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http://www.abbs.info e-mail:[email protected] ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(6): 780-785 CN 31-1300/Q |
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Short Communication |
Relationship
between a Novel Polymorphism of Hepatic Lipase Gene and Coronary Artery Disease
LIAO
Lin-Chuan1, XIAO Cui-Ying
(
Department of Medical Genetics, West China Hospital, Sichuan University,
Chengdu 610041, China
1Institute
of Forensic Medicine, West China Medical Center, Sichuan University, Chengdu 610041,
China
2Department
of Cardiology, West China Hospital, Sichuan University, Chengdu 610041,
China )
The
human HL gene has been assigned to chromosome 15q21[6] and
spans over 35 kb with 9 exons encoding a cognate mRNA of 1.6 kb that is
translated into a mature 476-amino acid protein[7]. Several
polymorphisms have now been described in the HL gene, including a number of mutation
associated with the rare HL deficiency condition[5, 8-10].
Recent studies demonstrated that polymorphisms in the promoter of the HL
gene are related to variants in plasma HDL-C concentrations, and the associations between HL
gene promoter variants and HL activity have been reported[11-15].
It seems clear that a reduction of HL activity by some mutations in HL
gene should lead to increased susceptibility of the body to coronary artery
disease(CAD). But the findings were contradictory, some studies reported lower
HL activity in patients with CAD than in health controls[16],
whereas others found that HL activity was similar in cases and controls[17],
or elevated in men with coronary disease[18].
The
present work was to study the polymorphisms of HL gene in Chinese Hans
which accounts for 95 percent Chinese population and test the relationships
between the polymorphisms and the CAD or plasma HDL cholesterol concentration.
1.1
Subjects
102
patients with coronary artery disease were from the First University Hospital
of West China of Medical Center Sichuan University. All of them were examined
by coronary angiography using the Judkins technique. For the coronary
score, main coronary artery
branches(left anterior descending, left circumflex artery, right coronary artery) having at least
one stenosis of ≥60%
were recorded. Meanwhile, 82 unrelated age-matched subjects selected via
health-screening at the same hospital free of any clinical and biochemical
signs of CAD were used as controls for the study.
1.2
Measurement of lipids and lipoproteins
Blood
samples were collected at baseline from patients and controls after an
overnight fast. Plasma separated from cells by centrifugation at 500 g
for 10 min at room temperature was used immediately for lipid and lipoprotein
analysis. The levels of plasma cholesterol and triglyceride were determined
with an enzymatic kit (Boehringer Mannheim) and calibrated with a serum
calibrator. HDL-cholesterol was measured in the supernatant after precipitation
of apoB-containing lipoproteins with a 4% sodium phosphotungstate solution
after centrifugation. LDL-cholesterol (LDL-C) was calculated by use of the
Friedewald Formula. The apolipoproteins apoA1 and apoB levels were determined
by immunonephelometric assay (Behring Nephelometer).
1.3
DNA preparation and PCR amplification
Genomic
DNA was prepared from peripheral blood leukocytes using the “salting-out”
procedure[19] and stored at 4 ℃.
Individual exon of the HL gene including all exon-intron boundaries was
amplified by PCR. Designing of the oligonucleotide primers (Table 1) for PCR
were based on GenBank M35425, M35426, M35427, M35428, M35429, M35430, M35431, M35432, M35433 and
X58779 information. Each PCR amplification mixture contained 0.1 mg
genomic DNA, 40 pmol of each primer, 25 pmol dNTPs and standard PCR buffer in a
total volume of 50 ml.
The reaction mixture was heated at 94 ℃
for 4 min. Subsequently, 0.4 u Taq polymerase was added. The 30 rounds
of PCR amplification strategy was denaturation for 45 s at 94 ℃,
annealing for 30 s at 55-61
℃ and extension for 30 s at 72 ℃.
The reactions were carried out in a Perkin Elmer GeneAmp 9600 PCR System
(Perkin Elmer).
1.4
Denaturing high performance liquid chromato-graphy(DHPLC)
The
search for single base change by DHPLC scanning was performed on an automated
HPLC instrument (Hewlett Packard Instrument) identical to that described by Su et
al[20]. PCR products were eluted with a linear acetonitrile
gradient of 1.8% per minute at a flow-rate of 0.8 ml/min, the start and end
points of the gradient were adjusted according to the size of the PCR products.
The temperature for successful resolution of heteroduplex molecules was
predicted by the DHPLC algorithm available
athttp://insertion.stanford.edu/melt.html. In the present work, the appropriate temperature of analysis
for each amplification was determined empirically by running it at different
temperatures until a good resolution between homo- and hetero-duplexes was
obtained. The temperatures of DHPLC for the 10 amplifications of HL gene
are 61 ℃,
55 ℃, 57 ℃,
56 ℃, 60 ℃,
59 ℃, 58 ℃,
55 ℃, 61 ℃
and 57 ℃,
respectively.
1.5
DNA sequencing
The
location and chemical nature of the mismatch was confirmed by sequencing of the
re-amplified product. The heterozygous and homozygous samples were cloned in
T-Easy vector(Promage), then sequenced in both directions on the “ALFexpress
DNA” automated sequencer, using the
dye-terminator cycle Thermal sequenase sequencing kit (Usb company).
1.6
Statistical analysis
The
lipid phenotypic data between the CAD patients and controls were age and sex
adjusted, and were statistically analyzed using the Student t-test.
Deviation of the genotype counts from the Hardy-Weinberg equilibrium were
tested with HWE using Linkage Utility Programs[21]. Differences
between the patients with CAD and the controls with respect to the allele
frequencies and genotype distributions were analyzed by Fisher exact test.
