A Novel Polymorphism A⁺⁸⁸⁴→G
in the Hepatic Lipase Gene and Its Association with Coronary Artery Disease

SU Zhi-Guang, ZHANG Si-Zhong*, ZHANG Li¹,
TONG Yu, XIAO Cui-Ying, HOU Yi-Ping², LIAO Lin-Chuan²

(Department of Medical
Genetics, West China Hospital, Sichuan University, Chengdu 610041, China; ¹Department
of Cardiology, West China Hospital, Sichuan University, Chengdu 610041, China; ²Institute
of Forensic Medicine, West China Medical Center, Sichuan University, Chengdu
610041, China)

Abstract       Hepatic
lipase (HL) activity may influence susceptibility to coronary artery disease
(CAD). Association between the single nucleotide polymorphisms (SNPs) in the HL
gene with the occurrence of CAD has been investigated thoroughly, but to date
most studies focused on the base variation in the promoter of HL gene, little
is known about the variation in the coding region. In present study, the SNP in
all exons of the HL gene were analyzed. All 9 exons with their flanking
sequences of the HL gene were amplified from the Chinese patients with CAD and
normal controls by PCR technique, and the PCR products were detected by
denaturing high performance liquid chromatography (DHPLC) and sequenced with a dideoxy
terminal termination method. As the result, a novel SNP A⁺⁸⁸⁴→G
within the sixth exon of HL gene was found, the 276 codon AAA was changed into
AGA and resulted in the substitution of arginine for lysine. Compared with the
control group, more CAD patients carried the G⁺⁸⁸⁴ allele (AG+GG)
(54.9% vs. 41.5%, χ2=6.164, df=2, P=0.046). The prevalence of the G+884
allele was significantly higher in the CAD patients than that in control
subjects (31.4% vs. 21.3%, χ2 =4.652, df=1, P=0.031). Data from the linkage
disequilibrium analysis showed that the A⁺⁸⁸⁴→G polymorphism was
strong in linkage disequilibrium with the T－2→C variation we identified
previously(D`=0.699, 0.742 in CAD patients and controls, respectively), and the
frequency of the C－2/G⁺⁸⁸⁴ haplotype (mutation) is significantly
higher in CAD patients than that in controls (0.253 vs. 0.172, P<0.05).

Key words   hepatic
lipase gene; single nucleotide polymorphism (SNP); linkage disequilibrium;
coronary artery disease

Coronary artery
disease (CAD) is one of the most severe cardiovascular diseases and a major
cause of death in many countries. In the vast majority of cases, a process
influenced by both environmental and genetic factors, underlies the development
of CAD[1]. Disorders of lipoprotein metabolism such as elevated low-density
lipoprotein (LDL) cholesterol and low high-density lipoprotein (HDL)
cholesterol are considered important risk factors in the pathogenesis of
atherosclerosis[2].

Hepatic lipase
(HL) is a key enzyme involved in lipoprotein metabolism[3]. Its catalytic
activity contributes to the remodeling of chylomicron remnants, intermediate
density lipoproteins, LDL, and HDL and participates in the reverse cholesterol
transport[4]. HL deficiency leads to elevation in HDL cholesterol, increased
levels of triglyceride in HDL and LDL, and impaired metabolism of post-prandial
glyceride-rich lipoproteins[5－7], and the latter are considered to be risk factors for premature
atherosclerosis. Although HL seems to be an important enzyme with multiple
functions, the exact role in lipoprotein metabolism has not yet been
established.

The human HL
gene has been assigned to chromosome 15q21 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[8－11]. Several
polymorphisms have now been described in the HL gene, including a number of
mutation associated with the rare HL deficiency condition[12－14]. 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[15－20]. It seems clear that a reduction of HL activity by some
mutations in HL gene should lead to increased susceptibility to CAD. But the
findings were contradictory, some studies reported lower HL activity in
patients with CAD than in health controls[21], whereas others found that HL
activity was similar in cases and controls[22], or elevated in men with
coronary disease[23].

We have shown
previously that the T^－2→C variant in the promoter of the HL
gene was associated with the variation of plasma HDL-C level and the
predisposition to CAD[24]. The aim of present work is to study whether any
other base substitution in the coding region of HL gene is associated with the
occurrence of CAD in Chinese Hans which accounts for 95% Chinese population.

1 Materials and Methods

1.1  
Subjects

The subjects
have been described previously[24]. In brief, 102 patients with CAD were
recruited from West China Hospital of 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

All lipid
analysis were performed by using procedures identical to that described
previously[15, 24]. 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[25] and stored at 4 °C. All the 9 exons including the exon-intron
boundaries of the HL gene were amplified by PCR. Primers for the PCR were used
as previously described[24]. PCR was performed in a total volume of 50 μl
containing 0.1 μg genomic DNA, 40 pmol of each primer, 25 pmol dNTPs and
standard PCR buffer. The reaction mixture was heated at 94 °C for 4 min.
Subsequently, 0.4 u Taq polymorase was added. The 30 rounds of PCR
amplification strategy was denaturation for 45 s at 94 °C, annealing for 30 s
at 55－61 °C and extension
for 30 s at 72 °C. The reactions were carried out in a Perkin Elmer GeneAmp
9600 PCR System (Perkin Elmer).

1.4 Denaturing high performance liquid
chromatography (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.[26]. The appropriate temperature of DHPLC for the 9 amplificons of HL gene
are 55 °C, 57 °C, 56 °C, 60 °C, 59 °C, 58 °C, 55 °C, 61 °C and 57 °C,
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
(Promega), 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 data were
analyzed using the SAS statistical software, the significance level for
statistical tests was taken to be 0.05.

