Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.
MINISTRY MINISTRY OF HEALTH OF EDUCATION AND TRAINING HANOI MEDICAL UNIVERSITY DANG DUY PHUONG CLINICAL, PARACLINICAL CHARACTERISTICS AND SCN5A GENE MUTATIONS IN PATIENTS WITH BRUGADA SYNDROME Specialized: Internal Medicine-Cardiology Code: 9720107 MEDICAL DOCTORAL THESIS HANOI - 2022 The Work has been successfully completed at: HANOI MEDICAL UNIVERSITY Science Instructors: Prof DO DOAN LOI, PhD, MD Assoc Prof TRAN HUY THINH, PhD, MD Reviewer 1: Assoc Prof Nguyen Oanh Oanh, PhD, MD Reviewer 2: Assoc Prof Phan Quoc Hoan, PhD Reviewer 3: Assoc Prof Ta Manh Cuong, PhD,MD The thesis has been defended at University-level Thesis Evaluation Council held in Hanoi Medical University At , 2022 This thesis may be found at: National Library Library of Hanoi Medical University LIST OF ANNOUNCED RESEARCH PROJECTS RELATED TO THESIS TOPIC Đặng Duy Phương, Nguyễn Quý Hoài, Nguyễn Minh Hà, Phạm Mạnh Hùng, Đỗ Doãn Lợi, Trần Vân Khánh, Trần Huy Thịnh (2019) Xác định đột biến gen SCN5A bệnh nhân hội chứng Brugada kỹ thuật giải trình tự gen Tạp chí Y Học TP Hồ Chí Minh, 23(4): 164-170 Đặng Duy Phương, Nguyễn Minh Hà, Đỗ Doãn Lợi, Trần Vân Khánh, Trần Huy Thịnh (2021) Phát đột biến D252N gen SCN5A bệnh nhân hội chứng Brugada Tạp chí Khoa học Cơng nghệ Việt Nam, 63(7): 1-6 Đặng Duy Phương, Nguyễn Minh Hà, Đỗ Doãn Lợi, Trần Vân Khánh, Trần Huy Thịnh (2022) Đột biến gen SCN5A yếu tố liên quan bệnh nhân hội chứng Brugada Việt Nam Tạp chí Y học Việt Nam, 512(1): 275-281 Đặng Duy Phương, Nguyễn Minh Hà, Đỗ Doãn Lợi, Trần Vân Khánh, Trần Huy Thịnh (2022) Khảo sát tính sinh bệnh đột biến gen SCN5A hội chứng Brugada Tạp chí Y học Việt Nam, 513(2): 216-224 Part A: INTRODUCTION BACKGROUND Brugada syndrome is an inherited disorder affecting the electrical conduction system of the heart, which leads to increased risk of sudden death The disease is caused by a mutation that reduces the function of at least one of 23 genes encoding voltage-gated ion channels in the myocardial cell membrane Among these genes, SCN5A mutations account for the largest proportion, 20-25% According to current guidelines, mutations of the SCN5A gene are the only group of mutation in which testing is recommended Despite this, the role of SCN5A gene testing in diagnosis, treatment orientation, and genetic counseling is still only based on expert opinion The pathogenicity of these mutations has not been considered as a risk factor in longitudinal follow-up studies There has not been any studies so far to investigate 23 related genes simultaneously Therefore, the correlation between the patient's history, clinical presentation and test results (phenotype) with the genetic mutation (genotype) remained unclear Vietnam is a Southeast Asian country, an area with the highest prevalence of Brugada syndrome in the world The number of studies on this disease is limited and no studies so far have identified the type of genetic disorder in Brugada patients OBJECTIVES The study "Clinical, paraclinical characteristics and SCN5A gene mutations in patients with Brugada syndrome" was carried out with the following objectives: Investigating the clinical presentation and test result characteristics of patients with Brugada syndrome Identifying SCN5A gene mutations and the correlation between gene mutations with clinical and test result characteristics URGENT NATURE OF THE STUDY Brugada syndrome is the leading cause of sudden cardiac death 90% of people affected are men and the average age of onset is 40 years old, which causes remarkeable social burden The number of clinical and genetic studies of Brugada syndrome has increased in recent years worldwide However, the role of SCN5A gene testing in diagnosis, treatment orientation, and genetic counseling for the syndrome is still only based on expert opinion Vietnam is a Southeast Asian country, an area with the highest prevalence of Brugada syndrome in the