Familial hypercholesterolemia (FH) is an inherited disorder of lipid metabolism. FH is characterized by raised serum LDL - C levels, leading to accelerated atherosclerosis and increased risk of premature coronary heart disease. More than 80% of the time, FH is caused by mutations in the LDL receptor. The LDL receptor consists of 18 exons, and the largest number of LDLR mutations are believed to be in exons 4 and 3. The aim of our study was to examine mutations in exons 3 and 4 of the LDLR gene in Vietnamese patients with FH. The sample group consisted of 50 Vietnamese patients diagnosed with FH, based on Medped (USA) criteria. Blood samples were taken after an overnight fast for determination of lipoprotein parameters and DNA extraction. Polymerase chain reactions were performed to amplify exon 3 and 4 of LDL receptor gene, after which direct sequencing was done. Of the 50 FH patients in our sample, 9 patients were found to have mutations in exons 3 and 4 of the LDL receptor gene. These mutations included: mutation D69N (exon3), and mutations D206E, D147Y,C201R (exon 4).
Trang 1Corresponding author: Pham Thi Minh Huyen,
Biochemis-try department, 108 hospital
Email: minhhuyenv108@gmail.com
Received: 02 November 2016
Accepted: 10 December 2016
IDENTIFICATION OF MUTATIONS IN EXONS 3 AND 4 OF THE LDL-RECEPTOR GENE IN PATIENTS WITH FAMILIAL
HYPERCHOLESTEROLEMIA
Pham Thi Minh Huyen 1 , Hoang Thi Yen 2a , Dang Quang Huy 2b , Nguyen Quynh Giao 2c , Dang Thi Ngoc Dung 3
1
Biochemistry department, 108 hospital; 2a General laboratory, Hanoi Heart hospital, 2b,2c, Department of medical laboratory science, Hanoi Medical University, 3 Biochemistry department, Hanoi Medical University Familial hypercholesterolemia (FH) is an inherited disorder of lipid metabolism FH is characterized by raised serum LDL - C levels, leading to accelerated atherosclerosis and increased risk of premature coronary heart disease More than 80% of the time, FH is caused by mutations in the LDL receptor The LDL receptor consists of 18 exons, and the largest number of LDLR mutations are believed to be in exons 4 and 3 The aim of our study was to examine mutations in exons 3 and 4 of the LDLR gene in Vietnamese patients with FH The sample group consisted of 50 Vietnamese patients diagnosed with FH, based on Medped (USA) criteria Blood samples were taken after an overnight fast for determination of lipoprotein parameters and DNA extraction Polymerase chain reactions were performed to amplify exon 3 and 4 of LDL receptor gene, after which direct sequencing was done Of the 50 FH patients in our sample, 9 patients were found to have mutations in exons 3 and 4 of the LDL receptor gene These mutations included: mutation D69N (exon3), and mutations D206E, D147Y,C201R (exon 4).
Key words: FH, LDL receptor mutation, D69N, D147Y, D206E, C201R
I INTRODUCTION
Familial hypercholesterolemia (FH) is one
of the most common metabolic inherited
disorders, affecting 1 in 500 people yearly
The prevalence of the disease is relatively
high in most communities worldwide [1],
particularly in African countries [2] FH is a
monogenic inherited disease caused by
mutations occurred mostly in genes that affect
the metabolic clearance of LDL - C More than
80% of FH cases are believed to be caused by
mutations in the LDLR gene, while other
genes such as the apolipoprotein B (apoB)
gene, the PCSK9 gene, and the LDLRAP1
gene are believed to be responsible for the majority of the other cases of FH [3] The LDL receptor (LDLR) gene locus is located on chromosome 19p13.1 - 13.3 and spans 45 kb
It contains 18 exons and 17 introns [4] Patients with malfunctions in the LDLR gene have elevated LDL - C levels LDL - C deposits in tissues and causes external manifestations of FH, such as tendinous
deposits in arteries can lead to premature
Individuals with untreated FH are at an approximately 20 - fold increased risk of having CHD compared to healthy control groups [7] Untreated men have a 50% chance
of a coronary event by the age of 50 and untreated women have a 30% chance of a
Trang 2coronary event by the age of 60 [8] In
contrast, early identification and treatment of
patients with FH may reduce the risk of the
development of early CHD [9] It is generally
difficult to diagnose young FH patients based
on the FH phenotype alone because the
various physical manifestation of FH, such as
the tendon xanthomas, do not develop until
later in life [10] Therefore, a molecular test
provides an unequivocal diagnosis prior to the
appearance of clinical symptoms In Vietnam,
there is a lack of research on the diagnosis of
FH based on genetic analysis, resulting in a