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 was tested with HWE
using Linkage Utility Programs. Differences between the patients with CAD and
the controls with respect to the allele frequencies and genotype distributions
were analyzed by Fisher exact test. Haplotype frequencies for pairs of alleles,
as well as χ2 values for allele associations, were estimated by the Estimating
Haplotype-frequencies software program[27] (Rockefeller University,
http://linkage.rockefeller.edu), LD coefficients D`=D/Dmax were calculated by
2LD program[28] (University of London, http://www.iop.kcl.ac.uk/IoP/Departments).

2 Results

2.1 Lipoprotein and apolipoprotein
profiles

The general
characteristics of the samples have been described before in detail and key
traits are presented in Table 1, the parameters used for HDL-cholesterol,
triglyceride and ApoAI were significantly different between the two groups (P<0.001).

Table
1 Comparison of serum lipids levels between control group and CAD

*NS, no significant difference. Data were represented as (x±s).

2.2 A novel polymorphism A⁺⁸⁸⁴→G
within exon 6 of the HL gene

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, any 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 A⁺⁸⁸⁴→G transition (sequence number according to GenBank NM－000236) within the sixth exon of the
HL gene was detected(Fig.1), which results in a substitution of the 276 codon
AGA for AAA and the substitution of Arg for Lys.

Fig.1 Sequence analysis of SNP within exon 6 of the HL gene

The
arrow indicates the A⁺⁸⁸⁴→G. (A) A allele. (B) G allele.

2.3 Distribution of the A⁺⁸⁸⁴→G
in CAD patients and controls

To determine the
prevalence of the A→Gsubstitution, we screened this variation in all the 102 CAD
patients and 82 controls. The genotype distribution and allele frequencies are
listed in Table 2. No deviation from Hardy-Weinberg equilibrium (χ2 =0.879,
df=1, P=0.348 for CAD group; χ2 =3.237, df=1, P=0.072 for
controls) was noted in both CAD and control groups. As the result, excess of
carriers of the A⁺⁸⁸⁴→G substitution were detected in
the CAD patients compared with the nonsymptomatic control subjects (54.9% vs.
41.5%, χ2 =6.164, df=2, P=0.046). The prevalence of the G⁺⁸⁸⁴
allele was significantly higher in the CAD patients than in control subjects
(χ2 =4.652, df=1, P=0.031).

Table 2 Frequency distributions of the HL gene in patients
with CAD vs. controls

2.4 Linkage disequilibrium between T^－2→C
and A⁺⁸⁸⁴→G polymorphisms in the HL gene

Recently we
identified T^－2→C polymorphism in the promoter of the HL gene[24]. Here, we also
analyzed the relation between T^－2→C and A⁺⁸⁸⁴→G
polymorphisms and their effects on CAD. The extent of D in pairwise
combinations of alleles in locus at the HL promoter and exon 6 was estimated by
means of maximum likelihood from the frequency of diploid genotypes in the CAD
and control groups. Haplotype frequencies and the coefficient of linkage
disequilibrium(D`) are given in Table 3. It is clear that the D` values for －2/+886 pairs differ significantly from
zero, and the frequency of the CG haplotype (mutation) is significantly higher
in CAD patients than that in controls (0.253 vs. 0.172, P<0.05).

Table
3 Estimate of pairwise haplotype frequencies and disequilibrium statistics

3 Discussion

In present
study, a novel base variation (A⁺⁸⁸⁴→G ) within exon 6 of HL gene
was found by DHPLC and DNA sequencing, which resulted in the 276 codon AAA
substituted by AGA and the substitution of Arg for Lys. This polymorphism was
present in about 54.9% of patients with angiographically established coronary
artery disease and in about 41.5% of nonsymptomatic control subjects. The G
allele was significantly more frequent in patients with CAD than in controls.

In previous
study[24], the T^－2→C polymorphism in the promoter of
the HL gene was identified and the frequency of the C allele was higher in CAD
patients than that in controls, and the association studies showed that the T^－2→C
variant was associated with the variation in plasma HDL-C concentration, at
least in the tested Chinese. Since the T^－2→C polymorphism are not located in
the regions containing putative regulatory elements[29], it is unlikely that
this promoter variant directly affect the hepatic lipase expression. This
suggests that the T^－2→C variant could be in linkage
disequilibrium with another polymorphism of the gene that may impact the enzyme
activity level. Results from this study showed that the A⁺⁸⁸⁴→G
variant was in strong linkage disequilibrium with the T^－2→C
polymorphism. This finding suggests that the substitution of Arg for Lys at
codon 276 may decreases the activity of hepatic lipase. Since we did not
measure the hepatic lipase activity in the present study, so it can be only
speculated that the A⁺⁸⁸⁴→G polymorphism may affect the activity of
this enzyme and thereby influence the occurrence of CAD.

In summary, we
have identified a novel base change (A⁺⁸⁸⁴→G, Lys²⁷⁶→Arg)
in the exon 6 of HL gene in Chinese CAD patients and normal controls, and it was
in strong linkage disequilibrium with the T^－2→C polymorphism identified in
previous study. The association between HL genotypes and CAD is significant at
the 0.05 level, which suggests that genetic variation at the HL locus is
involved in the determination of hepatic lipase activity and the predisposition
to CAD. Further studies are needed to elucidate the molecular mechanism by
which the activity of the hepatic lipase is influenced.

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________________________________________

Received: March 31, 2003     Accepted: May 6, 2003

This work was supported by the grants from
the National Natural Science Foundation of China (No. 30200161), 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]