world The number of studies investigating this disease is limited with no study so far has identified the type of genetic mutation in Brugada patients We aim to identify the rate of SCN5A gene mutation in patients with Brugada syndrome in our country and seek for any difference in clinical presentation and test result characteristics between the two groups with and without mutations These data will be the fundamental information for further studies on the genotype–phenotype association, diagnostic approach and risk stratification of Brugada syndrome in the future Therefore, research on SCN5A gene mutations in Brugada syndrome is of great interest NEW CONTRIBUTIONS FROM THE THESIS This is the first large-scale study in Vietnam which assesses both clinical presentations and molecular biology, investigating genetic disorders that leads to cardiac arrhythmias and heart conduction disorder in general and Brugada syndrome in particular Our study is an important foundation for building diagnostic approach and risk stratification in the future Our study dertermined the rate of SCN5A gene mutation in Brugada syndrome in Vietnam, and identified 10 new mutations which have not been published on biological databases The study also showed the initial correlation between the clinical and paraclinical characteristics and SCN5A gene mutation as well as detected gene carriers among family members The combination of modern molecular biology techniques in mutation analysis would assist clinicians in diagnosing the cause of disease, guiding genetic counseling and prevention, hence reduce the disease burden on the patients’ family and on society In addition, our study adds valuable data to the mutation map of the disease and helps to assess the genotype-phenotype relationship, paving the way for epidemiological management of the disease on the molecular level THESIS OUTLINE This thesis covers 125 pages, including: preamble (2 pages), the literature review (37 pages), materials and method (16 pages), results (33 pages), discussion (36 pages), conclusion (1 page), petition (1 page) It consists of 26 tables, charts, diagram, 27 figures, appendices and 133 references (8 in Vietnamese and 124 in English) Part B: THESIS CONTENT CHAPTER LITERATURE REVIEW 1.1 Brugada syndrome Brugada syndrome is characterized by disorder of the repolarization process, presenting on the electrocardiogram as right bundle branch block and ST elevation of ≥ mm on right precordial leads, with an increased risk of syncope and sudden death The prevalence of the disease is - 0.1% in the United States and Europe, and 0.1 - 1.4% in Southeast Asia 90% patients are men, and the median age of onset is 40 years old Brugada syndrome is diagnosed according to guidelines from the European Society of Cardiology in 2015 and is classified into types according to the features of ST segment elevation in V1-V3 on electrocardiogram Most patients are asymptomatic, detected incidentally or intentionally by electrocardiogram Symptoms of the disease are associated with life-threatening ventricular arrhythmias, including: syncope, paraxysmal nocturnal dyspnea, and sudden cardiac death The patient may have a relative who has been diagnosed with the disease, or had sudden cardiac death, or unexplained episodes of syncope The main tools to help diagnose the disease include electrocardiogram, flecanide test and electrophysiological profile The current treatment therapy is avoidance of triggers leading to ventricular arrhythmia, automated defibrillator implantation, radiofrequency ablation, and medication 1.2 SCN5A gene mutation in Brugada syndrome Brugada syndrome is caused by gene mutations on autosomes that results in structural or functional defects of the votage-gated ion channels in the myocardial cell membrane These are polygenic mutations, with several different pathological mechanisms: reducing the influx of sodium-calcium or increasing the efflux