Genetic factors associated with hyperalphalipoproteinemia (HALP; or high levels of high-density lipoprotein cholesterol) are incompletely understood. The aim of this study was to resequence 3 candidate genes, CETP, LIPC, and LIPG, which encode cholesteryl ester transfer protein, hepatic lipase, and endothelial lipase, respectively, in Thai subjects with HALP and compare them to normolipidemic controls. Sequence variants of CETP, LIPC, and LIPG were identified by sequencing exons and exon-intron junctions in 64 subjects with high-density lipoprotein cholesterol levels ≥2.59 mmol/L (100 mg/dl) and compared to those of 113 normolipidemic subjects. Two heterozygous frameshift mutations in CETP (p.Leu262ProfsX31 and p.Val411ArgfsX6) and two heterozygous missense mutations in LIPC (p.Gly141Ser and p.Val173Met) were found. One deletion mutation and 3 point mutations in the CETP promoter were also identified. Collectively, these rare mutations were found only in the HALP group but not in the control group (8% vs 0%, p = 0.0056). One common variant of CETP (p.Asp459Gly) was found at a higher frequency in the HALP group (23% vs 4%, p = 0.000074). Altogether, rare variants of CETP or LIPC and/or the common CETP p.Asp459Gly variant were found in 30% of the HALP group and 4% of the controls (p = 0.0000014). No rare variant of LIPG was identified. In conclusion, common and rare genetic variants in CETP and LIPC, but not LIPG, were more commonly found in the Thai HALP group, which could potentially contribute to high high-density lipoprotein cholesterol phenotypes in this population.
Hyperalphalipoproteinemia (HALP), characterized by high levels of high-density lipoprotein (HDL) cholesterol, is an uncommon and heterogenous condition caused by various genetic and environmental factors. Twin studies have shown that approximately 50% of HDL cholesterol levels are heritable. The genetic determinants of HALP in different populations are, however, complex and incompletely understood. In Japan, where the prevalence of HALP appears to be high, HALP is associated with cholesteryl ester transfer protein (CETP) deficiency due to mutations in the CETP gene. In Caucasians with HALP, however, mutations in CETP are extremely rare. Recent reports have demonstrated an association of HALP with variants in LIPC and LIPG, which encode hepatic lipase and endothelial lipase, respectively. To date, rare sequence variants in candidate genes involved in HDL metabolism have not been fully examined in HALP. Previously, our pilot study of 38 Thai subjects with HALP documented lower CETP and hepatic lipase activities compared to those of control subjects and identified a few novel genetic variants in CETP and LIPC. In this study, using a resequencing approach, we extended the study by examining the genetic variations in 3 candidate genes, CETP, LIPC, and LIPG, in a larger group of Thai subjects who had HALP and determined the association of these variants with HALP.
Methods
We recruited 64 ambulatory subjects who had documented HDL cholesterol ≥2.59 mmol/L (100 mg/dl) on ≥2 occasions measured from the central laboratory of King Chulalongkorn Memorial Hospital. A subset of 38 subjects was reported previously. Secondary causes of HALP and the use of medications known to affect lipid levels were excluded in all subjects. Normolipidemic control subjects, who were free of cardiovascular, hepatic, and renal diseases and were not taking medications known to affect lipid levels, were also enrolled from the same hospital. Each participant gave written informed consent, and the study protocol was approved by the ethics committee of the Faculty of Medicine at Chulalongkorn University. The study protocols conformed to the ethical guidelines of the 1975 Declaration of Helsinki, and procedures were performed in accordance with institutional guidelines.
