Nghiên cứu đa hình thái đơn gen MUC1 và PSCA trên bệnh nhân ung thư dạ dày tt

MINISTER OF EDUCATION MINISTER OF HEALTH HA NOI MEDICAL UNIVERSITY NGUYEN THI NGOC LAN STUDY ON SINGLE NUCLEOTID POLYMORPHISMS OF MUC1 GENE AND PSCA GENE IN GASTRIC CANCER PATIENTS Specialty Code : Biochemistry : 62720112 SUMMARY OF DOCTORAL THESIS IN MEDICINE HA NOI - 2020 The work was completed at: HA NOI MEDICAL UNIVERSITY Supervisor: Prof Ta Thanh Van Assoc Prof Dang Thi Ngoc Dung Review 1: Assoc Prof.PhD Bach Khanh Hoa Review 2: Assoc Prof.PhD Bach Vong Hai Review 3: Prof.PhD Tran Van Thuan The thesis was defended before the Council at the university level Meeting at: Ha Noi Medical University On: The thesis can be found in the library: - National Library - The Library of Ha Noi Medical University THE RESEARCH RELATED TO THE THESIS Nguyen Thi Ngoc Lan, Dang Thi Ngoc Dung, (2018), Research on pepsinogen concentration and some related factors in patients with stomach cancer, Vietnam Journal of Medicine, Specialized issue Topic (469), 87 - 94 Tran Van Chuc, Nguyen Thi Ngoc Lan, Dang Thi Ngoc Dung, Ta Thanh Van (2018), Single polymorphism rs2294008 PSCA gene and risk of gastric cancer, Journal of Medical Research, (115), 25-32 Nguyen Thi Ngoc Lan, Nguyen Van Tan, Ta Thanh Van, Dang Thi Ngoc Dung (2019), Identification the polymorphism rs4072037 of MUC1 gene in patients with gastric cancer, Journal of Medicine Research, (118 E4), 8-15 INTRODUCTION Gastric cancer (GC), which is statistically proven to be the fifth most common cancer and the third leading cause of cancer death, is a malignant disease The pathogenesis of GC is a complex process involving factors such as Helicobacter pylori (H.pylori) infection, diets, lifestyles and genetics With regard to genetic changes, Single Nucleotide Polymorphism (SNP) is one of the common types of genetic variants in the human genome Nowadays, a plenty of researchers in some countries with high prevalence of GC patients such as Japan, South Korea and China tend to carry out investigations on SNPs of MUC1 and PSCA, the promising findings of which indicated that these SNPs are potential markers for screening GC risks MUC1 encodes the mucin-1 protein, which prevents exogenous factors from causing damages to cells under normal conditions In addition, PSCA has been shown to be linked to many cancers including GC Although Vietnam is a country with a high rate of patients with GC, the number of studies on genetic variants such as SNPs on GC patients, especially the combination of these SNPs with the risk factors of GC is still small and with lots of limitations For the above reasons, the research "Study on Single Nucleotide Polymorphism of MUC1 and PSCA gene in gastric cancer patients" was conducted with the following objectives: 1) Identify single nucleotide polymorphisms of MUC1 and PSCA genes among patients with Gastric cancer 2) Assess the correlation between MUC1 and PSCA polymorphisms and z risk factors of gastric cancer 1.1 Urgency Single-nucleotide polymorphisms (SNP) are common genetic changes in the human genome In recent time, many scientists have shown their interests in studies on SNPs’ roles in the pathological risk especially in cancer and metabolic diseases Many SNPs including the ones of MUC1 and PSCA were reported to have relation with GC The studies on combination of these SNPs with risk factors including H.pylori infection, drinking history, or smoking history, etc was launched The risk of getting diseases in general and GC in particular is a combination of risk factors and genetic factors Research on SNPs of MUC1 and PSCA especially on their combination with risk factors in GC has not been studied in Vietnam These are the reasons pushing the research to be conducted New contributions of the thesis - It is identified that SNPs including AA genotype of rs4072037 and GG genotype of rs2070803, both belonging to MUC1 gene, increase the risk of GC development - No matter what risk factor is in combination with MUC1’s sensitive genotypes does the risk of GC development increase Especially when these genotypes combine with the family history of GC, patients have six times higher risks of suffering from it When these SNP genotypes combine with each other, only the combination of one