Association between rs2069718, rs280500, rs6443624 and rs1042522 variants and risks of essential thrombocythemia (khóa luận tốt nghiệp)

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -  - GRADUATION THESIS TITLE: ASSOCIATION BETWEEN RS2069718, RS280500, RS6443624 AND RS1042522 VARIANTS AND RISKS OF ESSENTIAL THROMBOCYTHEMIA Hanoi, November 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -  - GRADUATION THESIS TITLE: ASSOCIATION BETWEEN RS2069718, RS280500, RS6443624 AND RS1042522 VARIANTS AND RISKS OF ESSENTIAL THROMBOCYTHEMIA Student name : Pham Viet Nhat Student code : 634847 Class : K63CNSHE Faculty : Biotechnology Supervisor : Assoc Prof Nguyen Thi Xuan McS Nguyen Thanh Huyen Hanoi, November 2022 COMMITMENT The work on this thesis has not been submitted for a degree or diploma at any other institution of higher education I hereby declare that the work of this thesis is my own work To the best of my knowledge and belief, this thesis contains no material previously published or written by others unless referenced Hanoi, December 5th, 2022 Student Pham Viet Nhat i ACKNOWLEDGEMENTS First, I would like to express my gratitude to the director of Vietnam National University of Agriculture, board of dean and all teachers in Faculty of Biotechnology gave me possibility to complete this thesis I also express my heartfelt respect, gratitude and sincere appreciation to Assoc Prof Nguyen Thi Xuan, Department of Immunogenomic, Institute of Genome Research (IGR) for constructive criticisms and constant inspiration during the entire period of the study as well as the research, for your time to help me in writing and for provide me the opportunity to carry out my thesis in the laboratory of Institute of Genome Research I would like to thank all teachers from Department of Biology, Faculty of Biotechnology, especially McS Nguyen Thanh Huyen who encouraged me to enhance my knowledge and help me get the orientation of thesis I also wish to show my appreciation towards all the staff members of the Immunogenomics Laboratory, who supported me the laboratory techniques and statistical analysis Last but not least, I owe a debt of gratitude to my beloved parents, relatives and friends for their blessings, constant source of inspiration, all out sacrifice and moral support throughout the entire period of my academic life With warm and regards! Hanoi, December 5th, 2022 Student Pham Viet Nhat ii CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS x CHAPTER 1: INTRODUCTION 1.1 Preface 1.2 Objectives and Requirements 1.2.1 Objectives 1.2.2 Requirements CHAPTER 2: LITERATURE REVIEWS 2.1 Essential thrombocythemia overview 2.1.1 Definition of Essential thrombocythemia 2.1.2 Epidemiology of Essential thrombocythemia 2.1.3 Etiology and pathophysiologic mechanisms 2.2 Studies of Essential thrombocythemia in the world and Vietnam 2.2.1 Studies of Essential thrombocythemia in the world 2.2.2 Studies of Essential thrombocythemia in Vietnam 2.3 IFNG gene overview 2.3.1 Definition of IFNG 2.3.2 The location of IFNG gene 2.3.3 The function of IFNG gene 2.4 TYK2 gene overview 2.4.1 Definition of TYK2 2.4.2 The location of TYK2 gene 2.4.3 The function of TYK2 gene iii 2.5 PIK3CA gene overview 2.5.1 Definition of PIK3CA 2.5.2 The location of PIK3CA gene 2.5.3 The function of PIK3CA gene 2.6 TP53 gene overview 2.6.1 Definition of TP53 1.6.2 The location of TP53 gene 10 2.6.3 The function of TP53 gene 10 CHAPTER 3: MATERIALS AND EXPERIMENTAL METHODS 11 3.1 Materials 11 3.1.1 Objects of study 11 3.1.2 List of primers 11 3.1.3 Chemicals 12 3.1.4 Equipment 12 3.2 Methods 13 3.2.1 Genomic DNA extraction 13 3.2.2 PCR 14 3.2.3 SNP genotyping 15 3.2.4 Analysis of gene polymorphisms/mutations 18 3.2.5 Statistical analysis 18 CHAPTER 4: RESULTS AND DISCUSSION 21 4.1 DNA extraction 21 4.2 Association between IFNG gene rs2069718 variants and risk of essential thrombocythemia 21 4.2.1 Results of gene polymorphism analysis 21 4.2.2 Sequence analysis of some samples by sequencing method 23 4.3 Association between TYK2 gene rs280500 variants and risk of essential thrombocythemia 26 iv 4.3.1 Results of gene polymorphism analysis 26 4.3.2 Sequence analysis of some samples by sequencing method 27 4.4 Association between PI3KCA gene rs6443624 variants and risk of essential thrombocythemia 30 4.4.1 Results of gene polymorphism analysis 30 4.4.2 Sequence analysis of some samples by sequencing method 31 4.5 Association between TP53 