Genomic deoxyribonucleic acid was isolated from whole blood. Each exon and exon-intron junction of CETP (16 exons), LIPC (9 exons), and LIPG (10 exons) was individually amplified by polymerase chain reaction. Because a mutation in the CETP promoter has been associated with HALP, we additionally sequenced the 835-bp promoter region of the CETP gene in the HALP group. Data on the primers used are available in the supplementary material. The polymerase chain reaction products were purified with the Exo-SAP-IT kit (Amersham Biosciences, Buckinghamshire, United Kingdom) and sequenced using an ABI 3730XL DNA Analyzer (Applied Biosystems, Carlsbad, California) at Macrogen (Seoul, South Korea). Identified common and rare deoxyribonucleic acid variants were independently verified in another sequencing experiment. Screening of identified variants in the control group was performed using either direct sequencing or polymerase chain reaction restriction fragment length polymorphism. The nomenclature for the description of sequence variants was according to the recommendations of the Human Genome Variation Society ( http://www.hgvs.org/mutnomen/recs-prot.html ). The novelty of the variants was checked using the National Center for Biotechnology Information’s dbSNP and 1000 Genomes ( http://www.1000genomes.org ).
The functional consequences of the identified sequence variants were determined using the PANTHER ( http://www.pantherdb.org ), PolyPhen-2 ( http://genetics.bwh.harvard.edu/pph2/ ), and SNP3D ( http://www.snp3d.org ) programs. In the PANTHER program, a substitution position–specific evolutionary conservation score was calculated to determine whether a variant identified would have the deleterious effect on the protein function. The probability of a variant being deleterious, P deleterious , was calculated from a substitution position–specific evolutionary conservation score, and a substitution position–specific evolutionary conservation score < −3, which corresponded to P deleterious of 0.5, was found to be a cutoff for a functional significance. A higher value of P deleterious indicates more severe impairment. The PolyPhen-2 program predicts an impact of an amino acid substitution on the basis of multiple sequence alignments and protein 3-dimensional structures. In the SNP3D program, a support vector machine score was calculated. A negative support vector machine score indicates that the variant is deleterious.
Statistical analysis was performed using SPSS version 12 (SPSS, Inc., Chicago, Illinois). Statistical significance between the groups was evaluated using unpaired Student’s t tests or Fisher’s exact tests. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated according to Bland and Altman. A discounting technique was used to avoid an infinite OR. A p value <0.05 was considered statistically significant. Sidak correction was used to calculate the lower significance threshold to account for multiple testing involved.
Results
Clinical characteristics of the study subjects are listed in Table 1 . The HALP group had significantly higher levels of total cholesterol and HDL cholesterol and lower levels of triglyceride. The range of HDL cholesterol levels in the HALP group was 100 to 151 mg/dl.
| Variable | HALP (n = 64) | Controls (n = 113) | p Value |
|---|---|---|---|
| Age (years) | 54 ± 14 | 51 ± 17 | 0.22 |
| Women | 91% | 81% | 0.08 |
| Total cholesterol (mg/dl) | 246 ± 42 | 200 ± 42 | 1.6 × 10 −10 |
| Triglyceride (mg/dl) | 67 ± 32 | 99 ± 53 | 1.1 × 10 −6 |
| HDL cholesterol (mg/dl) | 115 ± 13 | 63 ± 13 | 3.0 × 10 −51 |
| Low-density lipoprotein cholesterol (mg/dl) | 117 ± 39 | 118 ± 37 | 0.83 |
Rare sequence variants in the candidate genes are listed in Table 2 . We found 2 heterozygous rare variants in the coding region of CETP. The first rare variant was a 4-bp deletion in exon 9, c.785–788delTCCC, which was predicted to cause a frameshift mutation resulting in a premature stop codon 31 amino acids downstream (p.Leu262ProfsX31). The second variant was a novel 5-bp duplication in exon 13, c.1226–1230dupAGACT, which was predicted to cause a frameshift mutation resulting in a premature stop codon 6 amino acids downstream (p.Val411ArgfsX6). These 2 rare variants were predicted to cause truncated proteins lacking the lipid-binding C terminus. Low plasma CETP activities were found in these 2 probands previously (7.2 and 15.2 pmol/μl/hour, respectively, vs 43.7 ± 2.6 pmol/μl/hour in the control group).
