2.1 Study Methodology
2.2 Study sitting and Study population
A prospective hospital-based study was carried out at public and private hospitals in Khartoum state. The hospitals included Ibin Sina specialized hospital, Soba teaching hospital, Modern Medical Centre and Al Faisal Specialized Hospital. A total of 121 individuals, who had been referred for endoscopy, were recruited. Out of that, 14 had gastric cancer, 27 had peptic ulceration, 64 had gastroduodenal inflammation, while 15 showed normal upper gastroduodenoscopic features. The diagnosis of gastroduodenal diseases had been made by an experienced gastroenterologist during the upper GI endoscopy procedure. While gastric cancer was diagnosed based on histology. Participant’s demographic and clinical data were obtained by structured questionnaire, personal interviews, and review of case records. The selection criteria included the Sudanese population from both sexes, no antibiotic or NSAIDS uses. All the participants were informed with the objectives and purposes of the study and the written informed consents were taken. The demographic characteristic of participants is presented in Table 1.
Table 1. Demographic characteristic of participants
Variables
|
Total
n=121
|
H. pylori (+ve)
n=61
|
H. pylori (-ve)
n=60
|
P-value
|
Age years ± Std. Deviation
(range)
|
44.55±17.44
(15-89)
|
44.30± 16.76
(15-85)
|
44.82±18.24
(17-89)
|
0.9959
|
Gender
|
Male
|
72 (59.50%)
|
37 (51.38%)
|
35 (48.61%)
|
0.7046
|
Female
|
49 (40.50%)
|
24 (48.98%)
|
25 (51.02%)
|
Residence
|
Urban
|
54 (44.63%)
|
24 (44.44%)
|
30 (55.56%)
|
0.132
|
Rural
|
67 (55.37%)
|
37 (55.22%)
|
30 (44.78%)
|
2.3 DNA extraction
Gastric biopsies were collected in 400µl phosphate buffer saline (PBS). For histological examination, the biopsies were transported in formalin. DNA extraction was carried out by using innuPREP DNA Mini Kit (analytikjena AG, Germany) according to the protocol given by the manufacturer, as previously described in (38).
2.4 PCR amplification of specific 16S rRNA of H. pylori
The specific 16S rRNA gene of H. pylori was amplified by using the following primers (primers: F:5'-GCGCAATCAGCGTCAGGTAATG-3') (R:5'-GCTAAGAGAGCAGCCTATGTCC-3').(39) The PCR condition was previously described.(40)
2.5 PCR amplification and sequencing of the IL1B promotor region
The IL-1B-511 and -31, promoter polymorphisms, were amplified using the following primers: F:5'- CATCCATGAGATTGGCTAG-3' and R:5'- AGCACCTAGTTGTAAGGAAG-3'.(41) The cycling conditions were an initial denaturation at 94°C for 5min, followed by 35 cycles of 94°C for 1min, 60°C for 1min and 72°C for 1min, with a final extension at 72°C for 7min. The amplified PCR product is 800bp and was located between -687bp upstream and +297bp downstream of the IL-1B gene.
Out of 14 PCR products, of H. pylori-infected subjects, which have the clearest bands, were sent for DNA purification and Sanger dideoxy sequencing. Both DNA strands were sequenced commercially by Macrogen Inc, Korea.
2.6 Sequence analysis and SNPs detection
The sequencing results, two chromatograms for each patient (forward and reverse), were visualized, checked for quality and analyzed using the Finch TV program version 1.4.0.(42) The nucleotide Basic Local Alignment Search Tool (BLASTn; https://blast.ncbi.nlm.nih.gov/) was used to assess nucleotide sequence similarities.(43)
To determine the SNPs in the IL-1B promoter region, multiple sequences alignment (MSA) for tested sequences with a reference sequence (NG_008851) were performed by using BioEdit software.(44)
2.7 Bioinformatics analysis of the IL-1B promoter region in H. pylori-infected subjects
2.7.1 in silico prediction of the promoter
The crucial element for initiating and regulating messenger RNA transcription is the promoter sequence which is generally located in the 5’ upstream region of a structural gene.(45) In this study, five computational promoter recognition tools were used: Berkeley Drosophila Genome Project (BDGP) (http://www.fruitfly.org/),(46) Promoter 2.0 Prediction Server (http://www.cbs.dtu.dk/),(47) FPROM, TSSG, and TSSW (http://softberry.com/) which are based on neutral network and linear discriminant approach.(48)
2.7.2 In silico analysis of the predicted promoter regions
2.7.2.1 Assessment for the presence of promoter associated features
In silico predicted promoter regions were additionally assessed for the presence of promoter associated features, including promoter-associated histone marks, broad chromatin state segmentation, transcription factor ChIP-seq, and DNase I hypersensitivity clusters, using the ENCODE data (https://epd.epfl.ch/cgi-bin/get_doc?db=hgEpdNew&format=genome&entry=IL1B_1).(49-51)
2.7.2.2 Prediction of CpG Islands
A CpG island is often regarded as a marker for the initiation of gene expression. It is a segment of DNA with high GC and CpG dinucleotide contents which located in the 5’ UTR (untranslated regions) of genes. In this study, MethPrimer(45, 52) and GpC finder software(http://www.softberry.com/berry.phtml?topic=cpgfinder&group=programs&subgroup=promoter) were employed to predict CpG islands in the promoter.
