PATHOLOGICAL ANATOMY OF THE HEART AND SCN5A GENE MUTATIONS IN YOUNG INDIVIDUALS WITH SUDDEN CARDIAC DEATH

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH HANOI MEDICAL UNIVERSITY NGUYEN TAT THO PATHOLOGICAL ANATOMY OF THE HEART AND SCN5A GENE MUTATIONS IN YOUNG INDIVIDUALS WITH SUD

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH

HANOI MEDICAL UNIVERSITY

NGUYEN TAT THO

PATHOLOGICAL ANATOMY OF THE HEART

AND SCN5A GENE MUTATIONS

IN YOUNG INDIVIDUALS WITH SUDDEN CARDIAC DEATH

Field of Study : Biomedical Science

SUMMARY OF THE DOCTORAL DISSERTATION IN MEDICINE

HA NOI - 2025

THIS DISSERTATION WAS COMPLETED AT

HANOI MEDICAL UNIVERSITY

Supervisors:

1 Dr Luu Sy Hung

2 Assoc Prof Dr Ta Van To

Reviewer 1: Assoc Prof Dr Nguyen Van Ha

Reviewer 2: Assoc Prof Dr Luong Thi Lan Anh

Reviewer 3: Dr Nguyen Sy Lanh

The dissertation will be defended before the Dissertation Evaluation Council at Hanoi Medical University

Time: … hour …, date … month … year 2025

The thesis can be found at:

Vietnam National Library

Hanoi Medical University Library

LIST OF PUBLISHED WORKS

1 Nguyen Tat Tho, Luu Sy Hung, Au Quoc Duong Uy, Sudden unexplained death in children with rare causes identifed by forensic medical examination, Journal of Medical Research, 162 (1), 2023: 214 - 220

2 Nguyen Tat Tho, Luu Sy Hung, Trinh Xuan Ha, Au Quoc Duong Uy Epidemiological and histopathological characteristics of cardiac lesions in young sudden cardiac death, Journal of Medical Research, 179 (6), 2024: 117 - 125

3 Nguyen Tat T, Lien NTK, Luu Sy H, Ta Van T, Dang Viet D, Nguyen Thi H, Tung NV, Thanh LT, Xuan NT, Hoang NH Identifying the Pathogenic Variants in Heart Genes in Vietnamese Sudden Unexplained Death Victims by

Next-Generation Sequencing Diagnostics 2024;14(17):1876

https://doi.org/10.3390/diagnostics14171876

1

DISSERTATION INTRODUCTION

1 Rational of the study

Sudden cardiac death (SCD) is defined as sudden and unexpected death from cardiac causes, occurring within one hour of symptom onset or within 24 hours of being declared healthy in people with known or unknown heart disease

Several pathological characteristics of the heart in SCD cases among patients which have young age, have been reported worldwide, including abnormal myocardial thickening, fibrotic scarring, and structural abnormalities of the coronary arteries Hypertrophic cardiomyopathy (HCM) is one of the leading causes of SCD in young individuals, with an estimated annual mortality rate of approximately 1% Other studies have indicated that about 10–20% of SCD cases

in young individuals involve myocardial scarring, which is a risk factor for arrhythmias and sudden death However, in Vietnam, no comprehensive and systematic study has been conducted on the pathological anatomy of the heart in SCD victims within the young age group

Forensic autopsies in Vietnam have shown that many SCD cases in patients who have young in age exhibit subtle histopathological changes in the myocardium, often insufficient to determine the exact cause of death Recent studies have focused on identifying genetic variants associated with cardiovascular diseases to elucidate the genetic mechanisms and pathophysiology underlying SCD in young individuals Preliminary findings have identified several genes related to inherited cardiac conditions, such as SCN5A, KCNQ1, and KCNH2, which are associated with long QT syndrome, while PKP2, DSP, and DSG2 are linked to hypertrophic cardiomyopathy A study conducted by Hager Jaouadi et al (USA) shown that there is about 40% of 173 SCD cases in young individuals which were associated with genetic variants Timothy F Simpson et al (Europe) studied

