LIPC, which is synthesized in liver, is secreted and bound to hepatocytes and hepatic endothelial surfaces via heparin sulfate proteoglycans (HSPGs). Active LIPC exists as a homodimer and has broad substrate specificity, catalyzing the hydrolysis of fatty acyl chains at the sn-1 position of phospholipids and of mono-, di-, and triacylglycerols associated with a variety of lipoproteins, including high density lipoprotein (HDL). LIPC may also facilitate binding and uptake of lipoproteins and selective uptake of cholesteryl esters from lipoproteins (summary by Brown et al., 2004). (from OMIM) A Deficiency of HL is characterized by abnormally triglyceride-rich low and high density lipoproteins as well as beta-migrating very low density lipoproteins. Familial human hepatic lipase deficiency is a rare recessive disorder that results from mutation in position 405 of the mature protein. The disease is characterised by premature atherosclerosis and abnormal circulating lipoproteins.
BACKGROUND: The incidental finding of severe hypertriglyceridemia (HyperTG) in a child may suggest the diagnosis of familial chylomicronemia syndrome (FCS), a recessive disorder of the intravascular hydrolysis of triglyceride (TG)-rich lipoproteins. FCS may be due to pathogenic variants in lipoprotein lipase (LPL), as well as in other proteins, such as apolipoprotein C-II and apolipoprotein A-V (activators of LPL), GPIHBP1 (the molecular platform required for LPL activity on endothelial surface) and LMF1 (a factor required for intracellular formation of active LPL). OBJECTIVE: Molecular characterization of 5 subjects in whom HyperTG was an incidental finding during infancy/childhood. METHODS: We performed the parallel sequencing of 20 plasma TG-related genes. RESULTS: Three children with severe HyperTG were found to be compound heterozygous for rare pathogenic LPL variants (2 nonsense, 3 missense, and 1 splicing variant). Another child was found to be homozygous for a nonsense variant of APOA5, which was also found in homozygous state in his father with longstanding HyperTG. The fifth patient with a less severe HyperTG was found to be heterozygous for a frameshift variant in LIPC resulting in a truncated Hepatic Lipase. In addition, 1 of the patients with LPL deficiency and the patient with APOA-V deficiency were also heterozygous carriers of a pathogenic variant in LIPC and LPL gene, respectively, whereas the patient with LIPC variant was also a carrier of a rare APOB missense variant. CONCLUSIONS: Targeted parallel sequencing of TG-related genes is recommended to define the molecular defect in children presenting with an incidental finding of HyperTG.
Familial combined hyperlipidemia (FCH) is a complex and common familial dyslipidemia characterized by elevated total cholesterol and/or triglyceride levels with over five-fold risk of coronary heart disease. The genetic architecture and contribution of rare Mendelian and common variants to FCH susceptibility is unknown. In 53 Finnish FCH families, we genotyped and imputed nine million variants in 715 family members with DNA available. We studied the enrichment of variants previously implicated with monogenic dyslipidemias and/or lipid levels in the general population by comparing allele frequencies between the FCH families and population samples. We also constructed weighted polygenic scores using 212 lipid-associated SNPs and estimated the relative contributions of Mendelian variants and polygenic scores to the risk of FCH in the families. We identified, across the whole allele frequency spectrum, an enrichment of variants known to elevate, and a deficiency of variants known to lower LDL-C and/or TG levels among both probands and affected FCH individuals. The score based on TG associated SNPs was particularly high among affected individuals compared to non-affected family members. Out of 234 affected FCH individuals across the families, seven (3%) carried Mendelian variants and 83 (35%) showed high accumulation of either known LDL-C or TG elevating variants by having either polygenic score over the 90th percentile in the population. The positive predictive value of high score was much higher for affected FCH individuals than for similar sporadic cases in the population. FCH is highly polygenic, supporting the hypothesis that variants across the whole allele frequency spectrum contribute to this complex familial trait. Polygenic SNP panels improve identification of individuals affected with FCH, but their clinical utility remains to be defined.
BACKGROUND: Variants in the CETP and the LIPC genes, encoding cholesteryl ester transfer protein and hepatic lipase, respectively, are associated with high levels of HDL-cholesterol or hyperalphalipoproteinemia (HALP). Recently, we have identified three novel variants in the CETP promoter and two novel variants in LIPC in Thai subjects with HALP. In this study, we investigated the functions of these 5 variants in vitro. METHODS: For CETP promoter variants, we used site-directed mutagenesis, transient expression in HepG2 cells and luciferase reporter assay. For LIPC variants, cDNA was cloned and mutagenesis for missense variants was performed before expression in HepG2 cells. RESULTS: The transcriptional activities of -49G>T,-70C>T, and -372C>T CETP promoter variants were markedly reduced (5%, 8% and 30%, respectively, compared to that of the wild-type, P<0.001). For LIPC variants, hepatic lipase activities in the lysates of cells transfected with c.421A>G (p.G141S) and c.517G>A (p.V173M) variants were 41% and 46%, respectively, compared to that of the wild-type (P<0.05). CONCLUSIONS: The recently-identified variants in the CETP promoter and in the LIPC gene may contribute to HALP. Our result may have a diagnostic application in the genetic evaluation of subjects with high HDL-cholesterol levels.
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.
