Nghiên cứu đột biến gen LDLR ở người tăng cholesterol máu có tính chất gia đình tt tiếng anh

Identify 4 mutations in 7/26 FH pediatric patients 26.92%: - Two mutations which were announced pathogenic: + Exon 4 mutation c.664 T>C: 5 pediatric patients, 4 of them are heterozygou

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH

HANOI MEDICAL UNIVERSITY

HOANG THI YEN

RESEARCH ON MUTATION OF LDLR GENE IN

RESEARCH IS COMPLETED AT HANOI

MEDICAL UNIVERSITY

Scientific instructor: Assoc.Prof Ph.D Dang Thi Ngoc Dung

Thesis defense committee members:

Research can be found in:

- National Library of Vietnam

- Hanoi Medical University Library

INTRODUCTION

1 Importance of research problem:

Familial hypercholesterolemia (FH) is a spontaneous disorder, characterized by lifelong increase of serum cholesterol related to low

density lipoprotein (LDL) FH patients with LDLR gene mutations

(accounting for 85% of LH patients) display a significant increase in total cholesterol (TC) and LDL-Cholesterol which leads to cholesterol deposition in the arteries at a very early age, consequently atherosclerosis and increased risk of myocardial infarction Especially with FH patients, myocardial infarction tends to develop faster and could cause sudden death, severe coronary heart disease (CHD) at a young age or other cardiovascular complications in the 40s or 50s of the patient’s life Death and coronary artery bypass grafting (CABG) rates

in adolescence are considerably high among FH patients However, FH patients are still not diagnosed and treated timely or receive deficient treatment Cholesterol level solely is insufficient to accurately diagnose

FH since cholesterol level in the blood may vary between ages, genders, population characteristics and it could be confused with acquired hypercholesterolemia Therefore, diagnostic criteria for FH consist of clinical symptoms and paraclinical examinations as well as family medical history of any dominant genotypes of early cardiovascular disease or hypercholesterolemia Currently in Vietnam, FH patients are not fully concerned, genetic examinations are rarely performed; many children have been admitted due to critical cardiovascular complications Researches on molecular biology identifying mutation

forms of genes, especially LDLR gene in FH patients are still scarce

This shortage hinders preventive consultation with the patients and

other family members in order to decrease the risk of early complications of coronary disease

4 New research contributions:

4.1 Identify 4 mutations in 7/26 FH pediatric patients (26.92%):

- Two mutations which were announced pathogenic:

+ Exon 4 mutation c.664 T>C: 5 pediatric patients, 4 of them are heterozygous mutation and one is homozygous

+ Exon 9 mutation c.1285G>A: 1 pediatric patient (also carries c.664 T>C mutation)

- Two new mutations have not been published but are likely to cause

- Determine the dominant genetic rule on the common chromosome

of FH disease in these 3 pedigrees

5 Scientific and practical importance of research:

This research has a high practical meaning; identify mutation of

LDLR gene in familial hypercholesterolemia patients help doctors

correctly diagnose the disease in order to perform medical treatment appropriately and timely as well as detect gene carriers, cascade screening, manage and consult patients and their families about preventing risks and complications related to atherogenesis Vietnam’s disease patterns are shifting towards non-infectious diseases and senescence; this circumstance emphasizes the necessity of researches related to these diseases, aiming to improve the quality of the population

This study has scientific meaning with suitable and systematic research design, logical layout and proper data processing methods Along with specialized research facility, advanced technology has been used for DNA sequencing to ensure the results are accurate and highly reliable

- Research includes 23 tables, 35 charts, figures and diagrams; 175 reference documents which include 3 Vietnamese documents and 172 English documents Appendix includes: Medical record sample, DNA clean-up and concentration results, examination techniques used in research, DNA sequence analysis results; List of patients participated in the study

Chapter 1: OVERVIEW

1 FH epidemiology:

Many studies have shown that FH is one of the most common gene inheritance disorders Global FH prevalence is estimated to be 1:500 to 1:300 (0.2-0.3%), which approximately 13 millions people all over the world and 600.000 Americans Some populations have even higher rate: Lebanese 1/85 European South African 1/100 to 1/72, Tunisian 1/165, French Canadian 1/270

2 Diagnostic criteria for FH:

Simon Broome diagnostic criteria:

(1) Total cholesterol greater than 7.5 mmol/L or LDL-C greater than 4.9 mmol/L in an adult; Total cholesterol greater than 6.7 mmol/L or LDL-C

greater than 4.0 mmol/L in a child aged younger than 16 years

(2) Associated symptoms: Tendon xanthomas or tendon xanthomas in first (parents, siblings) or second (grandparents, uncle,

aunt) degree relatives

(3) OR DNA-based evidence of an LDLR mutation, familial

defective apo B-100 or PCSK9 mutation

(4) Family history of myocardial infarction before age 50 years in

a second-degree relative or before age 60 years in a first-degree relative

(5) Family history of elevated total cholesterol greater than 7.5 mmol/L in an adult first or second-degree relative; greater than 6.7

mmol/L in a child, siblings age 16 years or younger

so even when no mutation is detected, FH is still possible

3 Mutation forms of LDLR gene and its effect on phenotype

LDLR gene resides on the short (p) arm on chromosome 19 at the

band 19p13.2, spans 45kb and includes 18 exons and 17 introns, consisted of 11.089.361 to 11.133.829 base pairs and decodes to a 5.4kb

mRNA Mutation of LDLR gene is the most common cause of FH (about 80%) There are 1700 announced mutation on LDLR gene, 1295

are independent mutations (1064 are pathogenic, 143 are

non-pathogenic, 88 unknown) FH caused by LDLR gene mutation shows

various clinical phenotypes depends on the mutation form and the remaining allele and LDLR gene activity Clinical phenotypes of FH

caused by LDLR mutation can be classified into severe mutation forms

(homozygous mutation, compound heterozygous mutations, nonsense mutation ) and heterozygous mutation

4 Polymorphism of LDLR gene

Single nucleotide polymorphism (SNP) or many SNPs could solely

or synergistically affect the haplotypes as well as pathogen risk If we assess SNP solely without considering the SNP-SNP interaction (SNP shows weak correlation with the estimated odds ratio), it will be difficult to examine the effect levels of SNPs on the carrier’s phenotype

5 Management program and screening strategy for FH patients

A few screening strategies for FH patients in the community: (1) Opportunistic screening (for initial patients)

(2) Systematic screening (for children and adolescents – NICE guidelines)

(3) Cascade screening (intentional screening for FH patient’s family) Cascade screening is valued as the most efficient method in detecting and timely treating FH in order to increase lifespan, decrease coronary disease risk and bring in economical benefit by reducing healthcare cost

Chapter 2 SUBJECTS AND METHODS OF THE STUDY

2.1 Research subjects

2.1.1 Pediatric FH patient group:

Children under 16 years of age are examined and diagnosed with

FH diseases at the National Pediatric Hospital according to the following selection and exclusion criteria

Selection criteria:

(1) TC is higher than 6.7 mmol/L or LDL-C above 4.0 mmol/L (2) PLUS: Tendon xanthomas in patient or a 1st degree relative (parent, sibling, child) or a 2nd-degree relative (grand parent, uncle, aunt)

- Parents or guardians of pediatric patients and FH patients agree to allow children to participate in the study

Exclusion criteria:

- Children with one of the following diseases: Hyperthyroidism, hypothyroidism, nephrotic syndrome, diabetes mellitus, chronic liver disease

2.1.2 Group of children's family members:

- Members of the pedigree of the three families of FH pediatric patients (MS02, MS03, MS15): Including 45 members

+ Of which 30/45 members in the genealogy of 3 families are related to the genetic lineage of FH disease

+ The members of the genealogy of 3 families agree to participate

- Technique for separating DNA from whole blood

- DNA quality testing: DNA purity measurement technique and DNA concentration measurement

- PCR technique to amplify exon 3, 4, 9, 13 and 14

+ Designing primers: Designing primers is crucial to the success of the research The purpose of primer design is to amplify the exon 3, 4,

9, 13, and 14 of LDLR gene The priming design is strictly cohered with

the primer design principle

We designed 4 pairs of primer for 5 exons: exon 3, exon 4, exon 9; exon 13 and exon 14 The team designed a primer pair to cover the 13-intron13-exon14 exon segment (431 bp) The sequences that cover the target sequence from NCBI are included in the Primer-BLAST software, selecting a minimum sequence length of 500 bp

Table 2.1 The sequence of primers amplified exon 3, 4, 9, 13 and 14

2.2.3 Genetic sequencing technique

After purification, PCR products were conducted genetic sequencing

on automatic sequencing machine Prism 3730xl - ABI (USA)

2.3 Ethics in research:

- This study was approved by the Ethics Council, according to Decision No.187/HĐĐĐHYHN, dated February 20, 2016 of the Medical Ethics Council, Hanoi Medical University

- Patients are fully voluntary to participate in the study and provide full and truthful information related to their diseases

- Patient is notified of the result of genetic mutation test by the treating Doctor

- The patient information, the diagnosis results are kept strictly confidential Research is conducted purely for scientific purposes, not for any other purpose