of potassium, hence creating the characteristic manifestations on electrocardiogram, as well as episodes of ventricular tachycardia, ventricular fibrillation Until recently, about 23 genes have been identified to be involved, and each type of mutation has distinctive features regarding prevalence, pathogenicity, the level of function of the encoded proteins Mutations in the SCN5A gene account for the highest proportion (20-25% in the Brugada syndrome group) The SCN5A gene is a gene located on chromosome consisting of 28 exons and coding protein Nav1.5 This protein is the alpha half-unit of the voltage-dependent sodium channel on the myocardial cell membrane It forms a channel hole, a potential sensing region, and many associated beta subunits that regulate channel activity SCN5A gene mutations disrupt the original physiological function of the protein, causing several types arrhythmias and conduction disorders Depending on the location of mutation on the SCN5A gene, structural changes in specific regions of the protein reduce the function of the membrane channels Determining the pathogenicity of each mutation is difficult due to its rarity, so it has only been predicted by structural analysis and prediction of mutant protein function Determining the genotype-phenotype correlation and disease risk to add in management practices is also challenging for the same reason above Current official guidelines only recommend testing for SCN5A gene because of its highest prevalence and proven pathogenic mechanism Gene mutation testing is performed by next-generation sequencing or Sanger sequencing, which aids in genetic counseling and diagnosis 1.3 Current studies of SCN5A gene mutation in Brugada syndrome in Vietnam In our country, from 2001 to present, there have been only reports in the area of Brugada syndrome These reports focus on clinical presentations, mainly clinical case reports, case series reports, or crosssectional studies There has been no studie with longitudinal follow-up cohort designated to assess cardiovascular events There also has been no published study about mutation genes, as well as evaluating the correlation between gene mutation and clinical characteristics Thus, the data gap in this topic in our country include: The prevalence of mutation in the SCN5A gene in patients with Brugada syndrome? Do the gene mutation carriers have any clinical and laboratory characteristics different from the ones without mutation? Which type of SCN5A gene mutation is pathogenic, and whether it helps prognose or predict the risk of ventricular arrhythmia events? What are the benefits of testing for SCN5A mutations in the diagnosis and management of Brugada syndrome? PART MATERIALS AND METHODS 2.1 Materials 117 patients with a confirmed diagnosis of Brugada syndrome according to the 2015 European Society of Cardiology diagnostic criteria and agreed to participate in the study 2.2 Study design Cross-sectional descriptive study, from 01/01/2017 to 04/30/2022 2.3 Study method 2.3.1 Study process - Patient selection: review from medical records retrospectively, contact and inform the patient about the study and obtain informed consent from patients to participate in the study - Collection of clinical characteristics and test results: from medical records or in-person interview during follow-up visits (to collect missing information) - Collection of mL of venous blood for SCN5A gene testing - Identify mutations (if any) of the SCN5A gene by next-generation sequencing technique Recheck mutation using Sanger sequencing The pathogenicity of mutations is classified according to the ACMG classification, which includes: pathogenic, possibly pathogenic, benign/neutral, unspecified Use ClinVar database and recognized online software to predict the pathogenicity of mutations (PolyPhen-2, Mutation Taster, Provean, SNP&GO) Pathogenicity conclusion is the final result based on the consensus of the softwares