gene rs1042522 variants and risk of essential thrombocythemia 34 4.5.1 Results of gene polymorphism analysis 34 4.5.2 Sequence analysis of some samples by sequencing method 35 4.5 Discussion 38 CHAPTER 5: CONCLUSION AND SUGGESTION 39 5.1 Conclusion 39 5.2 Suggestion 40 REFERENCES 41 v LIST OF TABLES Table 3.1: Primers used for amplification 11 Table 3.2: List of laboratoty equipment 12 Table 3.3: Components of PCR reaction 14 Table 3.4: Constituent reagent volumes for KASP genotyping reaction of wet DNA method for 384-well plate 16 Table 4.1: Distributions of genotypes of the IFNG gene rs2069718 between essential thrombocythemia and control groups 22 Table 4.2: Distributions of genotypes of the TYK2 gene rs280500 between essential thrombocythemia and control groups 26 Table 4.3: Distributions of genotypes of the PL3KCA gene rs6443624 between essential thrombocythemia and control groups 30 Table 4.4: Distributions of genotypes of the TP53 gene rs1042522 between essential thrombocythemia and control groups 34 vi LIST OF FIGURES Figure 1.1: Late excess mortality in essential thrombocythemia: a population-based study in the Netherlands, 2001–2018 Figure 1.2: IFNG Gene in genomic location: bands according to Ensembl, locations according to GeneLoc Figure 1.3: TYK2 Gene in genomic location: bands according to Ensembl, locations according to GeneLoc Figure 1.4: PIK3CA Gene in genomic location: bands according to Ensembl, locations according to GeneLoc Figure 1.5: TP53 Gene in genomic location: bands according to Ensembl, locations according to GeneLoc 10 Figure 3.1: PCR amplification heat cycle 14 Figure 3.2: SNP Genotyping – KASP system 15 Figure 3.3: Thermal cycle conditions for KASP genotyping reactions 16 Figure 4.1: Agarose gel electrophoresis of genomic DNA A 1-2: Total DNA of essential thrombocythemia patients, 3- 5: Total DNA of control subjects 21 Figure 4.2: Analytical results SNP genotyping, orange dots: wild type genotypes, green dots: heterozygous genotypes, blue dots homozygous genotypes A: Patients group B: Controls group 22 Figure 4.3: Agarose gel electrophoresis of PCR product of IFNG gene A 1-2: PCR product of essential thrombocythemia patients, 3-5: PCR product of control subjects 24 Figure 4.4: Result of the nucleotide sequence of representative individual carrying GG genotype of IFNG gene rs2069718 polymorphism 25 Figure 4.5: Result of the nucleotide sequence of representative individual carrying AG genotype of IFNG gene rs2069718 polymorphism 25 vii Figure 4.6: Result of the nucleotide sequence of representative individual carrying AA genotype of IFNG gene rs2069718 polymorphism 25 Figure 4.7: Agarose gel electrophoresis of PCR product of TYK2 gene 1-2: PCR product of essential thrombocythemia patients, 3-4: PCR product of control subjects 28 Figure 4.8: Result of the nucleotide sequence of representative individual carrying GG genotype of TYK2 gene rs280500 polymorphism 29 Figure 4.9: Result of the nucleotide sequence of representative individual carrying AG genotype of TYK2 gene rs280500 polymorphism 29 Figure 4.10: Result of the nucleotide sequence of representative individual carrying AA genotype of TYK2 gene rs280500 polymorphism 29 Figure 4.11: Agarose gel electrophoresis of PCR product of PL3KCA gene 1-3: PCR product of essential thrombocythemia patients, 4-7: PCR product of control subjects 32 Figure 4.12: Result of the nucleotide sequence of representative individual carrying AA genotype of PL3KCA gene rs6443624 polymorphism 33 Figure 4.13: Result of the nucleotide sequence of representative individual carrying CA genotype of PL3KCA gene rs6443624 polymorphism 33 Figure 4.14: Result of the nucleotide sequence of representative individual carrying CC genotype of PL3KCA gene rs6443624 polymorphism 33 Figure 4.15: Agarose gel electrophoresis of PCR product of TP53 gene 12: PCR product of essential thrombocythemia patients, 3-4: PCR product of control subjects 36 Figure 4.16: Result of the nucleotide sequence of representative individual carrying CC genotype of TP53 gene rs1042522 polymorphism 37 viii Figure 4.7: Agarose gel electrophoresis of PCR product of TYK2 gene 1-2: PCR product of essential thrombocythemia patients, 3-4: PCR product of control subjects After electrophoresis was finished, it could be seen that no incorrect DNA products was found All bands are bright, bold, and clear and no collateral band, therefore PCR conditions of thermal cycle and