of the two above mentioned sensitive genotypes increases the risk of GC A prognosis model of GC based on age, history of individual gastrointestinal disease, family history of GC, drinking history and rs4072037 (MUC1) was developed Model test results obtained AUC = 70%, accuracy = 0.63, Brier index = 0.231 Layout of the thesis - The 123-page- thesis includes the following parts: 2-page introduction, 29-page literature review, 16-page research participants and methodology, 34-page research findings, 40-page discussion, 1page conclusion - The thesis has 27 tables, 11 charts, 26 figures as well as231 references arranged in the order of appearance in the thesis Chapter LITERATURE REVIEW 1.1 Overview about Gastric cancer 1.1.1 Epidemiology According to GLOBOCAN in 2018, there were about million new cases of GC GC becomes the fifth leading malignancy after lung cancer, breast cancer, colon cancer and prostate cancer More than 70% of new cases are found in developing countries andhalf of the new cases in East Asia (mainly in China) Mortality caused by GC ranks the third position following lung cancer and colon cancer The age-standardized incidence rate among men is approximately twice as high as the one among women The prevalence of GC varies by geographic region, particularly between countries or between different regions within a country The proportion of GC patients increases in accordance with their age, with the highest rate among the age of 50-70 Vietnam is located in the region with the high prevalence of GC Although there have been no accurate epidemiology nationwide surveys, it is statistically proven that the rate of GC patients in the community is still fairly high According to GLOBOCAN 2018, Vietnam had about 164,000 new cases of cancer in both sexes, including about 17,000 GC, accounting for 10.6% and ranking the 3rd among common cancers, only following liver and lung cancer 1.1.2 Risk factors of Gastric cancer The complex pathogenetic mechanism of GC is a combination of risk factors and genetic changes H.pylori infection is considered as a Type I agent that causes GC Besides, tobacco is a risk factor of more than 14 types of cancer, including GC According to Yhokisazu (2006), smokers have 1.56 times higher risk of GC development The International Cancer Research Organization (IARC) told that alcohol consumption can increase the risk of GC development History of individual gastric disease and family history of GC are also thought to be related to risk of GC One pathogenetic factor causing three diseases including tomach ulcer, duodenal ulcer and GC is H.pylori infection Chen (2004) found a link between gastric ulcer and non-cardiac GC In addition to hereditary GC syndromes, the risk of developing gastric cancer among people with a family history of GC is three times as high as those without a GC history 1.1.3 Molecular Mechanism of Gastric cancer Many studies have shown a comprehensive picture of the molecular causes of GC including: gene mutations, non-gene mutations and SNPs SNPs are common genomic changes in humans, with about one million SNP sites found SNP has initially explained the phenomenon that individuals are exposed to same environmental conditions but the likelihood of disease development among them is very different This dissimilarity is attributed to the correlation of genetic traits such as SNP and environmental conditions In GC,many SNPs are proven to have relation and SNPs of MUC1 and PSCA are specifically focused on research 1.2 Polymorphism of MUC1 and PSCA genes In humans, the MUC1 gene is located in the long branch of chromosome 1, region 2, band (1q22), with a size of 4407 bp, including exons and introns The PSCA gene is on chromosome 8, band q24.2 The MUC1’s function is to encode proteins that act as a barrier against exogenous agents to the cell However, in cancer cells, MUC1 is also considered as a carcinogenic protein PSCA is responsible for differentiation, immune balance and T-cell activity The PSCA gene appears in the epithelium of the stomach and it partially reduces activity in gastric tissue with intestinal dysplasia According to the data of NCBI, there are many SNPs of the MUC1 and PSCA genes associated with the risk of GC, of which rs2070803 and rs4072037 belonging