302 SCD cases in young individuals and reported that 36% had inherited genetic variants

In Vietnam, apart from a few studies on Brugada syndrome and the SCN5A gene, no research has systematically combined pathological examination and genetic mutation analysis of cardiovascular-related genes in SCD cases The lack of comprehensive studies may limit forensic applications in determining the causes of sudden death This study aims to investigate the cardiac pathological features and genetic mutations associated with SCD in young people, thereby providing forensic experts with scientific evidence to diagnose the cause of death In addition, the findings can serve as a foundation for prevention and treatment strategies to improve survival rates,

For the aforementioned reasons, we conducted this study titled: "Pathological

Anatomy of the Heart and SCN5A gene mutations in young individuals with sudden cardiac

death " with the following two objectives:

Objective 1: To describe the cardiac pathological anatomy of the heart in young individuals with sudden cardiac death

Objective 2: To identify genetic variants associated with cardiovascular diseases in young individuals with sudden cardiac death

2

2 Novel contributions of the dissertation

This is the first study in Vietnam within the field of forensic medicine to utilize Generation Sequencing (NGS), combined with Sanger sequencing validation, ensuring comprehensive, accurate, and reliable identification of genetic variants in young SCD cases (aged 1–40 years)

Next-For the first time, the characteristics and prevalence of fundamental pathological lesions associated with cardiovascular diseases leading to SCD in young individuals have been systematically analyzed, documented, and described in detail in this study

The analysis of a large gene panel (166 genes) using Next-Generation Sequencing (NGS), with variant validation through Sanger sequencing and pathogenicity prediction using ClinVar and in silico tools, has led to highly valuable new conclusions, including:

- Among the 166 analyzed genes, 20 genes were found to carry mutations, identifying a total of 25 genetic variants

- Of the 25 identified variants, in addition to 7 previously reported pathogenic variants

in the ClinVar database, 12 novel variants were discovered, and 6 variants had been published in ClinVar but had not yet been classified for pathogenicity The discovery of these novel variants contributes valuable data to international genetic databases

- 20 out of 52 SCD cases (38.5%) carried at least one mutated gene

- 13 out of 52 SCD cases (25%) carried pathogenic variants, providing strong evidence that genetic mutations in these cases were directly associated with cardiovascular-related causes of death

- This study combined advanced molecular techniques like NGS sequencing with classical methods such as Sanger sequencing to accurately detect and confirm genetic variants By doing so, it helps refine the mutation landscape and offers a clearer understanding of how specific genetic changes relate to disease characteristics

This study provides important insights for forensic investigations involving SCD cases and sudden unexplained death (SUD) At the same time, it lays the foundation for applying molecular biology techniques in forensic research and diagnostic pathology

3 Structure of the dissertation

The dissertation is 127 pages long, with the following structure: 2 pages for the introduction, 35 pages for the literature review, 24 pages covering research subjects and methods, 28 pages presenting research findings, 36 pages for discussion, 01 pages for conclusions, and 1 page for recommendations It includes 35 tables, 19 figures, and 03

charts, with a total of 221 references

As part of this research, the doctoral candidate has published three articles in reputable scientific journals Two of them, written in Vietnamese, were published in the Journal of Medical Research by Hanoi Medical University, while one English-language article appeared in Diagnostics, an ISI/Scopus-indexed journal

3

CHAPTER 1

LITERATURE REVIEW 1.1 GENERAL UNDERSTANDING OF SCD

1.1.1 Definition and Related Terminology

Definition of the sudden cardiac death

Sudden cardiac death (SCD) is defined as a sudden, unexpected death occurring within one hour of symptom onset or in patients without prior symptoms within the preceding 24 hours, caused by arrhythmias or hemodynamic collapse

Many researches on SCD in general and SCD in young individuals have chosen "young" research subjects aging from 1 to 40 years old According to the 'Consensus Statement on Recommended Autopsy Techniques for Young Sudden Cardiac Death Victims', the young SCD population refers to individuals aged between 1 and 40 years old

1.1.2 Classification of sudden cardiac death

Classification by coronary artery disease (Based on P Markwerth’s study)

- Causes due to coronary artery disease;