high mortality rate among young patients
Therefore, this study aims to determine the
rate and types of mutations in exons 3 and 4
of the LDLR gene in patients with FH in
Vietnam
II SUBJECTS AND METHOD
1 Subjects
Fifty patients selected from 108 hospitals,
who were examined between October 2015
and April 2016 and who fulfilled the Medped
(USA) diagnostic criteria for FH (see below),
were included in this study
Inclusion criteria:
< 18 age: Cholesterol ≥ 7.0 and LDL-C ≥
5.2 (mmol/L)
18 - 29 age: Cholesterol ≥ 7.5 and LDL-C ≥
5.7 (mmol/L)
30 - 39 age: Cholesterol ≥ 8.8 and LDL-C ≥
6.2 (mmol/L)
≥ 40 age: Cholesterol ≥ 9.3 and LDL-C ≥
6.7 (mmol/L)
Exclusion criteria: a diagnosis of diabetes
mellitus, renal syndrome, and/or hypothyroid
2 Methods
- Study design: Cross-sectional study
- Study process:
Four mL of blood was obtained from each patient after an overnight fast Total plasma cholesterol, triglyceride, and HDL-C levels were measured using commercially available kits (Beckman coulter) Plasma LDL-C was
formula Genomic DNA was extracted from peripheral blood samples by the phenol/ chloroform method Exon 3 and exon 4 of the LDLR gene were amplified using the polymerase chain reaction (PCR) method with two pairs of oligonucleotide primers: Exon 3: 5’TTTAGTAGGACAGGGTTTCAC-TATATTGG 3’
3’CTGGTCAAGGGGGGATTTGA 5’ Exon 4: 5’TGATGGTGGTCTCGGCCCAT 3’ 3’ACACCTGGGGGAGCCCA 5’
The PCR reaction mix contained: 17.5 µlGoTaqmastermix, 1µl DNA, 0.5µl forward primer, 0.5µl reverse primer, and 5.5 µl water nuclease free The total volume for each PCR reaction mix was 25 µl The thermal cycling regimen consisted of 1 cycle of denaturation at
950for 5 minutes, followed by 35 cycles at 950 for 30 seconds, an annealing at 580 for 30 minutes, extension at 720for 30 seconds, and
a final extension of 720 for 5 minutes The amplified products were identified on 2% agarose gel and were stained with ethidium bromide which was visualized under UV light The PCR products were then sequenced
- Mutation analysis:
DNA sequencing analysis was performed using Prism ABI 330xl software and the
Trang 3result-ing sequences were compared with original
sequence on genebank
III RESULTS
1.General characteristics of the participants
- Gender and age
Table 1 Age and gender of participants
3 Research ethics
All patients after being clearly explained about the study
Sex:
Male Female
25 25
50 50 Patients’ ages ranged from 24 to 79 years old The mean age of all participants was 50.34 years
- Clinical characteristics of the FH patients included in this study
Table 2 Clinical characteristic
Seven patients (14%) had angina pectoris and five patients (10%) had hypertension There were no patients with xanthomas or myocardial infarction Average body mass index (BMI) was approximately 21.96 ± 1.32 (kg/m2) Patients’ BMI ranged from 18.97 to 24.46 (kg/m2)
Lipid profile characteristics
Table 3 Lipid parameters
Variables Cholesterol
(n = 50)
Triglycerides (n = 50)
HDL - C (n = 50)
LDL - C (n = 50)
Cholesterol/HDL ratio (n = 50)
X
X
Trang 4The average cholesterol concentration was 10.35 ± 1.31 mmol/L, with a range from 8.76 to 16.09 mmol/L Triglyceride concentration ranged from 0.58 to 4.44 mmol/L LDL-C concentration ranged from 6.43 to 11.9 mmol/L
2 Identification of mutations in exons 3 and 4 of the LDL receptor gene
- PCR
Picture 1 Electrophoresis photograph of exon 3 (477bp)
M: marker; (-) negative; 1 - 10: samples S1 - S10
Picture 2 Electrophoresis photograph of exon 4 (465bp)
M: marker; (-): negative; 1 - 9: samples S1 - S9
Electrophoresis bands were clear and their sizes were exact
- Sequencing results
Picture 3 D69N mutation in exon 3 of the LDLR gene in patient S35
Trang 5Picture 4 D206E mutation in exon 4 of the LDLR gene in patient S9
Picture 5 D147Y mutation in exon 4 of the LDLR gene in patient S4
Picture 6 C201R mutation in exon 4 of the LDLR gene in patient S19
Trang 6Mutation categories in our study
Table 4 Mutation categories in exon 3, 4 of LDLR gene
There was one mutation found in exon 3 (D69N) and three mutations found in exon 4 (D147Y, D206E, and C201R)
IV DISCUSSION
Our study group consisted of 50
Vietnam-ese FH patients who fulfilled the Medped
(USA) diagnostic criteria for FH The average
age of our patients was 50.34 years and the
range was from 24 to 79 years old Our
average patient age and overall age range are
consistent with previous studies of FH
patients One recent study of 605 FH patients
from the Netherlands saw an average age of
47.08 (years old) [11]; another study included
86 Asian FH patients (with 72 Chinese
patients, 13 Malaysian patients and 1 Indian
patient) with an average age of presentation