to the MUC1 gene and rs2976392 and rs2294008 belonging to the PSCA gene have been identified to have a strong connection with GC All SNPs cause structural changes of the proteins; as a result, the function of normal proteins changes accordingly Rs4072037 (MUC1) involves exon splicing processes thereby creating different variants as well as different functions of protein, which may reduce the mucosal protective function of the MUC1 protein And rs2070803 (MUC1) is a genetic variation in this region that is related to GC In vitro studies have shown that rs2294008 can reduce the replication activity of the PSCA gene because it is located at the beginning of this gene's replication, and the function of rs2976392 remains unclear The combination of these SNPs with histopathological characteristics or history of H.pylori infection, history of drinking, smoking or personal and family history of GC has been a focused and prioritized topic on research with an aim at shedding the light on the complicated mechanism of pathogenesis in GC Chapter 2: RESEARCH PARTICIPANTS AND METHODOLOGY 2.1 Research participants - Criteria for selecting the case group: All patients with gastric carcinoma were clinically examined, took diagnostic imaging tests and were diagnosed by histopathological criteria Any patients and their families who did not agree to join the study or simultaneously suffered from other cancers were excluded from the study - Criteria for selecting the control group: Any individual without stomach pathology abnormality or with acute gastritis confirmed by clinical examination and endoscopy, was paired with GC patients by age and gender 2.2 Study design: The study followed case – control design, with a sample size being calculated based on OpenEpi software and on the results of Fang Li et al (2012) studying MUC1 and PSCA gene polymorphisms in GC patients Combining data from the study and the above mentioned software calculation we obtained the minimum sample size of 139 cases and 139 controls needed for this study During the study, we selected 302 patients in the case group and 304 people in the control group in accordance with the above stated including and excluding criteria 2.3 Time and location of research - Research time period: From 1/2016 to 8/2019 - Research location: Sampling was taken at Hanoi Medical University Hospital, K Hospital, 108 Central Military Hospital Samples were analyzed at Center for Testing and Quality Assurance - University Hanoi Medical School and Department of Applied Biology (Kyoto Institute of Technology, Japan) using the same method 10% of the sample was analyzed at both centers to ensure the accuracy of the results Quantification of H.pylori IgG by ELISA method was performed at Gen - Protein Research Center and Department of Biochemistry - Hanoi Medical University And the determination of the Pepsinogen I, II level on the automatic immune system was conducted at the Laboratory Department Hanoi Medical University Hospital 2.4 Contents and research variables Smoking status: participants were divided into groups: smokers and non-smokers According to the concept of the Centers for Disease Control and Prevention (CDC), smokers are people who have smoked at least 100 cigarettes in their lifetime, whereas non-smokers are those who have never smoked or smoked less than 100 cigarettes in their lifetime Alcohol use was assessed based on WHO standards, in which a 10-gram-alcohol-containing drinking unit is considered to be a standard unit of alcohol One standard unit is equal to cup of spirits/ whiskey (40 degrees, 30 ml); glass of wine (13.5 degrees, 120 ml); 2/3 of a bottle or a can of beer (330 ml) The research questionnaire was developed based on the WHO's Audit C Test in which harmful drinking is defined when the total score of answered questions for men is ≥ points and for women is ≥ points And in this study, people who drank alcohol to such a harmful level were called ones with drinking history and the rest were called ones without drinking history Personal history of gastric disease: This information was collected based on an interview question about the history of gastric disease that was diagnosed by a doctor Family history of gastric disease: This information was gathered through