2.4 Data processing methods

The results of gene sequencing were analyzed by Sequencing Scanner 2.0 and compared with sequences of a.a on genebank using ApE software Analysis to identify mutations, type of mutations or SNPs, combined with software to predict the likelihood of mutation or SNP

Use SPSS 16.0 software for statistical analysis Test and compare the average value of the variables according to the normal distribution

by T-test, by Mann-Whitney test non-standard

Figure 2.4 Research design diagram

Chapter 3: RESEARCH RESULTS 3.1 Characteristics of blood lipid index of the research subjects:

Table 3.1 Comparison of lipid index of 2 groups of pediatric patients

(mmol/L)

LDL-C (2) (mmol/L)

HDL-C (mmol/L)

TG (mmol/L)

Group of 26

pediatric patients

12,44 ± 4,88

Table 3.1 The average index of TC and LDL-C increases at group

of 26 pediatric patients, the differences of the 2 indexes compare to the group of 9 pediatric patients with detected mutations in 3 pedigree is statistically significant with p<0,05; the HDL-C and TG index do not

3.2 Identify mutations on exon 3, 4, 9, 13, 14 of LDLR gene:

3.2.1 Results of the LDLR gene mutations

Heterozygous mutation on exon 4 (c.664 T>C)

Normal c.664T c.664 T>C

Figure 3.5 Mutation of c.664 T>C on exon 4 of LDLR gene

The mutation caused a.a change in position 222 from Cysteine

to Arginine This is a heterozygous transformation and was found in

4 patients from group of 26 FH pediatric patients (MS02, MS03, MS08, MS18)

Homozygous mutation on exon 4 (c.664 T>C)

Figure 3.6 Mutation of c.664 T>C on exon 4 of LDLR gene

The nucleotide in position 664 on exon 4 of LDLR gene was replaced

from Thymine to Cytosine This mutation caused the a.a change from Cysteine to Arginine This is a homozygous mutation, which was found

in MS15 patient from group of 26 FH pediatric patients

Heterozygous mutation on exon 9 (c.1285G>A)

Normal c.1285G c.1285 G>A

Figure 3.7 Mutation of c.1285G>A on exon 9 of LDLR gene

In this mutation, Guanin in position 1285 on exon 9 of LDLR

gene was replaced by Adenin

This mutation caused the a.a change in position 429 from Valine

to Methionine This mutation was published, named c.1285G>A in heterozygous mutation, it was found in MS02 patient in group of 26 FH pediatric patients

Heterozygous mutation on exon 9 (c.1335 C>T)

Normal c.1335 C c.1335 C>T

Figure 3.8 Mutation of c.1335 C>T on exon 9 of LDLR gene

Cytosine in position 1335 on exon 9 of LDLR gene was replaced

by Thymine This mutation did not cause the a.a alteration and has not been published before This heterozygous mutation was found in MS19 patient from group of 26 FH pediatric patients

Heterozygous mutation on exon 13 (c.1978 C>T)

(CAGUAG)

Figure 3.9 Mutation of c.1978 C>T

Cytosine in position 1978 on exon 13 of LDLR gene was replaced by

Thymine

This mutation caused the codon change in position 660, from CAG to UAG (UAG is a stop codon) This is a newly discovered mutation in the study, named c.1978 C>T - a heterozygous stop codon which is a nonsense mutation This mutation was found in MS23 patient in group

of 26 FH pediatric patients

3.2.2 Results of polymorphisms identification of the LDLR gene

Single polymorphism SNP rs1003723 (intron 9)

(a) Normal

(b) c.1359-30 C>T heterozygous (c) c.1359-30 C>T homozygous

Figure 3.10 Heterozygous SNP rs1003723 and homozygous Image b: The nucleotide in position 1359 on intron 9 of LDLR gene

(before exon 9 a distance of 30 nucleotides) was changed from Cytosine

to Thymine This is a published SNP as SNP rs1003723 in heterozygous transformation This SNP was found in 11 patients in group of 26 FH pediatric patients

Image c: SNP rs1003723 in homozygous transformation This is a

published SNP that was found in MS15 patient in group of 26 FH pediatric patients

Single polymorphism SNP rs5925 (exon 13)

(a) Normal

(b) c.1959T>C heterozygous (c) c.1959T>C homozygous

Figure 3.11 Heterozygous and homozygous SNP rs5925

Image (b): The nucleotide in position 1959 on exon 13 of LDLR gene

was changed from Thymine to Cytosine This is a published SNP as SNP rs5925 in heterozygous transformation, this SNP did not cause the a.a change (p.Val653Val) and was found in 7 patients in group of 26 FH pediatric patients

Image (c): SNP rs5925 in homozygous transformation This SNP was

published and found in MS15 patient in the study