around 54 years old, and a range from 31 to
71 years old [12] The male: female ratio in our
study was 1:1, similar to the study conducted
in the Netherlands, where of the 605 patients
included, 53% were female [11] Another study
conducted in 2002 among 200 Japanese FH
patients included 53.6% females [13] In
Malaysia, a study conducted in 2013 with 164
FH patients included 48.4% women [14]
Thus, our gender proportions are consistent
with previous studies conducted with FH
patients In our study, there were 7 patients
(14%) with angina pectoris and 5 patients (10%) with symptoms of hypertension There were no patient with xanthomas or myocardial infarction In the study referenced above with Japanese FH patients, 26% of the 200 pa-tients in the study had CHD and 75% had
Malaysian FH patients conducted in 2013, 68% of patients had CHD, 40.8% of patients had xanthomas, and 23.14% of patients had hypertension, among the 164 FH patients included in the study [14] In our study, we only selected patients who fulfilled MEDPED criteria (based on Cholesterol concentration matched with patients’ age) In the study conducted with Japanese patients [13], FH was diagnosed according to the Simon Broome criteria, which include: cholesterol levels ≥ 5.9 mmol/L with tendon xanthomas, or
FH among first-degree relatives In the study conducted in Malaysia, study subjects were patients who had elevated cholesterol levels and xanthomas or CHD in first degree rela-tives Thus, these studies saw higher
Trang 7xanthomas BMI measurements among the
patients in our sample averaged out to 23.0 ±
3.0 kg/m2, with a range from 18.97 to 24.46
(kg/m2) All BMI values among the patients in
our sample were within normal range
FH is characterized by elevated LDL - C
levels In our study, the average cholesterol
was 10.35 ± 1.31 mmol/L; the average
triglyc-eride level was 2.19±0.96 mmol/L; the average
HDL- C level was 1.6 ± 0.35 mmol/L; the
aver-age LDL - C level was 7.76 ± 1.19 mmol/L;
and the average ratio of total cholesterol to
HDL - C was 6.7 ± 1.45 mmol/L
Cholesterol and LDL-C levels among the
patients in our study were similar to the results
of a study conducted with 605 FH patients in
the Netherlands [11] However, because of
different FH diagnostic criteria used in our
study versus in other studies, cholesterol and
LDL-C levels among patients in our sample
were higher than those of patients in studies
conducted with FH patients in other Southeast
Asian countries, specifically Malaysia [12; 14]
To date, more than 1700 different
muta-tions of the LDLR gene have been reported
worldwide as the cause of FH [5] The largest
number of LDLR mutations has been found in
exons 3 and 4 of the LDLR gene [12 - 14]
Therefore, in our study, we analyzed exons 3
and 4 of the LDLR gene among the
Vietnamese FH patients included in our
sample Among 50 patients in our sample, we
identified nine patients who carried mutations
(one patient had a mutation in exon 3 and
eight patients had mutations in exon 4) All of
the mutations were heterozygous The
loca-tions of mutaloca-tions were: D69N (exon 3) and
D206E, D147Y, C201R (exon 4) Five patients
carried the C201R mutation and two patients
had D147Y mutation The D69N and D206E mutations were found in one patient each All
of the mutations were nonsynonymous mis-sense mutations, with nucleotide substitutions that changed one amino acid into another These mutation thus do not change the size of protein, but do affect the proteins’ functions [15] Based on the effects of the mutations on LDL metabolism, these mutations are in the 2B class They do not disrupt the synthesis of the LDLR, but they block transportation of LDLR proteins to the Golgi apparatus As a result, LDLR proteins reach the cell surface at
a considerably reduced rate and are rapidly degraded, leading to defections of LDLR on the cell surface and decreasing LDL - C clea-rance [15]
V CONCLUSION
We found 9 patients who carried mutations
in exons 3 and 4 of the LDLR gene, among the 50 FH patients included in our sample The mutations included the D69N mutation in exon 3, and the D147Y, D206E and C201R mutations in exon 4 Overall, the C201R mutation was the most common mutation, present in five of the nine patients with mutations in exons 3 and 4
Acknowledgements
We would like to acknowledge the physi-cians from the health laboratory quality exami-nation centre at the Hanoi Medical University for their technical assistance We would also like to acknowledge the contribution of the physicians from the chemistry departments from 108 hospitals in Vietnam who submitted blood samples from FH patients
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