- Causes not due to coronary artery disease

Classification by structural heart damage (Based on G.L Sumner’s study)

- SCD group that has structural heart damage, including:

+ Cause due to sudden cardiac

+ Causee due to non - sudden cardiac

- SCD group that non structural cardiovascular abnormalities

1.2 ANATOMY OF CORONARY ATHEROSCLEROSIS

1.2.1 Pathological morphology of coronary atherosclerosis

1.2.1.1 Fatty Streaks or Endometrial xanthoma:

Macroscopic: Yellowish dots (~1mm) or streaks; flat or slightly elevated

Microscopic: Foam cells containing lipids, elongated smooth muscle cells with vacuolated cytoplasm (lipid-laden), and lymphocytes located beneath the endothelium

1.2.1.2 Complete atherosclerotic plaque

Macroscopic: Oval, white or yellow-white lesions measuring 0.3–1.5 cm along the coronary artery Fibrous cap is white and firm; necrotic core is yellowish, soft

Microscopic: Lesion located in the intima with two main components: fibrous cap and necrotic core (atheroma)

1.2.1.3 Atherosclerotic progression

The formation of an atherosclerotic plaque goes through the following stages: fatty streaks/fatty dots → complete atherosclerotic plaque → progressing atherosclerotic plaque

1.2.2 Classification (Type) of coronary atherosclerosis

1.2.2.1 According to AHA 2000 classification, there are 8 Types (Type I to Type VIII):

- The first 3 Types (Type I, Type II and Type III) have histopathological images of

"Fatty streak", and are not considered "atherosclerotic plaque"

- Type IV is the intermediate type, the initial stage of lipid core formation

4

- From Type V onwards, it is considered “complete atherosclerotic plaque”

- Type VI, VII, VIII: Advanced lesions (including hemorrhage, calcification, and fibrosis)

1.2.2.2 Pathological morphology of coronary atherosclerosis types

There are 7 types of pathological lesions including: adaptive intimal thickening; fatty streaks; pathological intimal thickening; fibrous cap; thin fibrous cap of atherosclerotic plaque; calcified nodules and fibrotic plaques

1.2.3 Coronary artery thrombosis

1.2.3.1 Causes of coronary artery thrombosis formation

There are 3 major causes for the mechanism of coronary artery thrombosis formation: atherosclerotic plaque rupture, atherosclerotic plaque erosion and calcification

1.2.3.2 Progress of coronary artery thrombosis

Consisting of acute coronary thrombosis and chronic thrombosis

1.2.4 Some other of coronary artery lesions

1.2.4.1 Coronary Artery Stenosis

In forensic medicine, the percentage of 75 % in narrowing is considered sufficient to determine the cause of SCD

1.2.4.2 Myocardial Bridging

The myocardial bridging is a congenital anatomical variation, in which segment of the coronary artery runs within the myocardial tissues, instead of lying on the epicardial surface

1.2.4.3 Congenital Coronary Artery Anomalies

Including anomalous origins, abnormal pathways, and accessory arteries, which are associated with ischemia and coronary thrombosis

1.3 PATHOLOGICAL ANATOMY OF THE MYOCARDIAL INFARCTION 1.3.1 Figure of the myocardial infarction lesions

From 12 - 24 hours: The myocardial infarction zone appears palmer than the surrounding From 24 - 72 hours: The myocardial infarction zone has pale yellow, with hemorrhage at the periphery From 3 to 10 days: The myocardial infarction zone is softer and depressed, which have a distinct red border From day 10 onward: The scar formation begins at the center of the myocardial infarction zone

1.3.1 Microscopic figure of myocardial infarction

- Early stage: Myocyte degeneration due to ischemia

- Inflammatory infiltration stage: Infiltration by neutrophils, mononuclear cells, macrophages, and lymphocytes; necrosis of cardiomyocytes

- Granulation tissue stage: Lymphocytes with collagen fibers arranged loosely

- Scar formation stage: Collagen fibers replace necrotic cardiomyocytes and neovascularization

