Download APOC3 rs2854116 single nucleotide polymorphism

APOC3 rs2854116 single nucleotide polymorphism
in Hanoi primary school children
Nguyen Thi Hong Hanh1, Pham Tran Phuong2, Tran Quang Binh2
1
Hanoi National University of Education, Vietnam
2
National Institute of Hygiene & Epidemiology, Vietnam
Abstract
The apolipoprotein C3 (APOC3) gene is responsible in part for transport and clearance
of chylomicron remnants, very low-density lipoprotein and high-density lipoprotein
from the bloodstream. The single nucleotide polymorphism rs2854116 is located in the
insulin-responsive element of the APOC3promoter. The genotyping methods of this
polymorphism have been widely developed because of its significant association with
elevated plasma lipid concentrations, risk of coronary heart disease and metabolic
syndromein multiple populations. The study aimed to apply the restriction fragment
length polymorphism (RFLP) method for identifying the APOC3-rs2854116
polymorphism in Vietnamese children. The polymerase chain reaction method was
designed to amplify the APOC3 gene region containing rs2854116. The FokI restriction
enzyme was recruited to detect the polymorphism. The genotypes obtained by the RFLP
method were confirmed by sequencing analysis. The frequency of G allele was 0.41 in
Vietnamese children population. The frequency of A/A, A/G and G/G genotypes was
0.39, 0.40 and 0.21, respectively. In summary, the study reports the optimal RFLP
method for genotyping APOC3-rs2854116 polymorphism and the result of its
application on allele and genotype frequencies in Vietnamese samples.It is necessary to
apply it for genotyping the APOC3-rs2854116 polymorphism in large-scale studies in
Vietnam.
Keywords: APOC3 rs2854116, genotyping, restriction fragment length polymorphism,
Vietnamese children, single nucleotide polymorphism.
1. Introduction
Apolipoprotein (apo) C-III, a component of very low-density lipoprotein (VLDL) and
high-density lipoprotein (HDL), is encoded by the APOC3 gene. APOC3 is expressed in
the liver and intestine. APOC3 gene locates in an apolipoprotein gene cluster on
chromosome 11q23.3 between the APOA1 and APOA4 genes [6]. APOC3 is wellknown as inhibitor of lipoprotein lipase and hepatic lipaseactivity. Therefore, APOC3
gene was involved in transport and clearance of chylomicron remnants, VLDL and
HDL from the bloodstream [10]. It has been demonstrated that APOC3 has correlation
to coronary heart disease (CHD) [7], metabolic syndrome [12], type 2 diabetes [15] and
dyslipidemia [5]. Especially, APOC3 gene also have been found to be strong associated
with altered plasma lipid concentrations in children [14].
The single nucleotide polymorphism (SNP) rs2854116 (-455T>C) is located in the
insulin-responsive element (IRE) of the APOC3 promoter. This polymorphism has been
studied extensively because they could alter the nuclear transcript factors which mediate
the insulin response. The significant association of rs2854116 polymorphism with
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elevated plasma lipid concentrations [2], risk of CHD [8] and metabolic syndrome [13]
has been found in multiple populations. In spite of the important role of APOC3rs2854116 in metabolism, relatively few data exist in the literature regarding the
APOC3 genotypes in Vietmanese, especially in children.
Nowadays, some modern methods were used for APOC3 genotyping such as TaqMan
allelic discrimination assays [15] but this method is in general costly. The principal
method used to determine APOC3 genotypes was Polymerase chain reaction restriction
fragment length polymorphism (PCR-RFLP) because this method can be applied in
laboratory with uncomplicated and low-cost equipments includingPCR machine and
electrophoresis devise. Therefore, the study aimed to apply the PCR-RFLP method for
identifying the APOC3 rs2854116 polymorphisms in Vietnamese children.
