international journal of research in pharmaceutical sciences

Ghaidaa Raheem Lateef Al-Awsi et al., Int. J. Res. Pharm. Sci., 9(SPL1), 1-4

ORIGINAL ARTICLE

INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACEUTICAL SCIENCES

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Linkage between H. pylori infection and TNF-α polymorphism in pregnant women Ghaidaa Raheem Lateef Al-Awsi*1, Azhar Omaran Al-Thahab2, Eqbal Dohan Chalap Al- Grawi3 1Department of Biomedical Engineering, Al-Mustaqbal University College, Babylon, Iraq 2Department of Biology, College of Science, University of Babylon, Iraq 3Department of Pathological analysis, Al-Mustaqbal University College, Babylon, Iraq

Article History:

ABSTRACT

Received on: 28.03.2018 Revised on: 19.05.2018 Accepted on: 22.05.2018

A study was performed on 100 pregnant women in the outpatient department of gynecology and obstetrics of Maternity and Children Hospital in AlDiwaniya City during the period between (March to September 2016). One hundred blood samples (50 for patients and 50 for control) were collected under the supervision of the treating gynecologist. The detection of Helicobacter. pylori was done by the use of the serum antibody Rapid test. The results showed that fifty percent were considered positive and fifty percent were considered negative regarding the H. pylori in above method. For the purpose of extracting genomic DNA, all blood of patients and control samples were utilized, where the 107 bp PCR product size. Genotyping of the TNF-α -308 SNP(G/A) was implemented through restriction fragment length polymorphism PCR (RFLP-PCR). The products of polymerase chain reaction have been digested with restr NcoI iction enzyme. Individuals with the TNF-α -308 (GG) homozygote produced digested DNA bands at 80, and 20 bp bp. A heterozygous genotype of TNF-α -308(GA) produced 107 bp, 80 bp, and 20 bp bands. Individuals with the TNF-α -308 (AA) homozygote genotype generated a single band of 107 bp and it had no amplicon digested. An important difference existed in the frequency regarding TNF-α -308 (GG) genotype between negative and positive Helicobacter pylori groups (78%, 72% respectively). Also for GA genotype, a notable differences existed between negative and positive Helicobacter pylori groups (18%, 24% respectively). Concerning the frequency of the TNF-α -308 (AA) genotype between the Helicobacter pylori negative and the Helicobacter pylori positive groups, a notable difference existed between those 2 groups.

Keywords: H. pylori, TNF-α Polymorphism, Pregnant women, Diwaniyah

* Corresponding Author

INTRODUCTION

Name: Dr. Ghaidaa Raheem Lateef Al-Awsi Phone: 9647711788884 Email: [email protected]

The most widespread chronic bacterial infection is the Helicobacter pylori, which can be abbreviated to (H. pylori), it was common all over the world and in individuals with variety of ages. The H. pylori infection can be examined in gastric diseases even through the pregnancy; specifically, this Gramnegative bacterium appear to have some association with hyperemesis gravidarum, which is serious type of vomiting and nausea through the pregnancy (Karaer et al., 2008).

ISSN: 0975-7538 DOI: https://doi.org/10.26452/ijrps.v9iSPL1.1298 Production and Hosted by

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Many researches stated that there is a relationship between Helicobacter pylori infection and an increment in inflammatory gene response defined

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through the up-regulation of many genes like TNFα and IL-1β. These cytokines can play a significant part in etiology regarding gastric cancer (GC), also it is thought to be as a key mediators regarding gastric pathophysiology (Person et al., 2011; Bhagat et al., 2008).

technique was conducted based on that described by (Santos et al, 2012). Table 1: The Multiplex PCR primers with their sequence and amplicon size

MATERIALS AND METHODS Samples collection Several samples of blood (50) were gathered from each pregnant women suspected to be infected with H. pylori who visited Maternity and Children Teaching Hospital during the period from (March to September 2016). And 50 blood samples were collected from pregnant healthy women (control group).The samples were collected under the supervision of the treating gynecologist. Serum Antibody Rapid Test was used for detection of H. pylori in (100) blood samples.

RFLP-PCR master mix preparation This mix was carried out through utilizing (Accu Power PCR PreMix Kit), also it was implemented based on the instructions of the company as illustrated in the table below (2), Table 2: PCR Master Mix with their volume for TNF-α gene

Serum Antibody Rapid test The test is implemented for diagnosing bacteria in the samples of blood, as the interactions produces a change of color during the sample movement of membrane test strip in the control line area will always result in red color. The color red only appears on letter C (control line) when there is no bacterium infection. A letter T (result line) will have red package on it when there is infection in along with the package control in red. Molecular study Genomic Blood DNA Extraction Through the use of Genomic DNA Mini kit extraction kit (Frozen Blood) Geneaid, USA, the Genomic DNA from the samples of blood were extracted, also it was implemented with the instructions of the company. Genomic DNA estimation

