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ISSN 0582-9879 Acta Biochim et Biophysica Sinica 2004, 36(1):33-36 CN 31-1300/Q
A Novel Missense
Mutation (L296Q) in Cholesteryl Ester Transfer Protein Gene Related to Coronary Heart
Disease
Ke-Qin ZHENG, Si-Zhong ZHANG*, Li ZHANG1, De-Jia HUANG1, Lin-Chuan LIAO2, and Yi-Ping HOU2
( Department of Medical Genetics, West China Hospital; Division of Human
Morbid Genomics, Key Laboratory of Biotherapy ofHuman Diseases of Ministry of
Education, Sichuan University, Chengdu 610041, China;
1 Department
of Cardiology, West China Hospital, Sichuan University, Chengdu 610041, China;2 School of Basic and
Forensic Medicine, Sichuan University, Chengdu 610041, China )
Abstract Cholesteryl
ester transfer protein (CETP) is a key participant in the reverse transport of
cholesterol from the peripheral tissues to the liver. To understand further the
role that CETP gene plays in the pathogenesis of coronary heart disease
(CHD), the promoter region, all 16 exons and adjacent intronic regions of CETP
gene were screened for single nucleotide polymorphisms (SNPs) in 203 CHD
patients and 209 healthy volunteers by the combination of PCR, denaturing high
performance liquid chromatography (DHPLC), molecular cloning, and DNA
sequencing methods. A novel missense mutation in the CETP gene was
identified. This mutation (L296Q) was caused by a T → A conversion at codon
296 of exon 10 which resulted in the replacing of the codon for leucine (CTG)
with the codon for glutamine (CAG). Further studies found that there was a
significant increase in the mutant allele frequency in the CHD patients
compared with that in the controls (0.160 vs. 0.091, c2 = 9.014, P = 0.003), and the
odds ratio to develop CHD was 1.83 for the 296Q allele carriers vs.
296LL homozygotes. Statistical analyses also demonstrated that the mutant
296Q allele carrier patients displayed
significantly higher total cholesterol (TC) and low density lipoprotein
cholesterol (LDL-C) concentrations than noncarrier patients. All these results
suggest that the Q296 mutation in CETP
gene was closely related to CHD, and the identification of new mutations in
the CETP gene will afford the opportunity to investigate the
relationship between CETP gene and CHD.
Key
words cholesteryl
ester transfer protein gene (CETP gene); coronary heart disease (CHD);
missense
Cholesteryl
ester transfer protein (CETP) is responsible for the net transfer and
heteroexchange of triglycerides (TG) and cholesteryl ester (CE) among the high
density lipoprotein (HDL), the low (LDL), and very low density lipoproteins
(VLDL) fractions [1]. Plasma CETP is a highly hydrophobic glycoprotein containing
476 amino acids and 4 N-linked glycosylation sites [2]. The human CETP gene
has 16 exons encompassing 25 kb genomic DNA and is located on chromosome
16q21[3]. In human, CETP mRNA is highly expressed in the liver as well
as in spleen and adipose tissue, with lower levels expressed in the small
intestine, adrenal, kidney and heart [2,4].
Several
mutations in the CETP gene have been identified in the Japanese and
Caucasian populations [5–8]. The CETP D442G mutation that replaces an aspartic acid (D) with
a glycine (G) in the exon 15 was first reported in 1993 and was observed to be
associated with elevated high density lipoprotein cholesterol (HDL-C)
concentration [7]. Japanese subjects with homozygous G442 genotype showed a corneal opacity and coronary heart
disease (CHD) despite the increased HDL-C level. Other mutations
of CETP
gene were also detected in different ethnic groups and were noticed to be
related to the variation of the plasma lipid and lipoprotein values as well as
CHD status [5,6,8,9].
In present
study, we reported a novel missense mutation (T→A transversion) at the nucleotide
13,161 (relative to the transcription start site) in exon 10 of the CETP gene,
which was the result of screening for single nucleotide polymorphisms (SNPs) in
203 CHD patients and 209 controls. The association of this mutation with
biochemical and clinical manifestations of CHD was also studied.
Materials and Methods
Study
subjects
203 CHD
patients were selected from the West China Hospital, Sichuan University. The
patients had at least one coronary artery with a stenosis of more than 60
percent as documented by angiography. In addition, 209 unrelated age- and
gender-matched subjects, with no clinical or biochemical signs of CHD,
recruited at the same hospital via routine health examination, were used as
controls in the study.
Plasma
lipid and lipoprotein assay
Venous
blood was collected from all subjects after an overnight fast. Plasma was
separated from the blood cell by centrifugation and used immediately for lipid
and lipoprotein analysis. The levels of plasma total cholesterol (TC), HDL-C,
LDL-C, VLDL-C and triglyceride(TG) were determined with enzymatic kits
(Boehringer-Mannheim) according to the manufacturer’s instruction.
