Identification of the polymorphism rs4072037 of MUC1 gene in patients with gastric cancer

Gastric cancer, a malignant disease, is the fourth most common cancer and second most common cause of cancer death. The pathogenesis of gastric cancer is a complex process, including Helicobacter pylori infection, genetic factors as well as many other factors. Among them, Single Nucleotide Polymorphism (SNP) is one of the most interesting topics nowadays.

JOURNAL OF MEDICAL RESEARCH IDENTIFICATION OF THE POLYMORPHISM RS4072037 OF MUC1 GENE IN PATIENTS WITH GASTRIC CANCER Nguyen Thi Ngoc Lan, Nguyen Van Tan, Ta Thanh Van, Dang Thi Ngoc Dung Hanoi Medical University Gastric cancer, a malignant disease, is the fourth most common cancer and second most common cause of cancer death The pathogenesis of gastric cancer is a complex process, including Helicobacter pylori infection, genetic factors as well as many other factors Among them, Single Nucleotide Polymorphism (SNP) is one of the most interesting topics nowadays Few studies on rs4072037 polymorphisms of the MUC1 gene have been done in different populations, particularly in Asian populations, which have indicated its role and significance as a susceptibility factor to gastric cancerL Our objective was to see if this varitation can be detected in Vietname patients with gastric cancer Methods: 130 patients diagnosed of gastric cancer and 130 healthy controls were recruited from the largest hospitals in the North Vietnam Genetic polymorphism at rs4072037 was determined by PCR-RFLP Results: Three types of SNPs were found as the allele type (G vs A: OR = 0.42, 95% CI: 0.25-0.72), the heterologeneous gene type (AG vs AA, OR = 0.47, 95% CI: 0.28-0.77) and dominant gene type (AG + AA vs GG: OR = 0.67, 95% CI: 0.47-0.97) Conclusions: Allele G of rs4072037 associated with the reduction of gastric cancer risk Key words: gastric cancer, polymorphism, rs4072037, MUC1 gene I INTRODUCTION In 2012, there were about million new to other South- East Asian countries [1] The cases of gastric cancer (6.8% of all incident mortality rates of gastric cancer for both males cancer cases) As the third leading cause of and females were also among the highest of cancer death and the fifth most common cancer cancers in Vietnam, surpassed only by liver worldwide, gastric cancer can be considered cancer and lung cancer [13] This has been one of the world’s pressing medical challenges argued to be attributable to the high prevalence [2] More than 70% of gastric cancer cases of Helicobacter pylori (seen in as much as 75% occur in developing countries, especially of Vietnamese adults), in combination with in East Asia Vietnam had the highest rate other risk factors such as smoking, obesity, compared to other South-East Asian countries socioeconomic status and genetic factors [8], In Vietnam, the ASR has been reported to [16] A single nucleotide polymorphism, or be about 16.3 per 100,000 people of both SNP, is a variation at a single position in a DNA sexes, which was the highest rate compared sequence among individuals One variant gene is called SNP if more than 1% of a population Corresponding author: Nguyen Thi Ngoc Lan, Ha- does not carry the same nucleotide at a specific noi Medical University position in the DNA sequence SNP may occur Email: ngoclannguyen@hmu.edu.vn Received: 27/11/2018 Accepted: 12/03/2019 within coding or noncoding regions They can relate to cancer-causing mechanisms in JMR 118 E4 (2) - 2019 JOURNAL OF MEDICAL RESEARCH sporadic cancer and SNPs have a important significance for community health One-hundred and thirty patients with gastric cancer were selected by the following The rs4072037 A > G SNP located in the criteria: 1) being diagnosed with gastric cancer 5′ UTR of the 2nd exon of MUC1 gene is (histopathology confirmation); 2) agreeing predicted to have an effect on the splicing to participate in the study; 3) being able to of the primary transcripts, which in turn communicate with the interviewers One- determines the type of variants The results hundred and thirty subjects without tumor of researches demonstrated that the G allele (controls) were recruited from endoscopy belongs to variant 2, while the A allele belongs departments (endoscopy confirmation with to variant The structural differences between normal or only benign, acute gastritis) Medical these two variants lead to insertion/deletion records of patients and controls were collected of nine amino acids encoded by the second under the agreement of the administrative exon, which are involved in the N-terminal departments of the hospitals signal peptide This differential signal peptide SNP genotype may lead to a different function of the encoded variant protein Also, the A allele reduces the transcriptional activity, which may result in decreased MUC1 expression [7] The rs4072037 