Sources of variability in genetic association studies: insights from the analysis of hepatic lipase (LIPC)

Genetic association studies have been widely used to identify loci that influence plasma lipoprotein concentrations, but few of the associations reported have proved consistently reproducible across different study populations. This lack of consistency is a widely recognized limitation of association studies, and is often ascribed to inadequate statistical power, population substructure, and population-specific linkage disequilibrium. However, few studies have assessed the causes of variability underlying a given genotype-phenotype association. We have examined two possible sources of variability in the association between the -514 polymorphism in hepatic lipase (LIPC) and plasma HDL-C concentrations. First, we compared the association between this polymorphism and hepatic lipase activity in four populations. A single copy of the -514C allele was associated with a 10 mmol.hr(-1).l(-1) increase in hepatic lipase activity in white American and Turkish men but only approximately 5 mmol.hr(-1).l(-1) in Chinese and African-American men. Second, we tested the effects of a stanozolol-induced increase in hepatic lipase activity on plasma HDL-C concentrations in men with normal (< 150mg/dl) or elevated (150-300mg/dl) levels of plasma triglyceride. The increase in hepatic lipase activity was similar in the two groups, but the resulting decline in HDL-C levels was significantly greater in normolipidemic men. These data suggest that the effect of a polymorphism on gene expression can vary among individuals, and that the resulting phenotype may be further modified by interactions with other factors. Three novel LIPC polymorphisms were identified in the study (-1596insC, -2740A>G, and -2880G>C).     

新LPL基因复合杂合变异导致的一例新生儿脂蛋白脂肪酶缺乏症

目的明确1例脂蛋白脂肪酶缺乏症新生儿的遗传学病因。方法应用目标区域捕获测序技术对患儿进行遗传代谢疾病相关基因检测,并对可疑变异位点在患儿及其父母进行Sanger测序验证。结果基因检测结果显示患儿LPL基因存在c.347G>C(p.Argll6Pro)和c.472T>G(p.Tyrl58Asp)复合杂合变异,父亲携带c.347G〉C(p・Argll6Pro)杂合变异,母亲携带c.472T>G(p.Tyrl58Asp)杂合变异,因此患儿的变异分别来源于其父母。结论LPL基因c.347G>C(p.Argll6Pro)和c.472T>G(p.Tyrl58Asp)复合杂合变异可能是这例脂蛋白脂肪酶缺乏症新生儿的致病原因。     

Genotype-phenotype studies of six novel LPL mutations in Chinese patients with hypertriglyceridemia

We screened 160 unrelated Chinese hypertriglyceridemic subjects for sequence alterations in the promoter and the 10 exons of the lipoprotein lipase (LPL) gene. We identified one reported mutation (L252R), one common polymorphism (S447X), and six novel mutations: V181I, C283Y, S298R and S338F (found in single individuals), L252V (in two individuals), and A71T (in three individuals). Screening of family members of the above probands revealed a total of 19 mutation carriers, most of whom, though not all, displayed reduced LPL activity and mass when compared to normolipidemic control subjects. In in vitro expression studies, A71T, V181I, L252R, L252V and C283Y decreased the specific activity of the gene product. Interestingly, S298R had no effect on the catalytic activity while S338F increased it. A71T and C283Y reduced the secretion of the mutant proteins significantly while V181I, S298R and S338F had mild effects only. The total LPL mass of all the mutant constructs was reduced compared to that of the wild type construct, probably due to the instabilities of the mutant mRNA or the mutant protein. The heterogeneity in phenotypic effects of these mutations is a likely consequence of their variable effects on proteoglycan binding, conformation and interactions with other secondary genetic or environmental factors.     