DNA
isolation and PCR amplification
Genomic
DNA was extracted from leucocytes by the ‘salting-out’ method [10].
Fragments containing the 5′flanking region and individual exon of the CETP
gene, including all intron-exon boundaries were amplified by PCR.
Oligonucleotide primers for amplification were synthesized according to
published sequence data (GenBank accession No. AC010550). A sequence of 5501 bp
was amplified.
Denaturing
high performance liquid chromatography (DHPLC) screening
DHPLC
screening for single nucleotide polymorphisms was performed on an automated
HPLC instrument (HP1100, Hewlett Packard). The heteroduplex molecules are
generally eluted ahead of the homoduplex molecules, therefore the additional
following peaks or shoulders during DHPLC was interpreted as an indication of a
single base mismatch in heteroduplex DNA fragments.
DNA
sequencing
Both
the location and chemical nature of the mismatch were determined by sequencing
the reamplified product. Then the heterozygous and homozygous DNA samples were
cloned into the pMD18-T vector (TaKaRa), then sequenced in both directions on
the ABI prism 377 DNA sequencer using the BigDye terminators cycle sequencing
kit. The sequencing was done commercially in Sangon Company, Shanghai.
Statistical
analysis
All the
statistical analyses were carried out by using SPSS10.0 software. The data were
presented as x± s or as percentages. Differences in lipid and lipoprotein values of the
various genotypes were evaluated with student t test, and differences in
frequencies of alleles and genotypes of the mutation between the two groups
were detected by ÷2 test. The odds ratios (OR) for CHD were
derived from the logistic regression analysis.
Results
Subject
characteristics
The
biochemical features of the subjects are provided in Table 1. Compared with the
matched control group, the CHD group had significantly larger body mass index
(BMI), significantly higher plasma TG and LDL-C levels. On the contrary, the
level of HDL-C was significantly lower in patients than in controls.
Table 1
Characteristics of CHD patients and controls
|
Characteristics |
CHD patients |
Controls |
P
|
|
n |
203 |
209 |
－ |
|
Gender (M/F) |
137/66 |
140/69 |
－ |
|
Age (y) |
55.4±6.5 |
54.8±8.7 |
0.441 |
|
BMI (kg/m2) |
25.92±3.24 |
23.89±2.73 |
0.000** |
|
TG (mmol/L) |
1.51±0.85 |
1.26±0.79 |
0.001** |
|
TC (mmol/L) |
5.01±1.01 |
4.90±0.99 |
0.601 |
|
HDL-C (mmol/L) |
1.17±0.42 |
1.31±0.49 |
0.017* |
|
LDL-C (mmol/L) |
2.36±0.76 |
2.19±0.75 |
0.041* |
|
VLDL-C (mmol/L) |
0.89±0.48 |
0.94±0.42 |
0.534 |
|
TC / HDL-C |
4.12±1.25 |
4.01±1.12 |
0.651 |
*P < 0.05, **P < 0.1.
Identification
of DNA sequence change
Screening
of the fragments containing promoter region and all 16 exons of the CETP gene
with DHPLC detected a putative sequence variation showing double peaks in the chromatogram.
Sequencing analysis revealed a missense mutation (T→A transversion) at
nucleotide 13,161 in exon 10 of the CETP gene that results in amino acid
substitution of a polar glutamine (codon CAG) for a nonpolar leucine (codon
CTG) at residue 296 (Fig. 1).
Fig. 1 Detection of the L296Q mutation in the CETP gene
by DNA sequencing
The arrows indicate the T to A transversion. (A) 296L allele; (B) 296Q allele.
Effect of the mutation on risk of CHD and plasma lipid levels
Table 2
presents the frequencies of alleles and genotypes in CHD patient group and
control group. The patients displayed significantly higher mutated allele
frequency and mutation genotype frequency than controls. The odds x ratio of the development of CHD was
1.83 for the 296Q allele carriers versus 296LL homozygotes with the 95% confidence interval
1.04–2.87. Significant differences with regard
to
lipid or lipoprotein values were also observed between genotypes within CHD
group (Table 3). The 296Q allele carriers
showed significantly higher TC and LDL-C levels than 296LL genotype.