| Location | Variant Name | Predicted Effect at the Protein Level | Number of Carriers | ||||
|---|---|---|---|---|---|---|---|
| DNA Level | Protein Level (Common Name) | PANTHER (subPSEC Score) | PolyPhen-2 | SNP3D (SVM Score) | HALP (n = 64) | Controls (n = 113) | |
| CETP | |||||||
| Exon 9 | c.785-788 delTCCC | p.Leu262ProfsX31 | Frameshift with early truncation | 1 | 0 | ||
| Exon 13 | c.1226-1230 dupAGACT | p.Val411ArgfsX6 | Frameshift with early truncation | 1 | 0 | ||
| Promoter | g.4989-5006 delGGGCGGACATACATATAC | (−25_−42del GGGCGGACA TACATATAC) | — | — | — | 1 | 0 |
| g.4982G>T | (−49G>T) | — | — | — | 1 | 0 | |
| g.4961C>T | (−70C>T) | — | — | — | 2 | 0 | |
| g.4659C>T | (−372C>T) | — | — | — | 1 | 0 | |
| LIPC | |||||||
| Exon 3 | c.421A>G | p.Gly141Ser | Deleterious (−4.43) | Probably damaging | Deleterious (−1.58) | 1 | 0 |
| Exon 4 | c.517G>A | p.Val173Met | Deleterious (−6.43) | Possibly damaging | Deleterious (−0.53) | 1 | 0 |
Rare variants in the CETP gene promoter are also associated with HALP. We found 1 deletion mutation and 3 heterozygous point mutations in the CETP promoter in 5 subjects. The 18-bp deletion mutation was previously shown to be associated with a reduction in transcriptional activity. The other 3 point mutations, g.4982G>T (−49G>T), g.4961C>T (−70C>T), and g.4659C>T (−372C>T), were novel. Given lack of definitive proof that these 3 novel point mutations were dysfunctional, we treated the carriers of these mutations as noncarriers in subsequent analyses.
Two heterozygous rare variants in LIPC were found, p.Gly141Ser and p.Val173Met. Using 3 different computerized prediction programs, these variants were consistently predicted to have a damaging effect on the protein ( Table 2 ). Low postheparin plasma hepatic lipase activities were also found in these 2 probands (25.1 and 130.6 nmol/ml/min, respectively, vs 227.4 ± 16.5 nmol/ml/min in the control group). For LIPG, no rare sequence variants were found.
Three common variants were found in CETP, p.Val422Ile (commonly known as I405V), p.Asp459Gly (commonly known as D442G), and g.4402C>A (−629C>A), as listed in Table 3 . In our study, only p.Asp459Gly was significantly more prevalent in the HALP group compared to controls (OR 8.3, 95% CI 2.6 to 26.4; Table 4 ).
| Location | Variant Name | Allele Frequencies | p Value | |||
|---|---|---|---|---|---|---|
| DNA Level | Protein Level (Common Name) | SNP Identifier | HALP (n = 64) | Controls (n = 113) | ||
| CETP | ||||||
| Exon 14 | c.1264G>A | p.Val422Ile (I405V) | rs5882 | 0.430 | 0.447 | 0.824 |
| Exon 15 | c.1376A>G | p.Asp459Gly (D442G) | rs2303790 | 0.133 | 0.018 | 2.0 × 10 −5 |
| Promoter | g.4402C>A | (−629C>A) | rs1800775 | 0.492 | 0.385 | 0.057 |
| LIPC | ||||||
| Exon 3 | c.283G>A | p.Val95Met (V73M) | rs6078 | 0.352 | 0.296 | 0.287 |
| Exon 5 | c.644A>G | p.Asn215Ser (N193S) | rs6083 | 0.297 | 0.580 | 3.0 × 10 −7 |
| Exon 7 | c.1068C>A | p.Phe356Leu (L334F) | rs3829462 | 0.039 | 0.053 | 0.616 |
| LIPG | ||||||
| Exon 3 | c.332C>T | p.Thr111Ile | rs2000813 | 0.344 | 0.263 | 0.115 |
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