Association of an intronic haplotype of the LIPC gene with hyperalphalipoproteinemia in two independent populations

Hepatic lipase (HL) plays a major role in the regulation of plasma lipids. Several groups seeking to find association between the gene encoding HL (LIPC) and plasma concentrations of high-density lipoprotein cholesterol (HDLc) using various methods and populations have reported conflicting results. We have approached the problem of demonstrating a relationship between the LIPC locus and HDLc by means of haplotype association using four single nucleotide polymorphisms (SNPs) (rs12594375G/A, rs8023503C/T, rs4775047C/T, and rs11634134T/A) located in intron 1 of the LIPC gene in two independent Japanese populations consisting of 2,970 and 1,638 individuals, respectively. Significant association between hyperalphalipoproteinemia and a specific haplotype in this intron was detected in both populations. When HDLc levels among the three haplotypic categories were analyzed [haplotype rs8023503C/rs12594375G (haplotype-1; H1) homozygotes (H1H1), haplotype rs8023503T/rs12594375A (haplotype-2; H2) homozygotes (H2H2), and heterozygotes (H1H2)], HDLc levels were lowest among H1H1 [mean ± standard error (SE) = 58.4 ± 0.4 mg/dl], highest among H2H2 (62.5 ± 0.8 mg/dl), and intermediate among H1H2 (59.2 ± 0.4 mg/dl) (P = 0.00011), indicating that H2 haplotype elevates plasma HDLc levels. This association was validated in the second population (n = 1,638) (P = 0.00070). The results provide convincing evidence that the LIPC locus influences HDL metabolism.     

Pink‐creamy whole blood in a 3‐month‐old infant with a homozygous deletion in the lipoprotein lipase gene

Avis HJ, Scheffer HJ, Kastelein JJP, Dallinga‐Thie GM, Wijburg FA. Pink‐creamy whole blood in a 3‐month‐old infant with a homozygous deletion in the lipoprotein lipase gene.     

Common genetic variants of lipoprotein lipase that relate to lipid transport in patients with premature coronary artery disease

Two common coding sequence mutations of lipoprotein lipase (serine⁴⁴⁷-ter, producing a carboxy terminal truncation; and asp⁹-asn variants) were studied in 329 Caucasian subjects, of whom 243 had angiographic features of premature atheroscelerosis (220 with coronary artery disease; 23 with coronary and peripheral artery disease). As expected, the mean levels of cholesterol, triglycperides, LDL-cholesterol, ApoB and Lp(a) were significantly higher in the arterial disease group than in the controls. HDL levels were lower in the patient group. With regard to the common mutations, plasma triglycerides and VLDL-triglycerides were lower in subjects possessing the Serine⁴⁴⁷-Ter mutation (p=0.06 and <0.05, respectively). When the lipid distributions were analysed by tertiles, the Ser⁴⁴⁷-Ter mutation was significantly less frequent in the highest tertiles for triglycerides (p<0.02), and VLDL (p<0.04). The Asp⁹-Asn substitution was significantly more frequent in the lowest tertiles for ApoAI (p=0.05). Case-control analyses of genotypic distributions between the two groups with or without arterial disease did not show any significant differences. The possible functional effects of these common mutants of lipoprotein lipase are discussed.     

Association of pre-eclampsia with common coding sequence variations in the lipoprotein lipase gene

Marked dyslipidemia may contribute to endothelial cell dysfunction in pre-eclampsia. Carriers of N291S or D9N missense mutations in the lipoprotein lipase (LPL) gene exhibit reductions in LPL activity and are predisposed to dyslipidemia and cardiovascular disease. In Caucasians, the D9N variant is in strong linkage disequilibrium with the - 93T --> G promoter variant. A fourth LPL variant, S447X, is often associated with a beneficial lipid profile. We asked if the N291S and the combination D9N/- 93T --> G variants are more prevalent, and if the S447X variant is less prevalent, in Caucasian women with pre-eclampsia as compared with normal pregnancies. DNA amplification was followed by an allele-specific oligonucleotide ligation assay. Allele frequencies were analyzed with a chi2 table and Yates' correction. The N291S variant was identified in 11.1% of pre-eclamptics as compared with 2.9% of pregnancy controls (p = 0.008). All carriers of D9N were also carriers of - 93T --> G. The D9N/ - 93T --> G combined variant was found in 7.1% of pre-eclamptics as compared with 1.4% of pregnancy controls (p = 0.02). No individuals were carriers of both N291S and D9N/ - 93T --> G. Thus, 18.2% of pre-eclamptics had either of these LPL mutations compared with 4.3% of pregnancy controls (and 4.4% of population controls). The frequency of the S447X variant did not differ among groups. We conclude that carriers of N291S or combined D9N/ - 93T --> G mutations in the LPL gene are at substantially increased risk of pre-eclampsia.     