2.7.2.3 Prediction of Transcription Factor Binding Sites
One of the important steps in the chain of promoter analytical events is the prediction of the potentially functional transcription factor binding sites (TFBSs). Protein binding sites in a promoter represent the most important elements and the corresponding proteins are called transcription factors (TFs). In this step, the promoter region was analyzed for possible TFBSs using the following prediction software: Alggen Promo (http://alggen.lsi.upc.es/cgi-bin/promo_v3/),(53, 54) AliBaBa2 (http://www.gene-regulation.com/),(55) GPMiner (http://GPMiner.mbc.nctu.edu.tw/)(56), TF-Bind (http://tfbind.hgc.jp/),(57) and Tfsitescan (http://www.ifti.org).(58) These software were employed to predict possible TFBSs in the promoter region and corresponding TFs using binding sites from TRANSFAC®Public which provides data on eukaryotic TFs, their experimentally-proven binding sites, consensus binding sequences (positional weight matrices) and regulated genes.(59)
2.7.2.4 Prediction of composite regulatory elements
Composite regulatory element (CE) is the minimal functional unit, which can provide combinatorial transcriptional regulation of gene expression. Structurally a CE consists of two closely located DNA binding sites for distinct transcription factors. But its regulatory function is qualitatively different from regulation effects of either individual DNA binding sites. In this study, we identified the composite regulatory elements in our region by using MatrixCatch algorithm.(60)
2.7.2.5 Comparative analysis
Promoter region was analyzed for possible conservation using the ECR Browser (http://ecrbrowser.dcode.org),(61) NCBI BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and ClustalW(https://www.genome.jp/tools-bin/clustalw). Conservation was assessed in 11 species: chimpanzee (Pan troglodytes), rhesus monkey (Macacamulatta), mouse (Mus musculus), rat (Rattusnorvegicus), dog (Canisfamiliaris), cow (Bostaurus), opossum (Monodelphisdomestica), chicken (Gallus gallus), frog (Xenopuslaevis), zebrafish (Danio rerio), fugu pufferfish (Takifugurubripes), and spotted green pufferfish (Tetraodon nigrovoridis).
Also, conservation of SNPs was evaluated and the possible conservation of TFBSs at these SNP locations was screened with Multiple-sequence local alignment and visualization (Mulan) search engine (https://mulan.dcode.org/).(62)
2.8Detection of the IL-1B-31 C/T polymorphism using PCR with confronting two-pair primer (PCR-CTPP)
For detection of the IL-1B-31 polymorphism, PCR-CTPP was applied. The primers for the C allele were (F:5′-ACT TCT GCT TTT GAA GGC C-3′) and (R:5′-TAG CAC CTA GTT GTA AGG A-3′); and those for the T allele were (F:5′-AGA AGC TTC CAC CAA TAC T-3′) and (R:5′-CTC CCT CGC TGT TTT TAT A-3′).(63) 1µl of extracted DNA was used in a 25µl reaction mixture with a prepared Maxime PCR PreMix Kit (i-Taq) (iNtRON BIOTECHNOLOGY, Seongnam, Korea), 23μl of de-ionized sterile water, 0.25μl of each primer. PCR conditions were as follow: 5 min of initial denaturation at 94°C, followed by 25 cycles of 1min at 94°C, 1min at 54°C, and 1min at 72°C, and a 5min final incubation at 72°C. The PCR products were visualized by electrophoresis on a 2% agarose gel stained with ethidium bromide. Genotyping was performed as follows; 240, 155 bp for CC genotype, 240, 155, 122 bp for CT genotype, and 240, 122 bp for TT genotype.(63)
2.9 Statistical analysis
Deviations from Hardy-Weinberg equilibrium in control were examined by χ2test. According to prevalence of H. pylori infection, differences in distribution by age was assessed by Mann-Whitney test, while differences in distribution by categorical variables were examined by χ2test or Fisher's test. Odds ratios (ORs) were calculated and reported within the 95% confidence intervals (CIs). P<0.05 was considered to be statistically significant. The statistical analyses were performed using the GraphPad Prism 5.