Table 2 Frequencies of alleles and genotypes of L296Q mutation in CHD patients and
controls
|
Group |
Number |
Genotype (%) |
χ2 |
P
|
Allele (%) |
χ2 |
P |
OR |
|||
|
LL |
LQ+QQ |
296L 296Q |
|||||||||
|
Patient |
203 |
145(71.4) |
58(28.6) |
6.856 |
0.008 ** |
0.840 |
0.160 |
9.014 |
0.003 ** |
1.83 |
|
|
Control |
209 |
172(82.3) |
37(17.7) |
0.909 |
0.091 |
||||||
** P < 0.01.
Table 3 Comparisons of lipid and lipoprotein levels
between genotypes in CHD patients
|
Lipids (mmol/L) |
LL (n=145) |
LQ (n=51) |
QQ (n=7) |
P |
||
|
LL vs. LQ |
LL vs. QQ |
LQ vs. QQ |
||||
|
TG |
1.49 ± 0.79 |
1.51 ± 0.81 |
1.57 ± 0.59 |
0.854 |
0.687 |
0.637 |
|
TC |
4.88 ± 1.03 |
5.20 ± 0.92 |
5.81 ± 1.09 |
0.044* |
0.031* |
0.151 |
|
HDL-C |
1.16 ± 0.41 |
1.13 ± 0.30 |
1.21 ± 0.57 |
0.744 |
0.645 |
0.315 |
|
LDL-C |
2.26 ± 0.88 |
2.47 ± 0.67 |
2.89 ± 0.80 |
0.026* |
0.039* |
0.083 |
|
VLDL-C |
0.91 ± 0.37 |
0.88 ± 0.41 |
0.93 ± 0.51 |
0.884 |
0.891 |
0.583 |
P < 0.05.
Discussion
In the
present study, we have described a new mutation in the CETP gene, L296Q in exon 10. To our knowledge, this is
the first report on identifying novel CETP missense mutation in Chinese,
and the tenth mutation in the coding region of the CETP gene with
published allele frequency
data
[11,12].
Studies
have shown that genetic variability in the CETP gene is associated with
plasma lipid concentration and is a significant independent risk factor for CHD
[7,9]. The first reported CETP missense mutation, D442G within exon 15, was found to lead to
the reduction in CETP synthesis and increase of HDL-C. The 442G heterozygote had an increase of
3-fold in HDL concentration and markedly decreased plasma CETP mass and
activity. Cellular expression of mutant cDNA resulted in secretion of only 30%
of wild type CETP activity [7]. The results of study on Japanese- American men
demonstrated that the overall prevalence of definite CHD was higher in men with
CETP mutations than those without mutations, and the adjusted relative
riskof CHD was 1.61 (P=0.024) in men with the D442G mutation[9]. This result is
consistent with those studies in hich CETP transgenic mice with
hypertriglyceridemia expressing both human CETP and apo C-III genes
had reduced atherosclerosis [13]. Another study has shown a significant
relation between sequence variation at the CETP gene locus and the
progression of coronary atherosclerosis that is independent of plasma HDL-C
levels [14]. Clearly, the CETP gene may exert effects on cardiovascular
risk that are independent of HDL-C level. Another 2 missense mutations, A373P and R451Q, found only in Caucasians, were also
shown to be related to plasma lipoprotein levels [8,15].
Unlike
the D442G and R451Q that are close to the active site of CETP, L296Q mutation is far from the C-terminus
and leads to the increase in TC and LDL-C levels as well as CHD risks of the 296Q allele carriers. The mechanism
through which this mutation exerts its effect on the population is not clear.
With the aid of specific monoclonal antibodies [16] and site-directed
mutagenesis [17], the lipid transfer domain of CETP has been localized to the
C-terminal 12 amino acids. A putative hinge sequence [1], lipoprotein binding
[17] and HDL binding sites [18] have also been identified towards the
C-terminus of the protein. It seems that L296Q mutation can not change the structure of CETP
dramatically. On the other hand, leucine is a nonpolar amino acid while
glutamine is a polar amino acid, and they have different side chains. Such
amino acid substitution can change the hydrophobic nature of the protein and
hence may have different effects on the secondary structure of CETP. Therefore,
L296Q mutation
may modify the structure of CETP and somehow elevated TC and LDL-C levels which
are the well-known risk factors to CHD. Further studies of structure and
function of CETP in carriers with this genetic variant may help to elucidate
the correlation.
In
conclusion, a novel missense mutation L296Q in exon 10 of the CETP gene in
Chinese was identified in this paper. Association study revealed that this
mutation was related to CHD since a significantly higher 296Q allele frequency was found in CHD
patients than in controls. The mutation affected the levels of plasma lipids
with the 296Q allele carriers displaying
considerably higher TC and LDL-C concentrations than noncarriers. The identification
of mutations in the CETP gene will afford the opportunity to investigate
the role that CETP gene plays in the pathogenesis of CHD in Chinese.
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Received: September 1, 2003 Accepted: October 21, 2003
This work was supported by the grants from the National Natural Science
Foundation of China (No. 39993420), the National High Technology Research and
Development Program (863 program) (No. 2001AA224021-03) and the National Key
Science and Technology Program (2002BA711A08), Ministry of Science and
Technology
*Corresponding author: Tel, 86-28-85422749; Fax, 86-28- 85501518; E-mail, [email protected]