Mutations that are predicted differently between softwares will be classified as "unknown" - Data processing: according to the research objectives - Select 1-3 patients with mutations and their first-degree family members agree to screen for genetic pedigree - Summarize and present thesis results 2.3.2 Techniques used in the study Electrophysiology study; flecanide test; next-generation sequencing; Sanger sequencing 2.4 Data collection location Data collection at hospitals in Ho Chi Minh City: Thong Nhat Hospital, Tam Duc Heart Hospital, Heart Institute of Ho Chi Minh City; and hospitals in Hanoi: Bach Mai Hospital, Hanoi Heart Hospital Genetic mutation testing is performed at the Gene - Protein Research Center, Hanoi Medical University 2.5 Data processing Data processing and analyzing using SPSS 20.0 software The chisquare tool is used to compare the difference between groups, pG p.His184Arg (H184R) Possibly pathogenic c.754 G>A p.Asp252Asn (D252N) Pathogenic c.797 T>A p.Leu266His (L266H) Pathogenic c.980 G>A p.Gly327Glu (G327E) Pathogenic c.1100 G>A p.Arg367His (R367H) Pathogenic c.1890+14 G> A Intron splicing Inconclusive c.1712 G>C p.Ser571Thr (S571T) Possibly pathogenic c.1975 C>T p.Arg659Trp (R659W) Pathogenic Clinical features (group of pathogenic mutations) Male, 38 yo, had family member with sudden death < 45 yo; syncope, type ECG Male, 51 yo, had family member with sudden death < 45 yo; syncope, type ECG, flecanide test (+), ICD Male, 42 yo, had family member with sudden death < 45 yo; ventricular tachycardia, type ECG, flecanide test (+) Male, 44 yo, had family member with sudden death < 45 yo; type ECG Male, 36 yo, syncope, cardiac arrest, type ECG, ICD placed Male, 27 yo, had family member with sudden death < 45 yo; syncope, type ECG, ICD placed Male, 31 yo, had family member with sudden death < 45 yo; syncope; ventricular tachycardia, type ECG, ICD placed Male, 25 yo, syncope, type ECG, test flecanide (+), ICD placed Male, 38 yo, syncope; ventricular tachycardia, type ECG, ICD placed Male, 43 yo, had family 11 member with sudden death < 45 yo; syncope; ventricular tachycardia, type ECG, test flecainide (+), ICD placed c.2236 G>A p.Glu746Lys (E746K) Possibly pathogenic c.2678 G>A p.Arg893His (R893H) Pathogenic c.2893 C>T p.Arg965Cys (R965C) Pathogenic Benign Benign Possibly pathogenic c.3338 C>T c.3578 G>A c.4171 G>A p.Ala1113Val (A1113V) p.Arg1193Gln (R1193Q) p.Gly1391Arg (G1391R) c.4531 C>T p.Arg1511Trp (R1511W) Pathogenic c.4850_485 delTCT p.Phe1617del (F1617del) Pathogenic Female, 64 yo, had family member with sudden death < 45 yo; cardiac arrest, type ECG, electrophysiological profile (+), ICD placed Male, 23 yo, had family member with sudden death < 45 yo; syncope, type ECG, ICD placed Male, 35 yo, had family member with sudden death < 45 yo; syncope; paroxysmal nocturnal dyspnea, type ECG, electrophysiological profile (+), ICD placed Male, 57 yo, had family member with sudden death < 45 yo; type ECG, electrophysiological profile (+), ICD placed Male, 38 yo, syncope, type ECG, ICD placed Male, 46 yo, syncope, paroxysmal nocturnal dyspnea, type ECG, electrophysiological profile (+), ICD placed Male, 42 yo, had family member with sudden death < 45 yo; syncope, type ECG, ICD placed Male, 47 yo, syncope, type ECG, electrophysiological 12 profile (+), ICD placed Male, 49 yo, syncope, paroxysmal nocturnal dyspnea, type ECG, ICD placed c.5389 A>T c.5484 C>T c.5693 G>A p.Ile1797Phe (I1797F) p.Ala1828Ala (A1828A) p.Arg1898His (R1898H) Possibly pathogenic Benign Pathogenic Male, 57 yo, had family member with sudden death < 45 yo; syncope, type ECG, ICD placed (yo: years old) 3.2.2 Brugada syndrome patients with two mutations in the SCN5A gene simultaneously In 117 patients collected, 2/117 carried two types of mutations on the SCN5A gene, accounting for 1.71% Table Characteristics of cases with mutations in SCN5A gene simultaneously Characteristics Age SCN5A gene