also influences the transcriptional activity of the MUC1 gene promoter; the A allele associated with gastric cancer reduced the transcriptional activity, which may result in decreased MUC1 expression [10] Therefore, low expression of MUC1 may cause a reduction in its barrier function in the stomach and subsequently increases gastric cancer susceptibility The purpose of this study is to find whether rs4072037 is a risk factor in Vietnamese patients with gastric cancer II MATERIALS AND METHODS Study subjects A cross-sectional study was conducted from May 2016 to May 2018 in four hospitals in Hanoi, Vietnam, include: National Cancer Hospital, Hanoi Medical University Hospital, 108 Military Central Hospital, and Viet Duc Hospital JMR 118 E4 (2) - 2019 A 20 – minute interview via telephone or face-to-face was conducted with each patient, in which a research questionnaire was used to collect information socioeconomic regarding patient’s characteristics, disease characteristics The purpose, the benefits, the drawbacks, and the confidentiality aspect of patients in the study were introduced to the patients when they were asked to join the study Patients’s consent was ensured prior to the interview Patients were asked to self-report their information about gender, age, and medical history Blood collection for SNP analysis 2ml peripheral blood was taken from each patient and put into EDTA tube All collected samples were stored in appropriate condition and transported to the Medical Laboratory Quality Control – Hanoi Medical University for gene analysis SNP genotypes: Genomic DNA was peripheral blood using extracted from Exgene™ Blood JOURNAL OF MEDICAL RESEARCH SV (Gene All, Korea), according to the perform the repeated assays, and the results manufacturer’s PCR- were 100% concordant restriction fragment length polymorphism assay Statistical methods instructions The was used to detect the SNP rsA4072037G with the specific forward and reverse primers ’ - A A G G C C TAT G G G C A G A G A G A - ’ and 5’-ACGCTGCTGGTCATACTCAC-3’, respectively The 332-bp PCR products were digested overnight with the restriction enzyme AlwNI (New England BioLabs, Beverly, MA) and then separated on 2% agarose gel The A allele resulted in fragments of 223 and 109 bp, and the G allele produced fragment of 332 bp About 10% samples which without knowing the subject’s case or control status were Genotyped by direct sequencing method base on Sanger principle Additionally, 10% of the samples were also randomly selected to Student’s t-test or chi-square test was used to evaluate differences in the distributions of demographic characteristics, selected variables and genotypes of the rs4072037 between the cases and controls The associations between the genotypes of this SNP and risk of gastric cancer were estimated by computing the odds ratios (ORs) and their 95% confidence intervals (CIs) with unconditional logistic regression analyses All the above statistical analyses were performed with SPSS (v.16.0; IBM SPSS Statistics) Ethics approval This study was approved by the Ethics Council of Hanoi Medical University III RESULTS Table The socioeconomic characteristics of patients Case group   Control group n % n Male 79 60.8 69 53.1 Female 51 39.2 61 46.9 The average age Mean SD Mean SD 57.2 10.6 56.4 11.5   p %   Gender 0.21 0.51 Table describes the socioeconomic characteristics of patients There is no statistically significant difference in gender between case and control groups (p = 0.21) The mean age of the case group had no significant difference compared to the control group (p = 0.51) 10 JMR 118 E4 (2) - 2019 JOURNAL OF MEDICAL RESEARCH Figure Electrophoresis of productions treatedby enzyme AlwNI M: 100bp ladder; K1 đến K14: Samples of patients with gastric cancer; (+): Positive control and (-): Negative control Figure describes electrophoresis results after using PCR-RFLP with K3, K5 samples have GG genotype (332bp DNA fragment); K4, K7, K8, K11, K12: have AA genotype (223bp and 109bp DNA fragments); K1, K2, K6, K9, K10, K13 and K14 have AG genotype (332bp, 223bp, 109bp DNA fragments) Figure Sequencing results ofDNA fragment containing rs4072037 Figure describes the results of sequencing analysis of K4, K5, K6 samples after PCR-RFLP Product bands are clear and easy to determine the genotypes of these patients The results were 100% concordant Table Genotype and allele characteristics of rs4072037 Case n   p Control % n %   Genotype AA 68 52.3 44 33.8 AG 48 36.9 73 56.2 GG 14 10.8 13 10 0.006 Allele JMR 118 E4 (2) - 2019 11 JOURNAL OF MEDICAL RESEARCH Case n   p Control % n %   A 184 70.8 161 61.9 G 76 29.2 99 38.1 0.033 Table describes genotypes and alleles of rs4072037 between cases and controls In the case group, AA genotype has highest rate (52.3%) and was significantly differenent between the distribution