Functional variants of the lipoprotein lipase gene and the risk of preeclampsia among non-Hispanic Caucasian women

Hypertriglyceridemia is an important pathophysiologic feature of preeclampsia, a common vascular disorder of pregnancy. Three well-documented functional variants (N291S, S447X, and D9N) of the lipoprotein lipase gene were related to hypertriglyceridemia. Results from the only two studies concerning the relationship between these variants and preeclampsia risk have been inconsistent. We investigated this relationship in a case-control study including 144 preeclamptic and 290 normotensive pregnant women (all non-Hispanic Caucasians). We estimated odds ratios (OR) and 95% confidence intervals (CI) adjusted for maternal age, pre-pregnancy body mass index, and parity. After adjusting for covariates, women with the 291 N/S or S/S genotype had significantly increased risk of preeclampsia (OR 6.9, 95% CI 1.9-25.4) compared with women with the common 291N/N genotype. The 447 S/X or X/X genotype was not significantly associated with preeclampsia risk. The frequency of the 9N variant allele was 1.8% in controls, while this allele was not observed among cases. Haplotype 9D/291S/447S was strongly associated with higher risk of preeclampsia as compared with the most common haplotype 9D/291N/447S (adjusted OR 6.6, 95% CI 1.7-25.0). Results from our study support the thesis that abnormal lipid metabolism is important in the pathogenesis of preeclampsia.     

LPL在人脑星形细胞瘤中的表达及临床病理意义

目的探讨脂蛋白脂肪酶(lipoprotein lipase,LPL)在人脑星形细胞瘤中的表达及其与临床病理特征的关系,为星形细胞瘤的诊断和治疗提供分子生物学依据。方法采用免疫组化Max Vision法及RT-PCR法检测10例正常脑组织和60例脑星形细胞瘤中LPL蛋白、mRNA的表达,应用Western blot法检测星形细胞瘤患者和对照组脑脊液中的LPL水平,分析LPL蛋白表达与肿瘤病理分级、患者年龄、性别及3年生存率的关系。结果 LPL蛋白及其mRNA在星形细胞瘤中的表达水平明显高于正常脑组织(P0.05);Ⅰ、Ⅱ、Ⅲ、Ⅳ级各组星形细胞瘤中的LPL阳性率分别为8.20%、31.29%、62.00%、82.25%,等级相关性分析显示LPL表达与病理分级呈正相关(r_s=0.931,P0.001);LPL表达与星形细胞瘤患者性别、年龄无关;与3年生存率相关,生存期小于3年者LPL表达更强;脑脊液内LPL蛋白表达水平在星形细胞瘤患者中也有显著增高。结论 LPL表达与星形细胞瘤发生、发展密切相关,可作为预测星形细胞瘤患者病情进展潜在的生物学标志物。     

Pseudodominance of lipoprotein lipase (LPL) deficiency due to a nonsense mutation (Tyr302>Term) in exon 6 of LPL gene in an Italian family from Sardinia (LPL(Olbia))