1.4 PATHOLOGICAL ANATOMY OF CARDIOMYOPATHY

1.4.1 Hypertrophic cardiomyopathy

Macroscopic: thick heart wall, interventricular septum 1.3 times thicker that left ventricle Microscopic: large myocardial cells, structural disturbance Interstitial fibrosis, some myocytes having box-shaped nuclei

1.4.2 Myocarditis

Macroscopic: enlarged heart size, dilated heart chambers, flaccid myocardium Microscopic:

+ Lymphocytic and mixed myocarditis: predominantly lymphocytic

+ Giant cell myocarditis: microscopic images including lymphocytes, histiocytes

and multinucleated giant cells

5

1.4.3 Ischemic cardiomyocyte degeneration

There are 4 basic forms of ischemic myocardial cell damage: Wavy cardiomyocyte; Myocardial “contractile band necrosis”; Degenerated cardiomyocytes, loss of nucleus; Myocardial fiber fragmentation

1.4.4 Others pathological forms of cardiomyopathy

1.4.4.1 Myocardial fibrosis

- Interstitial Myocardial Fibrosis; Replacement Myocardial Fibrosis; Perivascular Fibrosis

1.4.4.2 Dilated Cardiomyopathy

Macroscopic: Enlarged heart, losing myocardial tone, ventricular walls thin

Microscopic: cardiomyocytes of varying sizes, with some cells appearing atrophic Interstitial fibrosis presence, with thickened fibrous bands diffusely surrounding myocardial cells Scattered inflammatory cells and subepicardial fatty infiltration

1.4.4.3 Fibrofatty Infiltration of the Myocardium

The myocardium is replaced by fatty tissue, which is accompanied by interstitial fibrosis

1.5 PATHOPHYSIOLOGICAL MECHANISMS OF SCD

Cardiac death is lethal arrhythmia Types include: ventricular fibrillation, atrial fibrillation, and ventricular tachycardia

Causes of arrhythmia can be primary or secondary

1.6 GENETIC VARIANTS ASSOCIATED WITH CARDIOVASCULAR DISEASES 1.6.1 Necessity of investigating genetic variants in SCD victims

SCD can be caused by genetic mutations affecting cardiomyopathies and ion channelopathies

1.6.1 Genes involved in hereditary cardiomyopathy

1.6.2.1.Genes associated with hypertrophic cardiomyopathy

About 40–60% of HCM cases are due to mutations in: MYH7, MYBPC3, TNNT2, TNNI3, and ACTC1

1.6.2.2 Genes associated with dilated cardiomyopathy

The main cause of dilated cardiomyopathy is mutations in genes related to the structure

of the myocardium: TTN (titin), LMNA (lamin A/C), MYH7, and SCN5A Mutations in the TTN gene have been recorded as the most common cause, accounting for about 25% of cases of dilated cardiomyopathy that have genetic origin

1.6.2.3 Genes associated with arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy is strongly associated with mutations

in genes: PKP2 (plakophilin 2), DSP (desmoplakin), DSG2 (desmoglein 2), and JUP

1.6.3 Genes associated with ion channel disorders

1.6.3.1 Genes Related to Long QT Syndrome (LQTS)

The primary genes associated with LQTS include: KCNQ1, KCNH2, and SCN5A,

which about 75 % of LQTS cases caused by mutations in these genes

1.6.3.2 Genes Associated with Brugada Syndrome

Around 20–25% of Brugada cases are associated with SCN5A mutations and Other implicated genes include CACNA1C, GPD1L, and KCNE3 Notably, CACNA1C gene associated of Brugada syndrome

6

CHAPTER 2 RESEARCH SUBJECT AND METHODOLOGY 2.1 RESEARCH SUBJECT

2.1.1 Research subject

206 SCD cases, aged from 1-40, were forensically examined from June 2021 to June

2024, at the Hanoi Forensic Center (57), Ho Chi Minh City Forensic Center (115) and the Military Forensic Institute (34)

2.1.2 Selection criteria

- SCD subjects who have full forensic records

- Correctly and fully obey the autopsy procedure and sample collection procedure;

- Permission for research from the Forensic Medicine Center

2.1.3 Exclusion criteria

- Have combined diseases; Decomposed body; Tested positive for drugs, COVID-19, HIV