interview questions, in which the person had a family history of gastric disease when GC runs into their families (diagnosed at the hospital) H.pylori infection history was assessed based on a patient's history of H.pylori infection diagnosis through interviewing, information gathered from medical records and quantitative results of IgG H.pylori test Patients have a history of H pylori infection if one of the above results is positive Patients have no history of H.pylori infection if all three results are negative 2.5 Equipment and chemicals: Using equipment and chemicals of reputable manufacturers: Gene All (Korea), NEB (UK), Sequencing kit (USA), quantifying Pepsinogen (Abbott - USA), quantification of IgG of H.pylori (Germany) 2.6 Research techniques DNA extraction: DNA was extracted from blood samples using the method of Exgene ™ Blood SV Kit (Gene All, Korea) PCR duplicates the genome containing SNP rs4072037, rs2070803, rs2294008 and rs2976392 with specialized primer pairs: SNP Rs4072037 Rs2070803 Rs2294008 RS2976392 Primer sequences 5’-AACCCAGGGGTTACTGAGGCTG-3’ 5’-AGTACGCTGCTGGTCATACTCAC-3’ (reverse) 5’-CTTAGCTGTCCGGGTGTGAAGT-3’ 5’-TGTGGTTCTAGGCAGGAGCAAC-3’ (reverse) 5’-TAGGCTCTGTCCTCCAGAG-3’ 5’-TCTGTCTACCTGCCCCCTAG-3’ (reverse) 5’-CTGGCCATCTGTCCGCAGCT-3’ 5’-CAGATGGAGGAGGATGGCTGGA-3’ (reverse) Size 332 bp 442 bp 545 bp 117 bp Performing restriction enzyme reaction: The standard PCR products were incubated with the corresponding specific enzymes including AlwNI for rs4072037, Taqa1 for rs2070803, NlaIII for rs2294008 and PvuII for rs2976392 Post-cut products were in electrophoresis on agarose gels of appropriate concentration to separate post-cut results The results of SNPs were read by observing the number and size of DNA bands on the electrophoresis Genetic sequencing: 10% of the samples were confirmed by direct sequencing 2.7 Statistical Analysis The study used Stata 12.0 data analysis software and R 3.6.2 software in calculating the OR ratio (odds ratio) using the OR algorithm with 95% CI confidence interval; the ratios were compared with the Chi square algorithm; the average was compared with tstudent test; the relationship of risk factors and GC was assessed by univariate and multivariate regression models Especially, algorithms on R software were used to develop GC prognosis model 10 Table 3.4: Analysis of the influence of risk factors on the research participants on logistic regression model OR P 95%CI Gender (Male vs Female) 1.56 0.10 0.92 – 2.64 Age group (≥ 60y vs < 60 y) 0.72 0.09 0.50 – 1.05 Smoking history (Yes vs No) 1.05 0.84 0.66 – 1.65 Drinking history (Yes vs No) 0.50 0.04* 0.26 – 0.95 H.pylori infection history (Yes vs No) 0.42 0.00* 0.29 – 0.61 PG ratio (PGI/II ≤ vs PGI/II >3) 2.56 0.07 0.93 – 7.09 Gastric disease history (Yes vs No) 1.42 0.06 0.99 – 2.04 Family GC history (Yes vs No) 3.69 0.00* 1.78 – 7.66 * Statistical significance Table 3.4 analyzes risk factors of GC by using multivariate regression including age group, sex, smoking history, drinking history, H pylori infection history, PGI/II ratio, personal history of gastric disease and family history of GC in both case and control groups The results showed that the rate of male suffering from GC were higher than the one of women with OR = 1.56, the ratio of PGI/II ≤ compared with> with OR = 2.56 The number of patients with history of gastric disease was higher than the one without history with OR = 1.42 The number of patients with family history of GC was higher than the one without history with OR = 3.69 The rest factors had OR GG 0.58 0.04* 0.35 – 0.97 AA>AG 2.09 0.00* 1.48 – 2.96 AA>AG+GG 1.85 0.00* 1.33 – 2.56 AA+AG>GG 1.19 0.45 0.75 – 1.91 A>G 1.32 0.02* 1.04 – 1.67 * Statistical significance Table 3.6 analyzes the correlation between RS4072037 genotypes and GC risk with the method of calculating the odds ratio and the risk of GC Results showed that people with AA genotype had a higher risk of developing GC than people with AG genotype with OR = 2.09 (95% CI: 1.48 - 2.96) Similarly, people with the AA genotype were at higher risk of developing GC than those with the AG + GG genotype with OR = 1.85 (95% CI: 1.33 - 2.56) The risk of developing GC for the AG genotype was lower than for the GG genotype with OR = 0.58 (OR