chemical composition and concentrations was optimized Accordingly, we succeeded in amplifying the DNA fragment of TYK2 gene All PCR products were purified for further DNA sequencing technique in this study ❖ Sequence analysis of TYK2 gene To confirm the accuracy of the PCR products amplified from TYK2 gene, the PCR products have been purified and sequenced by DNA sequencing technique First, we conducted direct DNA sequencing from PCR products containing DNA fragments of TYK2 gene that was compared with a reference sequence from GenBank The genotyping success rate was 100% in our study cohort After sequencing, the samples were analyzed for the same results as the SNP genotyping results Typically, the 40th, 41st control sample of both methods gave GG genotype of TYK2 gene rs280500 28 Figure 4.8: Result of the nucleotide sequence of representative individual carrying GG genotype of TYK2 gene rs280500 polymorphism Figure 4.9: Result of the nucleotide sequence of representative individual carrying AG genotype of TYK2 gene rs280500 polymorphism Figure 4.10: Result of the nucleotide sequence of representative individual carrying AA genotype of TYK2 gene rs280500 polymorphism Thus, after analyzing the IFNG rs2069718 gene polymorphism by genotyping and sequencing, there was no association between the TYK2 gene rs280500 and essential thrombocythemia 29 4.4 Association between PI3KCA gene rs6443624 variants and risk of essential thrombocythemia 4.4.1 Results of gene polymorphism analysis Table 4.3: Distributions of genotypes of the PL3KCA gene rs6443624 between essential thrombocythemia and control groups Genotype CC CA AA C A Patient Control (n=85) (n=107) 64 75 (75.29%) (70.09%) 17 27 (20.00%) (25.23%) (4.71 %) (4.67%) 145 177 (85.29%) (82.71%) 25 37 (14.71%) (17.29%) OR (95% CI) P-value 0.7378 0.9375 0.3692 1.4747 0.2415 3.6398 0.3895 0.9257 0.8248 0.4745 1.4337 0.4947 AA genotype was found in both essential thrombocythemia patient and control group with frequencies of 4.71% and 4.67%, respectively And P>0.05 wasn’t considered statistically significant This result illustrated that the difference of this genotype carried by both essential thrombocythemia and control groups was relatively small (Table 4.3, adjusted OR = 0.9357, 95% CI = 0.2415 – 3.6398) In this study, we found that the frequency of CA genotype in essential thrombocythemia patients was 20% and the control group was 25.23 % (Table 30 4.3, OR=0.7378 and 95% CI=0.3692 – 1.474) And P>0.05 wasn’t considered statistically significant This result illustrated that the difference of this genotype carried by both essential thrombocythemia and control groups was relatively small, and CA genotype frequency is less in patients In our study, the frequencies of CC genotype carried by both essential thrombocythemia patient and control groups were extremely rare and similar each other (75.29% and 70.09 %, respectively) (Table 4.3) The results of statistical analysis in (Table 4.3) suggested that the individuals carrying A allele in the PL3KCA gene were lower in essential thrombocythemia patients compared to healthy controls (OR=0.8248, 95% CI= 0.4745 – 1.4337), whereas no difference was observed in both essential thrombocythemia and control individuals carrying C allele And P>0.05 wasn’t considered statistically significant The data suggested that the C>A of rs6443624 SNP could not be a risk factor of essential thrombocythemia attack 4.4.2 Sequence analysis of some samples by sequencing method We observed that the most optimized annealing temperature in PCR reaction which amplified the DNA fragment of PL3KCA gene was 63°C Then, the correct DNA fragments of PCR products were purified and sequenced by DNA sequencing technique The components and thermal cycles of PCR reaction for amplifying the DNA fragment of TP53 gene were both shown in (Table 3.3, Figure 3.1) The final PCR products were purified and tested by electrophoresis using 0.8% agarose gel, then compared with 1kb DNA ladder under UV light after staining with EtBr (Figure 4.13) 31 Figure 4.11: Agarose gel electrophoresis of PCR product of PL3KCA gene 1-3: PCR product of essential thrombocythemia patients, 4-7: PCR product of control subjects After electrophoresis was finished, it could be seen that no incorrect DNA products was found All bands are bright, bold, and clear and no collateral band, therefore PCR conditions of thermal cycle and chemical composition and concentrations was