Thereafter, those PCR master mix components which were mentioned in the previous table are set in standard AccuPower PCR PreMix Kit which have all the other components that were required to PCR reaction like (loading dye, stabilizer, KCL, MgCL2, Tris-HCl pH: 9.0,dNTPs and Taq DNA polymerase), then each PCR tube is being transferred to Exispin vortex centrifuge at 3000rpm for three minutes, after that it will be placed in PCR Thermocycler (Mygene. Korea). PCR Thermocycler Conditions The conditions for the PCR thermo cycler were implemented for all genes in an independent way as illustrated in the table below, Table 3: PCR thermocycler conditions for TNFα

Through the uses of Nanodrop spectrophotometer (THERMO, USA), the extracted blood genomic DNA was checked by measuring the DNA concentration (ng/μL), also it checked the purity of the DNAthrough reading the absorbance at (260 /280 nm).

Primers TNF-α -308 gene polymorphisms primers were designed by (Santos et al., 2012), also (Bioneer Company, Korea) provided those primers as in the tables 1.

PCR product analysis

RFLP-PCR Technique

1. Through the use of 1X TBE, one percent of agarose gel were prepared, then dissolved in water bath at temperature of 100 Celsius and for fifteen minutes, then it will be left to cool at a temperature of 50 Celsius.

In the steps below, an analyzing process to the PCR products will be examined through agarose gel electrophoresis:

This method was implemented for genotyping and detection IL-1β single-nucleotide polymorphism in healthy samples of blood and in blood samples of Helicobacter pylori gastric diseases patients. This 2 © Pharmascope Publications | International Journal of Research in Pharmaceutical Sciences


2. After that, the agarose gel solution will have 3μL of ethidium bromide stain added into it. 3. The agarose gel solution was poured in tray following to fixing the comb in the right position, then it is left to solidify for fifteen minutes at room temperature, after that, the comb was taken away in a gentle way from the tray and each comb well will have 10μl PCR product added into it and 10ul of (100bp Ladder) in the first well. 4. The gel tray was filled by 1X TBE buffer and fixed in electrophoresis chamber. An electric current of 100 volts and 80 AM were performed for an hour. 5. A 304 bp PCR product for IL-1β was visualized by using UV trans illuminator. RFLP Mix for (TNF-α gene)

Through the use of NcoI restriction enzyme (New England Biolabs. UK), the RFLP-PCR mix was set, this mix was performed in an independent way based on the instructions of the company as illustrated in the table below, Table 4: RFLP master mix with their volume for TNF-α

Thereafter, the mix will be placed in Exispin vortex centrifuge at 3000rpm for two minutes, and it will be transferred to incubation at 37 Celsius all night, then through using 3% of agarose gel electrophoresis technique, the RFLP-PCR product will be analyzed. The tumor necrosis factor alpha -308 G/G genotype resulted in 80/20-bp bands following to amplicon digestion. Persons with tumor necrosis factor alpha -308 A/A genotype generated single band of 107 bp, also they did not have amplicon digestion. Three bands 107/80/20 were resulted in from the heterozygous tumor necrosis factor alpha -308 G/A genotype. RESULTS Detection of H. pylori by Serum Antibody Rapid Test This test is utilized for the purpose of diagnosing bacteria in the samples of blood. The color red only appears on letter C (control line) when there is no bacterium infection. A letter T (result line) will have red package on it when there is infection in along with the package control in red as demonstrated in (Figure 1). The result of this study revealed that (50) (100%) of patient blood samples

was positive for this test and all control blood samples (50) (100%) gave a negative result for this test.

Figure 1: Serum antibody rapid test A: A letter T will have red color on it when there is infection along with the red color on Letter C. B: The color red only appears on letter C when there is no infection RFLP-PCR for TNF-α Gene All blood specimens of patients and control were utilized to extract genomic DNA to detect TNF-α gene which appears in 107bp PCR product size . This result is in accordance with Santos work (2012). In this study the TNFα polymorphism -308(G/A) created a restriction site for NcoI. The amplicon of 107bp was digested with the restriction enzyme NcoI. The results showed TNF-α-308(GG) homozygote genotype produced 80bp and 20bp bands after amplification digestion. The heterozygous TNFα -308(GA) genotype resulted in 3 bands 107bp,80bp, and 20bp in length, while persons with the TNFα (AA) homozygote genotype generated a single band of 107bp and they did not have amplicon digestion. This result demonstrated that TNF-α-308(G/G) and TNFα -308 (G/A) alleles were associated in H. pylori positive patients. RFLP-PCR for Genotyping of TNFα SNP (G/A) The genotype frequencies of polymorphisms studied. The frequency of TNFα (GG) polymorphism differs significantly (p