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http://www.abbs.info e-mail:[email protected] ISSN 0582-9879
ACTA BIOCHIMICA et
BIOPHYSICA SINICA 2003, 35(7): 606-610
CN 31-1300/Q |
A Novel Polymorphism A+884→G
in the Hepatic Lipase Gene and Its Association with Coronary Artery Disease
(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)
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
|
Index |
Control (n=82) |
CAD (n=102) |
P |
|
TC (mmol/L) |
5.31±0.87 |
5.16±1.03 |
*NS |
|
TC (mmol/L) |
1.26±0.53 |
1.53±0.18 |
0.0023 |
|
LDL-C (mmol/L) |
3.18±0.80 |
3.33±0.93 |
*NS |
|
HDL-C (mmol/L) |
1.54±0.38 |
1.14±0.32 |
0.0001 |
|
ApoAI (g/L) |
1.38±0.33 |
1.15±0.28 |
0.0001 |
|
ApoB100 (g/L) |
1.10±0.26 |
1.11±0.30 |
*NS |
*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<sup>+884</sup>→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
|
Group |
Number |
Genotype |
χ2 |
P |
Allele (%) |
χ2 |
P |
|||
|
AA |
AG |
GG |
A |
G |
||||||
|
CAD |
102 |
46 |
48 |
8 |
6.164 |
0.046 |
68.6 |
31.4 |
4.652 |
0.031 |
|
Control |
82 |
48 |
33 |
1 |
78.7 |
21.3 |
||||
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
|
Polymorphic sites and subjects |
Estimated haplotype frequency |
D` |
P |
|||
|
T－2C/ A+884G |
TA |
TG |
CA |
CG |
|
|
|
CAD patients |
0.586 |
0.100 |
0.061 |
0.253 |
0.699 |
0.000 |
|
Controls |
0.703 |
0.084 |
0.041 |
0.172 |
0.742 |
0.000 |
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]