mutation type and predicted pathogenicity Symptoms, history ECG type Test results (*) Treatment Patient BrS14 35 − N109del: Pathogenic − R1193Q: Possibly pathogenic Asymptomatic, discovered incidentally Not performed Patient BrS57 41 − A1113V: Possibly pathogenic − G1391R: benign Asymptomatic, discovered incidentally Electrophysiology study (+) ICD placed Not treated (*) including: flecanide test, electrophysiological profile These are cases with complex combination of mutations - two simultaneous mutations in the SCN5A gene Both patients had the Brugada ECG pattern typical for type of the disease, and both had no clinical symptoms Predicting the combined in silico pathogenicity of these two cases has not been able The case of patient with N109del and R1193Q has not expressed phenotype and did not receive specific treatment 13 3.2.3 Results of family member mutation screening for the SCN5A gene The study analyzed pedigrees of patients Table Summary of pedigree analysis results Number of Type of member mutation screened Pedigree 01: Patient BrS14 members -Brother; -03 children N109del (pathogenic) + R1193Q (benign) Mutation result Note -Patient: (+), pathological phenotype -Brother: (-) -Two daughters: (-) - One son: (+), has not shown pathological phenotype -Not able to perform genetic testing for the patient's parents It inconclusive whether this is an inherited mutation or a novel mutation - The patient’s son inherited the same type of mutation from the patient but has not shown any pathological phenotype and should be investigated for disease risk -Patients: (+), pathological phenotype; -Father: (-) -Brother: (-) -Sister: (-) -Son: (+), has not shown pathological phenotype -2 nephews: (-) - The patient's mother and brother have not been tested It is not possible to conclude whether this is an inherited or novel mutation in the patient's generation -No gene testing has been performed for the nephew (son of the brother with sudden death) since he is currently abroad, and phenotype is also unclear - The son inherited the same type of mutation from the patient but has not shown any pathological phenotype, and should be investigated for disease risk Pedigree 02: Patient BrS117 members -Father -Brother -Sister -Son -2 nephews R659W (pathogenic) 14 3.2.4 Comparison of clinical characteristics between genetic mutation groups Table Comparison of clinical characteristics between genetic mutation groups With mutation (n = 30) Without mutation (n = 87) P value Male sex 29 (96,67) 85 (97,70) 1,000 Age 43,6 ± 10,6 48,3± 9,8 0,0560 Had family member with sudden death < 45 yo 20 (66,67) 17 (19,54) < 0,00001 Symptomatic 13 (43,33) 42 (48,28) 0,6399 Unexplained syncope 19 (63,33) 26 (29,89) 0,0012 Paroxysmal nocturnal dyspnea (13,33) (8,05) 0,3922 Ventricular tachycardia, ventricular fibrillation (20,00) (2,30) 0,0035 Resuscitated cardiac arrest (13,33) (3,45) 0,0490 Not treated (30,00) 21 (24,14) 0,5260 ICD placed 21 (70,00) 65 (74,71) 0,6140 Other arrhythmias (3,33) (6,90) 0,6759 Atrial fibrillation (3,33) (3,45) 0,5804 Other diseases (26,67) 15 (17,24) 0,2626 14 (46,67) 29 (33,33) 0,1915 Frequency, percentage (n, %) Symptoms Treatment Comorbidity cardiovascular Other diseases 15 3.2.5 Comparison of paraclinical characteristics between genetic mutation groups Table Comparison of paraclinical characteristics between genetic mutation groups Frequency, percentage (n, %) ECG type Type Type Type Flecanide test (+) Electrophysiological profile (+) With mutation (n = 30) Without mutation (n = 87) P value 23 (76,67) (10,00) (13,33) (23,33) 50 (57,47) 17 (19,54) 10 (11,49) (2,30) 0,0613 0,2313 0,7890 0,001 10 (33,33) 45 (51,72) 0,0818 3.2.6 Relationship between clinical characteristics and SCN5A gene mutation Table Relationship between clinical, paraclinical characteristics and SCN5A gene mutation (n=30) Characteristic Odds ratio 95%CI P value Had family member with sudden death < 45 yo 8,1 3,2 - 20,5