Adjusted odds ratio(OR) for CAD were derived from the logistic equation.
2.1
Lipoprotein and apolipoprotein profiles
Plasma
lipid levels were compared between CAD and control groups. As seen in Table
2, the parameters used for
HDL-cholesterol, triglyceride and
ApoAI were significantly different between the two groups (P<0.001).
2.2
A novel polymorphism T→C(HL-2)
in the HL promoter
Screening
for base variant of the entire coding region, as well as the flanking regions of every exon of the HL gene
with DHPLC in CAD patients and controls revealed that there was a variation in
some samples. As is known, anny
mismatched base pair in a heteroduplex molecule is generally eluted ahead of
the homoduplex, resulting in one
additional DHPLC peak (data not shown). The character of varied base was then
identified by sequence analysis. As the result, a new base variation,
namely -2T→C
transition was discovered (Fig.1).
Fig.1 Sequence analysis of SNP in the
promoter region of HL gene
The
arrow indicates the -2T→C.
(A) T allele; (B) C allele.
2.3
Distribution of the T→C(HL-2)
in CAD patients and controls
To
determine the prevalence of the T→C
substitution, we screened this
variation in all the 102 CAD patients and 82 controls. The genotype
distribution and allele frequencies are listed in Table 3. No deviation from
Hardy-Weinberg equilibrium (c2
=0.016, df=1, P=0.899 for CAD group; c2=0.884,
df=1, P=0.347 for controls) was noted in both CAD and control groups. As
the result, excess carriers of the -2T→C
substitution were detected in the CAD patients compared with the nonsymptomatic
control subjects (57.9% versus 42.7%,
c2
=4.181, df=2, P=0.041). The prevalence of the -2C allele was
significantly higher in the CAD patients than in control subjects (c2
=3.988, df=1, P=0.046). The OR of -2C allele associated with the risk of
CAD is 1.58 (95% confidence interval: 1.01-2.47).
2.4
Association between T→C
substitution and plasma lipids
Studies
on the relation between T→C
(HL-2) and plasma lipid showed that neither cholesterol and
triglyceride, nor LDL-cholesterol
was different significantly between subjects with or without this gene variant.
However, HDL cholesterol levels
did differ among different genotypes(P<0.05). The subjects homozygous
for the C allele (HL-2) had the highest HDL-cholesterol values [(1.13±0.24
) mmol/L] and subjects homozygous for the -2T allele had the lowest [(0.91±0.34)
mmol/L], while the heterozygote
had the intermediate value [( 0.98±0.43)
mmol/L, see Table 4].
3
Discussion
In
present study, a novel base variation (-2T→C
) in the HL promoter region was found through DHPLC and DNA sequencing.
This polymorphism was present in about 58% of patients with angio-graphically
established coronary artery disease and in about 43% of nonsymptomatic control
subjects. The T→C
allele was significantly more frequent in the patient with CAD than in the
control subjects.
There
is considerable evidence that hepatic lipase activity is an important
determinant of plasma HDL-C concentrations. Clinical studies have consistently
found an inverse relationship between hepatic lipase activity measured in post
heparin plasma and plasma HDL-C concentrations[22-24].
Association studies showed that the -2T→C
variation may account for the variation in plasma HDL-C concentration, at least
in the tested Chinese. Since we did not measure the hepatic lipase activity in
the present study, so we can only speculate that the -2T→C
polymorphism may affect the activity of this enzyme and thereby influence the
plasma HDL-C. Given the well established inverse relationship between hepatic
lipase activity and HDL-C concentrations, however, it seems very likely that the -2C is associated with low
hepatic lipase activity by directly affecting hepatic lipase expression or
through linkage disequilibrium with another polymorphism that directly
decreases the enzyme activity. Since no linkage of the -2T→C
variant with other polymorphisms in the first 668 bp of the HL promoter
was found, it suggests that the
base substitution may lead to a lowered hepatic lipase expression.
The
promoter sequence variant -514T in the HL gene has been shown to be
significantly associated with low post-heparin hepatic lipase activity[11,
12]. Some studies have also found that the -514T variant is associated
with elevation in plasma HDL-cholesterol[13-15].
We tested for associations of the same HL -514T with plasma lipoprotein
traits in Chinese CAD patients and normal controls. The HL -514T allele
frequencies in these two groups were 0.224 and 0.315, respectively, no significant association was found between HL
-514T and plasma HDL-cholesterol, after adjusting for covariates including
gender and body mass index, although the plasma HL activity was not available
for analyses. There was no consistent relationship between the population mean
plasma HDL-cholesterol concentration and the population HL -514T
frequency. Our findings are consistent with the idea that the common promoter
variation in HL, which has been reported to be associated with variation in
post heparin HL activity and HDL triglyceride concentration, is not always
associated with variation in plasma HDL-cholesterol concentration, possibly due to yet unspecified
environmental or genetic factors.
In
summary, we have identified a novel base change in the promoter of HL
gene in Chinese CAD patients and normal controls, The association between HL
genotype and HDL was significant at the 0.05 level, which suggests that genetic
variation at the HL locus is involved in the determination of lipid and
lipoprotein profiles and the predisposition to CAD. Further studies are needed
to elucidate the molecular mechanism and the transcription factors involved.
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Received:
April 1, 2002
Accepted: May 31, 2002
This
work was supported by grants from the National Natural Science Foundation of
China (No.39993420), and the National High Technology Research and Development
Program of China (863 Program) (No.2001AA224021-03)
*Corresponding
author: Tel,86-28-85422749; Fax,86-28-85501518; e-mail, [email protected]