We analyzed the molecular defect in the lipoprotein lipase (LPL) gene of a young boy from Sardinia who had primary hyperchylomicronemia, pancreatitis, and a complete LPL deficiency in post-heparin plasma. Analysis of LPL gene was performed by using single strand conformation polymorphism (SSCP) and direct sequencing of SSCP-positive region. The proband was homozygous for a C > A transversion in exon 6, which converts the codon for tyrosine at position 302 into a termination codon and eliminates an RsaI restriction site; this allowed the rapid screening of the proband's family members, among whom nine heterozygotes and one additional homozygote were identified. The homozygote was the proband's paternal grandmother who had shown the first clinical manifestation (recurrent pancreatitis) of LPL deficiency at the age of 54 years. LPL mutation carriers showed a mild dyslipidemic phenotype characterized by a reduction of high density lipoprotein-cholesterol (HDL-C) levels, HDL-C/total cholesterol ratio, and low density lipoprotein (LDL) size, associated with a variable increase of triglyceride levels. Five of these carriers were also heterozygotes for beta-thalassemia (Q39X mutation). In these double mutation carriers, plasma HDL-C levels were higher and plasma triglycerides tended to be lower than in carriers of LPL mutation alone. The Tyr302 > Term mutation encodes a truncated protein of 301 amino acids that is probably not secreted by the LPL producing cells. This is the first mutation of LPL gene found in Sardinians.     

C-MYC和LPL基因与前列腺癌的研究进展

前列腺癌中最常见的细胞遗传学改变是8号染色体异常,定位于8q24的C-MYC基因和8p22的LPL基因在前列腺癌的发生发展中起重要的作用。它们可能是新的前列腺癌预后因子和有前途的治疗靶点。     

Genetic study of common variants at the Apo E,Apo AI,Apo CIII,Apo B,lipoprotein lipase (LPL) and hepatic lipase (LIPC) genes and coronary artery disease (CAD): variation in LIPC gene associates with clinical outcomes in patients with established CAD

Background  

Current evidence demonstrates that positive family history and several alterations in lipid metabolism are all important risk factors for coronary artery disease (CAD). All lipid abnormalities themselves have genetic determinants. Thus, objective of this study was to determine whether 6 genetic variants potentially related to altered lipid metabolism were associated with CAD and with lipid abnormalities in an Italian population. These genetic variables were: apolipoprotein E (Apo E), Apo AI, Apo CIII, Apo B, lipoprotein lipase (LPL) and the hepatic lipase (LIPC) genes. Furthermore, an 8 years prospective analysis of clinical cardiovascular events was related to the various genetic markers.     

Association between variants of lipoprotein lipase and coronary heart disease in a Tunisian population

Objective

Coronary artery disease (CAD) is a complex multifactorial disease due to the interaction of multiple genes variations and environmental factors. Genetic variants of lipoprotein lipase (LPL), a key enzyme in the hydrolysis of triglyceride rich particles, may contribute to CAD. We analysed here the frequency of LPL variants (p.Asp9Asn, p.Asn291Ser and p.Ser447X) in a Tunisian population as well as their association with circulating lipid level and risk of CAD.

Patients and methods

LPL variations were investigated by PCR-RFLP and lipid parameters were measured in 135 patients and 109 controls.

Results

The frequency of the p.Asp9Asn variation was 10.37% in CAD patients versus 3.66% in controls. The frequency for the p.Ser447X variation was 8.8% in CAD patients versus 13.7% in controls. There was no significant association between these two variants and CAD. The p.Asn291Ser mutation variation was absent in this population. In healthy subjects, heterozygote carriers of the p.Asp9Asn substitution had a significant increase level of total cholesterol (4.2 ± 0.9 mmol/L vs 5.6 ± 1.2 mmol/L; P = 0.01) and a decreased level of HDL-cholesterol (1.36 ± 0.3 mmol/L vs 0.93 ± 0.1 mmol/L; P = 0.045).

Conclusion

There was no significant association between genetic variants of the LPL gene and CAD in this Tunisian population. The very low frequency of the p.Asn291Ser variation may be an ethnic specificity of Tunisians.     