optimized Accordingly, we succeeded in amplifying the DNA fragment of PL3KCA gene All PCR products were purified for further DNA sequencing technique in this study ❖ Sequence analysis of PL3KCA gene To confirm the accuracy of the PCR products amplified from PL3KCA gene, the PCR products have been purified and sequenced by DNA sequencing technique First, we conducted direct DNA sequencing from PCR products containing DNA fragments of PL3KCA that was compared with a reference sequence from GenBank The genotyping success rate was 100% in our study cohort After sequencing, the samples were analyzed for the same results as for the SNP genotyping Typically, the 20th and 21st control samples of both methods gave the AA genotype of PL3KCA gene rs6443624 32 Figure 4.12: Result of the nucleotide sequence of representative individual carrying AA genotype of PL3KCA gene rs6443624 polymorphism Figure 4.13: Result of the nucleotide sequence of representative individual carrying CA genotype of PL3KCA gene rs6443624 polymorphism Figure 4.14: Result of the nucleotide sequence of representative individual carrying CC genotype of PL3KCA gene rs6443624 polymorphism 33 Thus, after analyzing the IFNG rs2069718 gene polymorphism by genotyping and sequencing, there was no association between the PL3KCA gene rs6443624 and essential thrombocythemia 4.5 Association between TP53 gene rs1042522 variants and risk of essential thrombocythemia 4.5.1 Results of gene polymorphism analysis Table 4.4: Distributions of genotypes of the TP53 gene rs1042522 between essential thrombocythemia and control groups Genotype GG GC CC G C Patient Control (n=97) (n=128) 30 36 (30.93%) (28.13%) 41 56 (42.27%) (43.75%) 26 36 (26.80%) (28.13%) 101 128 (52.06%) (50%) 93 128 (47.94%) (50%) OR (95% CI) P-value 0.8786 0.8667 0.4679 1.6498 0.4306 1.7443 0.6872 0.6884 0.9208 0.6339 1.3375 0.6648 CC genotype was found in both essential thrombocythemia patient and control group with frequencies of 26.8% and 28.31%, respectively And P>0.05 wasn’t considered statistically significant Accordingly, the CC genotype tend to be not increased risk of essential thrombocythemia (Table 4.4, adjusted OR = 0.9208, 95% CI = 0.4306 – 1.7443) 34 In this study, we found that the frequency of GC genotype in essential thrombocythemia patients was 42.27 % and the control group was 43.75 % (Table 4.4, OR=0.8786 and 95% CI=0.4679 - 1.6498) And P>0.05 wasn’t considered statistically significant This result illustrated that the difference of this genotype carried by both essential thrombocythemia and control groups was relatively small In our study, the frequencies of GG genotype carried by both essential thrombocythemia patient and control groups were extremely rare and similar each other (30.93% and 28.13 %, respectively) (Table 4.4) The results of statistical analysis in (Table 4.4) suggested that the individuals carrying C allele in the TP53 gene were lower in essential thrombocythemia patients compared to healthy controls (OR=0.9208, 95% CI= 0.6339 – 1.3375), whereas no difference was observed in both essential thrombocythemia and control individuals carrying G allele And P>0.05 wasn’t considered statistically significant The data suggested that the G>C of rs1042522 SNP could not be a risk factor of essential thrombocythemia attack 4.5.2 Sequence analysis of some samples by sequencing method We observed that the most optimized annealing temperature in PCR reaction which amplified the DNA fragment of TP53 gene was 63°C Then, the correct DNA fragments of PCR products were purified and sequenced by DNA sequencing technique The components and thermal cycles of PCR reaction for amplifying the DNA fragment of TP53 gene were both shown in (Table 3.3, Figure 3.1) The final PCR products were purified and tested by electrophoresis using 0.8% agarose gel, then compared with 1kb DNA ladder under UV light after staining with EtBr (Figure 4.18) 35 Figure 4.15: Agarose gel electrophoresis of PCR product of TP53 gene 1-2: PCR product of essential thrombocythemia patients, 3-4: PCR product of control subjects After electrophoresis was finished, it could be seen that no incorrect DNA products was found All bands are bright, bold, and clear and no collateral band, therefore PCR conditions of thermal cycle and chemical composition and concentrations was optimized Accordingly, we succeeded in amplifying the DNA fragment of TP53 gene All PCR products were purified for further DNA