2. Materials and methods
2.1. Samples
A total of 406 Kinh-ethnic children aged 6-11 years were randomly recruited from 31
primary school in Hanoi. Genomic DNA was extracted from 300 μL peripheral blood
treated with EDTA by using Wizard® Genomic DNA Purification Kit (Promega
Corporation,USA) according to the manufacturer’s instructions. The extracted DNA
was stored at -20°C until analysis. The study was approved by The Ethics Committee of
the National Institute of Nutrition, Vietnam. A comprehensive verbal description of the
nature and purpose of the study were explained to the parents of all subjects. Informed
consent to participate in the study was given by the parents of all subjects. Children with
HIV, under medication at the time of the study were excluded.
2.2. Amplification of APOC3 sequences
Genotyping of APOC3-rs2854116 was performed by polymerase chain reaction and
restriction fragment length polymorphism (PCR-RFLP). The sequences of the forward
and backward primers used for the APOC3-rs2854116 detection designed by our group
were
5’-TTCACACTGGAATTTCAGGCC-3’
and
5’GGATTGAAACCCAGAGATGGAGGTG-3’, respectively.
Each reaction system of a total volume of 12.5 μL, comprised 2 μL of genomic DNA;
0.8 μL of each primer (8 pmo1); 2.9 µL nuclease-free water, 6.0 µL Dream Taq Green
PCR Master Mix (constituent: DreamTaq DNA polymerase, 2X DreamTaq Green
buffer, dATP, dCTP, dGTP and dTTP, 0.4 mM each, and 4.0 mM MgCl2) (Thermo
Corporation, USA). For the amplification, initial denaturation at 94°C for 3 minutes was
followed by 34 cycles of denaturation at 94°C for 30 seconds, annealing at 56 or 58 or
60 or 62°C for 30 seconds, and extension at 72°C for 30 seconds, with final extension at
72°C for 8 minutes. After electrophoresis on a 2.5% agarose gel with 0.05 μL/mL
Redsafe for 30 minutes at 100V, the 230-bp amplified products were visualized under
ultraviolet (UV) light.
2.3. Restriction of APOC3 sequences
Each restriction enzyme reaction was performed with 5.0 μL of amplified DNA product;
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4.1 μL of nuclease-free water and 0.65 μL of 10 × buffer solution and 0.25 μL of the
Fastdigest FokI restriction enzyme in a total volume of 10.0 μL digested for 15 minutes
at 37oC. The digestive products were separated by electrophoresis on agarose gelpoststained with 0.05 μL/mL Redsafe for 30 min at 100V. The length of each digested DNA
fragment was determined by comparing migration of a sample with that of standard
DNA marker. The gel was imaged with ultraviolet light and a gel documentation
system.
2.4. Determination of genotypes
FolkI restriction enzyme was recruited to detect the polymorphism with cleavage map
. Each genotype possessed unique combination of FokI
fragment size. As a result, the A/A homozygote pattern contained 130 and 100-bp
fragments, indicating the presence of FokI restriction. The G/G homozygote pattern was
only 230-bp fragments reflecting the absence of the FokI restriction. The A/G
heterozygote pattern contained 230, 130 and 100-bp fragments.
3. Results and discussion
3.1. Selection of appropriate annealing temperature
The determination of the appropriate annealing temperature is important for PCR
reslult. There is no exact annealing temperature of all PCR reaction. The annealing
temperature chosen for PCR relies directly on length and composition of the primers. So
we ran a gradien PCR with rising annealing temperature to find the best annealing
temperature from 56°C to 62°C. The amplified 230-bp PCR products were checked in
2.5% agarose gel. As shown in Figure 1, the bands weresimilar at all annealing
temperature. We selected the medium annealing temperature of 58°C for further
experiments.
Figure 1. Electrophoresis result of 230-bp products by PCR at 4 annealing temperatures.
Lanes 1, 2: annealing at 56°C; lanes 4, 5: annealing at 58°C; lanes 7, 8: annealing at 60°C;
lanes 10, 11: annealing at 62°C; lanes 3, 6, 10, 13: N, negative control (H2O); lane 7:phiX174
DNA/BsuRI (HaeIII) Marker.