A common truncation variant of lipoprotein lipase (Ser447X) confers protection against coronary heart disease: the Framingham Offspring Study

Genetic variation at the lipoprotein lipase (LPL) locus has been shown to influence plasma lipids and to modulate risk of coronary heart disease (CHD). Recently, we found that the most frequent variant at this locus, involving a C-terminal truncation of two amino acids (Ser447X), was associated with both higher LPL activity and high density lipoprotein cholesterol (HDL-C) in patients with CHD. However, the impact of this S447X variant on lipids and CHD in the general population was hitherto unknown. We, therefore, analyzed a total of 1114 men and 1144 women randomly ascertained from the Framingham Offspring Study (FOS) for the presence of this LPL variant. Carrier frequency of the S447X allele was 17%, and in men carrier status was associated with higher total cholesterol (delta = 6.2 mg/dl, p = 0.03). higher HDL-C (delta = 2.3 mg/dl, p = 0.01), and lower triglyceride (TG) levels (delta = -19.4 mg/dl, p = 0.02). Moreover, in men, the S447X allele conferred significant protection against CHD (odds ratio: 0.43; p = 0.04). These effects on lipids and CHD were not seen in women. Our study represents the first report on the impact of this mutation on CHD in men from the general population, and we conclude, therefore, that the S447X variant may confer significant protection against high TG levels, low HDL-C, and premature CHD in these subjects.     

Two common mutations (D9N, N291S) in lipoprotein lipase: a cumulative analysis of their influence on plasma lipids and lipoproteins in men and women

We assessed the effect of two common mutations in the lipoprotein lipase gene (LPL), D9N and N291S, which have been shown to modulate plasma lipids in a wide spectrum of patients. A total of 1114 men and 1 144 women from the Framingham Offspring Study (FOS) were analyzed for these two LPL variants. Subsequently, the association with fasting plasma lipids and risk of coronary artery disease (CHD) was determined. We extended our study by calculating weighed means of lipids and lipoproteins in carriers and non-carriers for these LPL mutations in patients with genetic dyslipidemias, CHD patients and healthy controls. In the FOS sample, the D9N and N291S alleles were associated with lower high-density lipoprotein-cholesterol (HDL-C) (delta = - 0.07 mmol/ 1, p = 0.03) and a trend towards increased triglycerides (delta = 0.25 mmol/ 1, p = 0.07). In women, a trend towards the high triglyceride, low HDL-C phenotype was evident (delta = - 0.02 mmol/1 for HDL-C and delta = 0.14 mmol/l for triglycerides, respectively). Cumulative analysis of other studies of male carriers of the D9N and N291S revealed higher levels of triglycerides (D291N; 2.60(1.85) mmol/l vs. 1.62(1.18) mmol/l: p < 0.0001) (D9N; 1.94 (1.19) mmol/l vs. 1.74(1.17) mmol/l: p < 0.001) and lower HDL-C (N291S; 1.04(0.32) mmol/l vs. 1.15(0.28) mmol/l: p < 0.0001) (D9N; 1.08(0.24) mmol/l vs. 1.16(0.28) mmol/l: p < 0.0001). In females, results differed with higher TG levels (N291S; 1.70(0.99) mmol/l vs. 1.10(0.63) mmol/l: p < 0.001) (D9N; 1.08(0.76) mmol/l vs. 0.96(0.51) mmol/l: p < 0.01) and lower HDL-C levels (N291S; 1.27(0.33) mmol/l vs. 1.51(0.32) mmol/l: p < 0.0001); however, the HDL-C levels for D9N carriers were similar to non-carriers (D9N; 1.52(0.29) mmol/l vs. 1.53(0.35) mmol/l: p = 0.83). Our data provide evidence that common variants of the LPL gene are significant modulators of lipid and lipoprotein levels in both men and women.