghdad, and it was approved by the university's Ethics Committee (Gastroenterology Hospital.No.13/September/551, dated 9/2/2020) of the University of Baghdad, and the experiment carried out according to the terms of the Helsinki Agreement.Written consent was obtained from all individuals participating in the study.The examination was conducted for infection with Helicobacter pylori in the laboratories of the Gastroenterology Hospital, Baghdad by the specialized consultant doctor using the tissue test process.

DNA Extraction
Genomic DNA was isolated from gastric mucosal biopsies carried out during endoscopy according to the protocols for DNA kit (Qiagen/German).Tetra primer ARMS-PCR was utilized to detect the mutation (A>G) in the Kruppel Like Factor 14 (KLF14) gene at the SNP rs972283 as described previously (19).The primers used in amplifying the KLF14 gene products are given in Table 1.

Polymerase chain reaction (PCR)
All of the PCR reactions were performed in a volume of 25μl, consisting of 5μl of DNA template, 0.5µl of forward inner primer (G allele), 0.5µl of reverse inner primer (A allele), 1µl for each of the outer primers, 12μl green master mix (Promega/USA) and 6 μl nuclease free water.The conditions for the PCR amplification were for 3 minutes at 95 ˚C before going through 30 cycles, however, denaturation for 1 minute at 95 ˚C, then at 60 ˚C for 1 minute in order to primer annealing, and at the extension was for 1 minute at 72˚C.The last extension continuously for ten minutes at a temperature of 72˚C.Electrophoresis was used to separate the PCR DNA samples after they had been run through 1.5 % agarose that had been stained with 0.5 µl of ethidium bromide.Under UV transillumination, the picture on the gel was visualized, and then it was photographed.The presence of two bands (437 and 221 bp) indicated the presence of the wild homozygous allele (GG), the presence of two bands (437 and 274) indicated the presence of the allele (AA), and the presence of three bands (437, 274 and 221 bp) indicated the presence of the heterozygous allele (GA) as shown in Fig. 1.

Statistical analysis
In order to determine which factors had a significant impact on the overall percentage, we ran our data through SAS (Statistical Analysis System, 2018).A statistically significant difference in the percentages was looked for using the Chi-square test (0.05 and 0.01 probability).Odds ratio and 95% CI were computed for this investigation (20).

RESULTS
Seventy-one patients with peptic ulcers were grouped further based on age and gender as shown in Table 2.
As seen, the incidence of peptic ulcers was observed to increase with increasing age with the highest seen among the age group 51-60 (Table 2).A statistical significant difference between age groups with increase in the incidence of peptic ulcer was also seen (P≤0.0001).Based on the gender, a statistically significant difference in peptic ulcer incidence was observed between men and women (Table 2).

Prevalence of genotypes for SNP rs972283 of KLF14 gene
In this study, Peptic ulcer sufferers and healthy people were compared in this research, and the researchers found that there was a statistically significant difference in the genetic variants associated with the KLF14 gene rs972283.Table 3 shows the proportion of the GG, AG genotypes for rs972283 prevalent in peptic ulcer patients as well as healthy controls.As seen, the GG wild type was most prevalent, followed by the AA and AG genotypes with a significant difference seen for the distribution of these genotypes in patient and control groups (Table3).Likewise, the allele frequency for the SNP rs972283 showed the 'G' allele frequency to be higher as compared to the 'A' allele frequency in both groups.

KLF14 gene polymorphism and Helicobacter pylori infection
In acute and chronic peptic ulcer patients the distribution of GG was seen to be highest followed by AA and AG genotypes (Table 4).A statistically significant difference was observed between the peptic ulcers patients suffering from acute and chronic H. pylori infection.The difference was highly significant for the homozygous (GG) and the heterozygous (A/G) genotypes (P≤0.01 and OR=1.218;Table 4).The prevalence of 'G' allele frequencies was observed to be higher in both groups as compared to the 'A' allele (Table 4).

DISCUSSION
In this study, adults over the age of 50 were found to have a higher chance of developing peptic ulcers, with a significant difference between males and females.Our findings are consistent with prior research that has demonstrated that peptic ulcers are more common and established among elderly (those >60 years) (4) in contrast to people aged 40-49 years were more likely to get peptic ulcers, while those aged 30-39 years were less likely to develop peptic ulcers (21).Contrary, a study showed age to have no statistically significant association to the development of peptic ulcers (22).Similarly, studies undertaken to associate gender and age with incidence of peptic ulcers reported no statistically significant difference between these factors (22,23).
Observations in this study pointed to the AG and AA genotypes of the KLF14 gene to be a significant risk factor for peptic ulcer disease.Additionally, the KLF14 SNP rs972283, which causes an A/G or A/A mutation in the gene, was found to be strongly related with peptic ulcers when the bacteria H. pylori was present.Much earlier research has found a connection between Helicobacter pylori infection and peptic ulcer disease, and our findings are consistent with those findings (24,25).Further results for allelic frequency within SNP rs972283 in this study showed that the frequency of the G allele (84.6%) was high in patients with H. pylori infection.This high frequency of the G allele in patients with H. pylori infection probably indicates that this allele is important in increasing the susceptibility to H. pylori infection.Similarly, the prevalence of the A allele was high (39%) in patients who had a persistent infection, which provides further evidence that the A allele plays a role in the progression of peptic ulcers to more advanced stages.This study also revealed that there is a positive effect of each of the genotype AG (O.R = 1.218; chi-square = 6.319;P≤0.01) and AA (O.R = 0.944; chi-square = 5.422; P≤0.05) when infection and transition to chronic infection in the case of H. pylori.In yet another investigation, a substantial connection was found between H. pylori infection and the genetic variants rs4986790, rs16944, and rs4986791, whereas rs352140 TT and 1143627 AG were significantly higher in patients with chronic gastritis (6).The variant of the NOD1 gene known as rs2075820 E266K has been shown to have a substantial connection with the risk of H. pylori infection as well as an elevated risk of peptic ulcer disease (26).It has been demonstrated that the polymorphisms rs4986791 and rs4986790 have a significant relationship with the onset of chronic gastritis (7).Therefore, the observation that was found in this investigation for genotypes within SNP rs972283 is significant, and it confirms the findings of earlier studies that demonstrated the influence of genetic factors as a risk factor in the acquisition of H. pylori infection and the development of peptic ulcers (21,26,27).

CONCLUSION
These results indicate the possibility that the KLF14 gene polymorphism is a risk factor for H. pylori infection and increased susceptibility to peptic ulcer.A broader study should be conducted to include more patients and study several genes to determine the associations affecting peptic ulcer disease.

Table 1 :
Primers used in KLF14 gene amplification by ARMS PCR

Table 2 :
Distribution of peptic ulcer patients (n=71) based on age and gender

Table 3 :
Genotypes prevalent for SNP rs972283 of KLF14 gene among the study population

Table 4 :
Genotype distribution among peptic ulcer patients in association to acute and chronic H. pylori infection