3.2. Selection of appropriate concentration of restricted enzyme
Enzyme volume depends on DNA concentration. We conducted an experiment in 7
samples to indentify the effectiveness of Fastdigest FokI which can digest completely
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the amplified PCR products. Follow the manufacturer's recommendation, 1 L
Fastdigest FokI is used for a reaction with total volume of 30 L. Figure 2 shows the
electrophores is results of PCR products before and after digestion by 0.25 L of FokI.
The bands were clear and could be identified exactly, PCR products were digested
completely at this concentrations of enzyme.
Figure 2. Electrophoresis result of PCR products before and after digestion by FokI.
A. Electrophoresis of PCR products of 7 samples (lanes 1-7) and 1 negative control (lane 8).
B. Electrophoretic separation of digested PCR products at 100V for 30 min. Lanes 1-7: sample
No 1-7 incubated with 0.25 μL of FastdigestFokI; lane 8:phiX174 DNA/BsuRI (HaeIII) Marker.
The samples No 1 and 7 had A/A genotype as contained 130 and 100-bp FokI
fragments. The samples No 2 and 3 had G/G genotype as contained only the 230-bp
fragment. The sample No 4, 5, and 6 had A/G genotype as contained 230 as well as 130
and 100-bp.
In this study, we describe the RFLP method for typing the APOC3-rs2854116
polymorphism and frequencies of alleles and genotypic of this polymorphism. The
demand for APOC3-rs2854116 genotype analysis will therefore increase, making a fast,
reliable assay for APOC3 genotyping essential. Methods for determining APOC3
genotypes were known along time ago. Methods for genotyping APOC3 have been used
since 1990s [3]. The major method used to genotype this polymorphism was PCRRFLP. Several studies used similar method in many countries with some modifies, such
as India [11], Canada [1] and USA [3]. Nowadays, some modern methods have been
used for genotyping APOC3-rs2854116 polymorphism as TaqMan allelic discrimination
assays [15]. However, this method require expensive cost.
Until now, to our knowledge, there has been no report on genotyping APOC3rs2854116 polymorphism in laboratories in Vietnam. To improve the APOC3 genotype
assay for routine use, we designed the PCR-RFLP protocol to a simple and fast method
for the unequivocal determination of A and G alleles based on restriction-enzyme typing
with FokI. Moreover, we used a non-toxic dye instead of ethidium bromide for safe
purpose. Another strength of our method was reducing enzyme level less than that
recommended by the manufacturer.
3.4. The genotypic and allelic frequencies of APOC3-rs2854116 in Hanoi primary
school children
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The genotype and allele distribution of APOC3-rs2854116 polymorphism in the study
population is shown in Table 1. The frequency of G and A allele in the study population
was 0.41 and 0.59, respectively. The frequency of A/A, A/G and G/G genotype were
0.39, 0.40 and 0.21, respectively. The genotype and allele distribution was not in HardyWeinberg Equilibrium (P < 0.05)
Table 1. The genotypic and allelic frequencies of APOC3-rs2854116 in Hanoi
primary school children
n
Frequency
Hardy-Weinberg
Equilibrium
Genotypic
A/A
158
0.39
A/G
163
0.40
G/G
85
0.21
0.007
Allelic
A
479
0.59
G
333
0.41
The genotypic and allelic frequencies of APOC3-rs2854116 has not appeared on the
HapMap database. Based on the results of the 1000 Genomes Project and the published
studies, we compared the allelic frequencies with other populations (Figure 4).
100%
0.36
80%
0.41
0.46
0.48
0.44
Note:
0.41
AFR: Afirican populations
60%
0.77
CEU: Utal European
G
A
40%
0.64
0.59
0.54
0.52
0.56
0.59
20%
CHB: Han Chinese in Beijing, China
JPT: Japanese in Tokyo, Japan
KHV: Kinh in Ho Chi Minh city,
Vietnam
0.23
0%
AFR CEU CHE CHB
CHE: Han Chinese in East China
JPT
KHV KHNV
KHNV: Kinh in Hanoi, Vietnam
Figure 4. The allelic frequencies of APOC3-rs2854116 in Hanoi children population
compare with some populations [16], [4]
The allelic frequencies of APOC3-rs2854116 in Vietnamese children population is
similar to the Han Chinese population in East China. Allele A frequency is significantly
higher than that of the Afirican populations and slightly higher than that of the Han
Chinese populations in Beijing, China; Japanese populations in Tokyo, Japan and Kinh
populations in Ho Chi Minh city, Vietnam but lower than Utal Europeans populations.