sequencing technique in this study ❖ Sequence analysis of TP53 gene To confirm the accuracy of the PCR products amplified from TP53 gene, the PCR products have been purified and sequenced by DNA sequencing technique First, we conducted direct DNA sequencing from PCR products containing DNA fragments of TP53 gene that was compared with a reference sequence from GenBank The genotyping success rate was 100% in our study cohort After sequencing, the samples were analyzed for the same results as for the SNP genotyping Typically, the 30th and 31st control samples of both methods gave the CC genotype of TP53 gene rs1042522 36 Figure 4.16: Result of the nucleotide sequence of representative individual carrying CC genotype of TP53 gene rs1042522 polymorphism Figure 4.17: Result of the nucleotide sequence of representative individual carrying TG genotype of TP53 gene rs1042522 polymorphism Figure 4.18: Result of the nucleotide sequence of representative individual carrying GG genotype of TP53 gene rs1042522 polymorphism 37 Thus, after analyzing the IFNG rs2069718 gene polymorphism by genotyping and sequencing, there was no association between the TP53 gene rs1042522 and essential thrombocythemia 4.5 Discussion Essential thrombocythemia or primary thrombocythemia that happens when abnormal stem cells in your bone marrow make too many platelets Essential thrombocythemia is a chronic disease with no cure If you have a mild form of the disease, you may not need treatment If you have severe symptoms, you may need medicine that lowers your platelet count, blood thinners or both Essential thrombocythemia is an acquired genetic condition, meaning it happens when certain genes mutate or change In this study we observed nucleotide changes in the IFNG gene rs2069718, and a small change in the TYK2 gene rs280500, while the remaining genes: PI3KCA gene rs6443624 and TP53 gene rs1042522 remained unchanged That proves the association between the common polymorphism of the IFNG gene rs2069718 and the susceptibility to essential thrombocythemia in the Vietnamese population Specifically, in the IFNG gene rs2069718, the GG genotype frequency was increased in the patient and control groups In other words, the rs2069718 variant is associated with essential thrombocythemia To confirm the role of this variant on the susceptibility of essential thrombocythemia attack in Vietnamese population, the larger number of samples are needed to perform Therefore, this is a new direction for us to continue to develop our study focused on not only essential thrombocythemia, but also other cancer in Vietnamese population 38 CHAPTER 5: CONCLUSION AND SUGGESTION 5.1 Conclusion ❖ INFG Results of nucleotide sequence analysis (patients=103, healthy=113) obtained three genotypes including AA, AG and GG of IFNG gene rs2069718 polymorphisms in patient and control groups with the frequencies of (66, 64.08%; 0, 0% and 37, 35.92%) versus (71, 62.83%; 39, 34.51% and 3, 2.65%), through it, GG genotype of the IFNG rs 2069718 gene has a risk of causing essential thrombocythemia And P0.05 wasn’t considered statistically significant ❖ PI3KCA Results of nucleotide sequence analysis (patients=85, healthy=107) obtained three genotypes including CC, CA and AA of PI3KCA gene rs6443624 polymorphisms in patient and control groups with the frequencies of (64, 75.29%; 17, 20 % and 4,4.71 %) versus (75, 70.09%; 27, 25.23% and 5, 4.67%), respectively P>0.05 wasn’t considered statistically significant ❖ TP53 Results of nucleotide sequence analysis (patients=97, healthy=128) obtained three genotypes including GG, GC and CC of TP53 gene rs1042522 39 polymorphisms in patient and control groups with the frequencies of (30, 30.93%; 41; 42.27 % and 26, 26.8 %) versus (36, 28.13%; 56,43.75% and 36, 28.13%), respectively P>0.05 wasn’t considered statistically significant 5.2 Suggestion A larger sample size is necessary for further study on determining the risk of these variants of INFG, TYK2, PI3KCA, TP53 gene on the essential thrombocythemia in Vietnamese population 40 REFERENCES 1.https://my.clevelandclinic.org/health/diseases/24031-essentialthrombocythemia Meier B, Burton JH (2017) Myeloproliferative Disorders Hematol Oncol Clin North Am 31(6):1029-1044 Mehta J, Wang H, Iqbal SU & Mesa R (2014) Epidemiology of myeloproliferative neoplasms in the United States Leuk Lymphoma 55(3):595600 Nangalia J, Green AR (2017) 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