2.2 METHODOLOGY

2.2.1 Study design

A descriptive cross-sectional study, combining prospective and retrospective data collection

2.2.2 Sample collection and size

- Sample collection: Convenient, comprehensive and purposive sampling

- Total sample size: 206 cases of SCD victims

2.2.3 Research time and location

- Time: From 01/2022 to 09/2024

- Location: Department of Forensic Medicine - Hanoi Medical University; Military Forensic Institute; Hanoi Forensic Center; Ho Chi Minh City Forensic Center

2.3 RESEARCH CONTENT

2.3.1 Research variables and indicators

- Age group, gender, occupation, physical condition, time of death

- Characteristics of macroscopic and microscopic cardiac pathology

- Characteristics of mutant genes and gene variants

2.3.2 Process techniques used during research

- Autopsy process

- Histopathological examination process

- ADN analysis process (NGS, Sanger)

- Process of analyzing genomic data and predicting pathogenicity of variants

2.3.3 Data management and analysis

Data entry using Epidata software Statistical analysis using SPSS version 27.0

2.4 RESEARCH EQUIPMENT AND CHEMICALS

- Chemical equipment for pathological research

- Genetic testing equipment and chemicals

7

CHAPTER 3 RESEARCH RESULTS 3.1 CHARACTERISTICS OF RESEARCH SUBJECTS

3.1.1 Number of research subjects according to each objective

Table 3.1 SCD research subjects

Investigation of the pathological characteristics of the heart

and coronary arteries

Observation: A total of 154 cases (74.8 %) exhibited specific pathological lesions 52 cases (25.2 %) have to do genetic testing, with the following results: 23 exhibited detectable mutations (44.2%), while 29 showed no mutations (55.8%)

3.1.2 Gender distribution characteristics

Figure 3.1 Gender Distribution of SCD Cases

Observation: Among the 206 study subjects, 181 cases were male (87.9 %), and 25 cases were female (12.1 %) The male-to-female ratio is approximately 7:1

3.1.3 Age distribution by gender

Table 3.2 Age Distribution of SCD Cases

Observation: Results from Table 3.2 indicate:

- The average age of SCD patients is 30,33 ± 8 Among these results, the 31 - 40 age group has the highest percentage (49.5 %) There is no statistically significant difference among the age groups (p > 0.05)

- The average age of males is 30,82 ± 7,6; which is higher than females (26,84 ± 10,2), and this difference is statistically significant (p = 0.02)

Male Female

8

3.2 PATHOLOGICAL CHARACTERISTICS

3.2.1 Some Macroscopic Characteristics

3.2.1.1 Body type

Observation: Majority of cases exhibited average body constitution (66%)

3.2.2 Pathological characteristics of the coronary arteries

Table 3.7 Distribution of Atherosclerotic Coronary Artery Lesions by Age Group

Pathological

Anatomy

No Atherosclerosis of the Coronary Arteries

Atherosclerosis of the Coronary

Atherosclerosis of the Coronary Arteries (OR; CI) P

9

3.2.3 Pathological characteristics of the myocardium

3.2.3.1 Histopathological patterns of myocardial lesions in SCD subjects

Table 3.12 Type myocardial pathology

3.2.3.2.Pathological characteristics of Hypertrophic cardiomyopathy

Table 3.13 Distribution of the severity of hypertrophic myocardial damage by age group

3.2.3.3 Distribution of the severity of myocarditis

Table 3.14 Distribution of the myocarditis damage by age group

3.2.3.4 Pathological characteristics of myocardial fat infiltration

Table 3.15 Distribution of the degree of myocardial fat infiltration by age group

10

3.2.3.5 Pathological characteristics of myocardial fibrosis

Table 3.16 Distribution of myocardial fibrosis types by age group

Observation: 55 cases of myocardial fibrosis, interstitial fibrosis has the highest percentage (45.5%)

3.2.3.6 Pathological characteristics of ischemic myocardial degeneration

Table 3.17 Distribution of ischemic myocardial degeneration types by age group

Myocytes with nuclear degeneration p

Fragmented myocardial fibers p

stage p Inflammatory cell stage p Scar formation stage p