These inconsistent results may be explained partly by the differences in lifestyle and
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environmental factors, risk-factor profiles, and genetic background. The reconstruction
of human demographic history is of direct biological and anthropological interest.
Additionally, the history of effective population size has a profound effect on important
quantities such as the extent of linkage disequilibrium and is therefore important for
medical association studies. There have been many attempts for demographic inference
from contemporary molecular data representing different molecular mutation systems
[9].
4. Conclusions
The study reports the optimal protocol for genotyping APOC3 gene using RFLP method
in Vietnamese children population. The appropriate annealing temperature for PCR
reaction was 58oC. Restriction enzyme Fastdigest FokI concentration can be reduced to
0.25 L per reaction. The frequencies of A/A, A/G and G/G genotypes were 0.39, 0.40
and 0.21, respectively in the Vietnamese children. This simple and useful assay could be
widely applied in community-based studies on metabolic disorders in Vietnam.
Acknowledgements
The authors would like to thank Dr. Nhung BT and colleagues at the National Institute
of Nutrition and Hanoi National University of Education for their kind help and
support.This study was supported by grant No. B2014-17-47 from the Ministry of
Education and Training, Vietnam and grant No. 01C-08/05-2011-2 from Hanoi
Department of Science and Technology.
Đa hình đơn nucleotide APOC3 rs2854116 ở trẻ em tiểu học Hà Nội
Nguyễn Thị Hồng Hạnh1, Phạm Trần Phương2, Trần Quang Bình2
1
Hanoi National University of Education, Vietnam
2
National Institute of Hygiene & Epidemiology, Vietnam
Tóm tắt
Gen apolipoprotein C3 (APOC3) có vai trò trong việc vận chuyển và giải phóng
chylomicron dư, lipoprotein tỷ trọng rất thấp (very low-density lipoprotein) và
lipoprotein tỷ trọng cao (high-density lipoprotein) ra khỏi dòng máu. Đa hình đơn
nucleotide (single nucleotide polymorphism, SNP) rs2854116 nằm ở vùng phản ứng với
insulin thuộc promotercủa gen APOC3. Những phương pháp xác định kiểu gen của đa
hình này khá phong phú do đa hình này đã được xác định có mối liên quan với nồng độ
lipid huyết tương, nguy cơ mắc bệnh tim mạch và hội chứng chuyển hoá ở nhiều quần
thể khác nhau trên thế giới. Chúng tôi tiến hành nghiên cứu này với mục đích xây dựng
quy trình xác định kiểu gen của đa hình APOC3-rs2854116 bằng phương pháp đa hình
chiều dài đoạn cắt (restriction fragment length polymorphism, RFLP) ở trẻ em tiểu học
Hà Nội. Phản ứng PCR (polymerase chain reaction) được thiết kế để khuyếch đại đoạn
gen chứa SNPrs2854116. Enzyme giới hạn FokI được sử dụng để phát hiện các kiểu
gen khác nhau thuộc đa hình này. Kiểu gen được xác định bằng phương pháp RFLP
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được so sánh với kết quả giải trình tự gen. Theo đó, tần số alen G ở 406 trẻ em tiểu học
tại Hà Nội là 0.41. Tần số kiểu gen A/A, A/G và G/G lần lượt là 0.39, 0.40 và 0.21. Như
vậy, nghiên cứu của chúng tôi đã xây dựng được quy trình RFLP tối ưu để xác định đa
hình APOC3-rs2854116 và kết quả phân tích alen, kiểu gen của đa hình này ở người
Việt Nam. Đây là cơ sở cho các nghiên cứu xác định kiểu gen của đa hình này với quy
mô lớn ở Việt Nam.
Từ khoá: APOC3 rs2854116, xác định kiểu gen, đa hình chiều dài đoạn cắt, trẻ em tiểu
học, đa hình đơn nucleotid.
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