genotypes between cases and controls (p = 0.006) Allele A in case group was higher than in control group with p = 0.033 Table3 Gastric cancer risk of genotype and allele of rs4072037 Genotype and allele Case Control OR 95% CI AG vs AA 48/68 73/44 0.42 0.25 - 0.72* GG + AG vs AA 62/68 86/44 0.47 0.28 - 0.77* G vs A 76/184 99/161 0.67 0.47 - 0.97* *p < 0.05 Unconditional logistic regression analysis revealed that the rs4072037 AG heterozygote was associated with a significant decrease in the risk of gastric cancer (OR: 0.42, 95% CI: 0.25 – 0.72), and the rs4072037 GG + AG was associated with a significantly decrease risk (OR: 0.47, 95%CI: 0.28 - 0.77) and the rs4072037 A allele was ascociated with a significantly decreased risk (OR = 0.67, 95%CI: 0.47 - 0.97) (Table 3) IV DISCUSSION In this study, the association of genetic variants of rs4072037 in MUC1 gene with gastric cancer susceptibility was found in 130 gastric cancer cases and 130 controls in Vietnamese population The results show that MUC1 rs4072037 G allele was significantly associated with decreased risk of gastric cancer This suggests the rs4072037 SNP can be considered as a marker for diagnosis and screening for Vietnamese patients with gastric cancer risk The mean age of the case group was similar to other research population in Vietnam and East Asia region Le Viet Nho et al’s research (2014) had average age 58.9 ± 13.8, and 59.2 12 ± 11.9 in Jeong O et al’s research (2011) [5], [3] The gender ratio in our research was 1.55/1 which was lower than the studies conducted by Trinh Hong Son and Wanebo (1.75/1) [14], [15] In the case group, the rate of AA, AG and GG genotypes was as 52.31%, 36.92% and 10.77%, respectively In control group, the highest rate was the AG genotype with 56.15%, followed by AA and GG with 33.85% and 10%, respectively There was a stastistically significant difference between genotype distributions between these groups Genotype distribution in our research was similar to other researches such as Hye-Rim Song (2014), JMR 118 E4 (2) - 2019 JOURNAL OF MEDICAL RESEARCH Hanze Zhang et al (2011), AA genotype was the most common genotype in research population 79.2% and 74.2%, respectively, less common were AG genotype 19.1% and 23%, respectively [12], [17] The genotype rate in control group was similar to research of Yanbin Jia et al (2010) Both groups have AG genotype with highest common rate, up to 51.6% [4] Our results show that rs4072037 G allele leads to reduce gastric cancer in the allele type (G vs A: OR = 0.42, 95% CI: 0.25 - 0.72), in the heterologous gene type (AG vs AA, OR = 0.47, 95% CI: 0.28 - 0.77), in dominant gene type (AG + AA vs GG: OR = in the stomach, and subsequent increases in gastric cancer susceptibility This strongly demonstrated rs4072037 G allele was a protective factor in gastric cancer These findings suggested that this SNP may be used as biomarker to screen Vietnamese patients with gastric cancer risk V CONCLUSION The SNP rs4072037 G allele has a significant association with reduced risk of gastric cancer and this SNP may be used as biomarker to screen Vietnamese patients with gastric cancer risk 0.67, 95% CI: 0.47 - 0.97) These results were Acknowledgments similar to the results of research groups from This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106-YS.02-2015.37 We wish to thank clinicians from National Cancer Hospital, Hanoi Medical University Hospital, 108 Military Central Hospital, and Viet Duc Hospital for their excellent assistance in recruitment of patients We thank scientists and technichians of Biochemistry Department and Quality Control Center for Medical Laboratory at Hanoi Medical University for their support during the research Japan, Korea and China with OR arrange from 0.26 - 0.69 [9], [17], [6] As we know, MUC1 is a member of mucin family and is a transmembrane mucin It is a multi-functional protein involved in mucosal lubrication, protection from pathogens, signal transduction and cell-cell interaction MUC1 was considered as oncogene because it has role in cell growth, anchorage independence, cell migration, anti-apoptosis and drug resistance of cancer cells [10], [11] The rs4072037 A > G SNP effects on the splicing of the primary transcripts and determines the type of variants The G allele of this SNP determines variant 2, while the A allele belong to variant Two variants differences about nine amino acids encoded by the second exon and lead to a different function of the encoded variant protein Also, the A allele reduces the transcriptional activity and decreased MUC1 expression [7] The rs4072037 A allele associated with gastric cancer reduced the transcriptional activity of the gene product, which may result in decreased MUC1 expression, reduction in barrier function JMR 118 E4 (2) - 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