Tom tat LATS (tieng anh): 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.Nghiên cứu đặc điểm lâm sàng, cận lâm sàng và đột biến gen SCN5A ở bệnh nhân hội chứng Brugada.

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

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

2 Đặng Duy Phương, Nguyễn Minh Hà, Đỗ Doãn Lợi, Trần Vân Khánh, Trần Huy Thịnh (2021) Phát hiện đột biến mới D252N

trên gen SCN5A ở bệnh nhân hội chứng Brugada Tạp chí Khoa học và Công nghệ Việt Nam, 63(7): 1-6

3 Đặng Duy Phương, Nguyễn Minh Hà, Đỗ Doãn Lợi, Trần Vân

Khánh, Trần Huy Thịnh (2022) Đột biến gen SCN5A và các yếu

tố liên quan ở bệnh nhân hội chứng Brugada Việt Nam Tạp chí Y học Việt Nam, 512(1): 275-281

4 Đặng Duy Phương, Nguyễn Minh Hà, Đỗ Doãn Lợi, Trần Vân Khánh, Trần Huy Thịnh (2022) Khảo sát tính sinh bệnh của đột

biến gen SCN5A trong hội chứng Brugada Tạp chí Y học Việt Nam, 513(2): 216-224

Part A: INTRODUCTION

1 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

2 OBJECTIVES

The study "Clinical, paraclinical characteristics and SCN5A gene

mutations in patients with Brugada syndrome" was carried out with

the following objectives:

1 Investigating the clinical presentation and test result characteristics of patients with Brugada syndrome

2 Identifying SCN5A gene mutations and the correlation between gene mutations with clinical and test result characteristics

3 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

4 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

5 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, 3 charts, 1 diagram, 27 figures, 5

appendices and 133 references (8 in Vietnamese and 124 in English)

Part B: THESIS CONTENT

CHAPTER 1 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 ≥ 2 mm on right precordial leads, with an increased risk of syncope and sudden death The prevalence of the disease is 0 - 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 3 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 3 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 7 reports in the area of Brugada syndrome These reports focus on clinical presentations, mainly clinical case reports, case series reports, or cross-sectional 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 2 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

- Collection of clinical characteristics and test results: from medical records or in-person interview during follow-up visits (to collect missing information)

- Collection of 4 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 3 hospitals in Ho Chi Minh City: Thong Nhat Hospital, Tam Duc Heart Hospital, Heart Institute of Ho Chi Minh City;

and 2 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 square tool is used to compare the difference between groups, p<0.05 is considered to be statistically significant Calculate the odds ratio to assess the possible correlation between the recorded characteristic and

chi-the SCN5A gene mutation

2.5 Ethics in medical research

The study was conducted based on voluntary participation of patients, ensuring confidentiality and fairness in recruitment The study has been granted a certificate of ethical approval for biomedical research by the Ethical Council of Hanoi Medical University (No 48/HĐĐĐĐHYHN, January 12th, 2017)

Study processing algorithm flowchart

PART 3 STUDY RESULTS

117 patients diagnosed with Brugada syndrome were collected, clinical and laboratory data and mutations in the SCN5A gene were recorded Males accounted for the majority of our samples, 97.44% compared to females with only 2.56% The average age was 47.5 ± 12.4 years old, the oldest was 79 years old and the youngest was 23 years old

3.1 Clinical and paraclinical characteristics

3.1.1 Clinical characteristics

Table 1 Clinical features

Clinical features Frequency

(*) Each patient may have more than one symptom or comorbidity Other associated arrhythmias reported were atrial fibrillation (4 cases),

ventricular extrasystoles (1 case) and II-III degree AV block (1 case)

3.1.2 Paraclinical characteristics

Table 2 Paraclinical characteristics

Laboratory features Frequency

(*) All of these 11 cases were performed at the Heart Institute of

Ho Chi Minh City

3.2 SCN5A gene mutation and the relationship between clinical characteristics and SCN5A gene mutation in patients with Brugada

syndrome

3.2.1 SCN5A gene mutations

33 mutations were identified in 30 Brugada patients, including 20 different types of mutations These mutations are all heterozygous and the inheritance pattern is autosomal dominant Of the 28 exons of the SCN5A gene, the most common mutation occurs in exon 13 and 17 (12.12%); followed by mutation on exon 28 (9.09%) No mutations were detected on exons 4, 6, 10, 11, 15, 19, 21-23, 25 Mutation on intron 12 accounted for 6.06% Mutations in coding regions (coding

exons) were 84.85% and noncoding regions were 15.15% (including introns; exon 1 and the first part of exon 2 - the 5' untranslated region (5' UTR); and exon 28 – the 3' UTR)

Table 3 Characteristics of SCN5A gene mutations

SCN5A gene mutation features Frequency Percentage

(*) excluding 2 intron mutations

Figure 1 Illustration of the sequence of the H184R mutation of the

SCN5A gene

Evaluation of the pathogenicity of mutations based on ClinVar database and in silico predictive software (Table 4) The clinical and laboratory features of the patients are widely diverse and there is nearly

no pathogenicity principle observed based on the data

Table 4 Pathogenicity of SCN5A gene mutations

Fre-Predicted patho- genicity

Clinical features (group of pathogenic mutations)

45 yo; syncope, type 2 ECG, flecanide test (+), ICD c.551 A>G p.His184Arg

Possibly pathogenic

G> A Intron splicing 2 Inconclusive

c.1712 G>C p.Ser571Thr

Possibly pathogenic

Male, 25 yo, syncope, type 2 ECG, test flecanide (+), ICD placed

Male, 38 yo, syncope; ventricular tachycardia, type 3 ECG, ICD placed

Male, 43 yo, had family