Hepatic lipase (HL) deficiency is a rare genetic disorder that has been associated with premature atherosclerosis despite high plasma high-density lipoprotein (HDL) cholesterol concentrations in the affected individuals. The authors describe the clinical and biochemical features of HL deficiency in a young male of Middle-Eastern-Arabic origin. This is the first report of cholesterol ester transfer protein (CETP) activity and mass in HL deficiency in a patient from this ethnic group. While the CETP mass was high, its activity was low, a discrepancy likely due to the abnormal composition of patient's HDL particles.
Title: Two novel mutations and functional analyses of the CETP and LIPC genes underlying severe hyperalphalipoproteinemia Plengpanich W, Siriwong S, Khovidhunkit W Ref: Metabolism, 58:1178, 2009 : PubMed
Previous studies have shown that CETP and LIPC mutations contribute to hyperalphalipoproteinemia (HALP) in some populations. We investigated whether activities in cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) contribute to HALP in the Thai population and performed genetic analyses of the CETP and LIPC genes. We recruited 38 individuals with high-density lipoprotein cholesterol (HDL-C) levels of at least 2.59 mmol/L (100 mg/dL) (HALP group) and an equal number of individuals with normal serum HDL-C levels (control group). The CETP and HL activities were determined in both groups. Genetic analyses covering all the coding regions and exon-intron junctions of the CETP and LIPC genes were performed in subjects who had low CETP activity and HL activity, respectively. The mean CETP and HL activities were significantly lower in the HALP group than in the control group (34 +/- 4 vs 44 +/- 3 pmol/[microL h], P = .04 and 150 +/- 17 vs 227 +/- 16 nmol free fatty acid/[mL min] P = .002, respectively). Of the 38 individuals with HALP, 19 and 16 were found to have low CETP activity and HL activity, respectively. Of the 19 subjects with low CETP activity, 6 subjects were found to be heterozygous for a known functionally relevant c.1325A>G (D442G) mutation. The other subject was found to be heterozygous for a novel deletion mutation, c.734_737delTCCC mutation. Of the 16 subjects with low HL activity, 8 and 2 subjects were found to be heterozygous for known variants, c.283 G>A (V73M) and c.1068A>C (L334F), respectively. These variants have previously been shown not to be associated with HALP. Another subject was found to be heterozygous for a novel missense mutation, c.421G>A (G119S). Its amino acid change, absence in controls, evolutionary conservation, occurrence in functionally important domain, and predicted damaging function suggested that the G119S mutation is functionally relevant. Two novel mutations in the CETP and LIPC genes found in this study are likely to be the causes of low enzyme activities and elevated HDL-C levels.
We previously selected a group of hypertension candidate genes by a key word search using the OMIM database of NCBI and validated 525 coding single nucleotide polymorphisms (SNPs) in 179 hypertension candidate genes by DNA sequencing in a Japanese population. In the present study, we examined the association between 61 non-synonymous SNPs and blood pressure variations and hypertension. We used DNA samples taken from 1,880 subjects in the Suita study, a population-based study using randomly selected subjects. Analyses of covariance adjusting for age, body mass index, hyperlipidemia, diabetes, smoking, drinking, and antihypertensive medication revealed that 17 polymorphisms in 16 genes (APOB, CAST, CLCNKB, CTNS, GHR, GYS1, HF1, IKBKAP, KCNJ11, LIPC, LPL, P2RY2, PON2, SLC4A1, TRH, VWF) were significantly associated with blood pressure variations. Multivariate logistic regression analysis with adjustment for the same factors revealed that 11 polymorphisms in 11 genes (CAST, CTLA4, F5, GC, GHR, LIPC, PLA2G7, SLC4A1, SLCI8A1, TRH, VWF) showed significant associations with hypertension. Five polymorphisms in five genes, CAST(calpastatin), LIPC (hepatic lipase), SLC4A1 (band 3 anion transporter), TRH (thyrotropin-releasing hormone), and VWF (von Willebrand factor), were significantly associated with both blood pressure variation and hypertension. Thus, our study suggests that these five genes were susceptibility genes for essential hypertension in this Japanese population.
OBJECTIVE: The goal of this study was to characterize the effect of microcoated fenofibrate (160 mg/day for 6 months) on plasma lipoprotein composition and kinetics in 2 patients with complete hepatic lipase (HL) deficiency. METHODS AND RESULTS: Fenofibrate treatment normalized the plasma lipoprotein profile of patients with complete HL deficiency, as evidenced by significant reductions in the plasma concentration of cholesterol (-49%) and triglycerides (-82%) and a significant increase in low-density lipoprotein (LDL) size (251.5+/-1.8 versus 263.5+/-0.7 A). The in vivo kinetics of very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL apolipoprotein (apo)B-100 and plasma apoA-I and apoA-II were studied using a primed-constant infusion of L-[5,5,5-D3]-leucine for 12 hours in the fasted state. Fenofibrate treatment in complete HL-deficient patients substantially decreased plasma concentrations of VLDL, IDL, and LDL apoB-100 attributable to important increases in VLDL (+325%), IDL (+129%), and LDL (+218%) apoB-100 fractional catabolic rates (FCR). IDL apoB-100 FCR nevertheless remained 60% lower after treatment compared with values obtained in controls (n=5). The kinetics of plasma apoA-I and apoA-II as well as the capacity of total plasma and of high-density lipoprotein particles to efflux cellular cholesterol from normal human skin fibroblasts was not altered by fenofibrate. CONCLUSIONS: Fenofibrate therapy exerts a pronounced antiatherogenic effect on triglyceride-rich lipoproteins even in the complete absence of HL.
A heritable deficiency of hepatic lipase (HL) provides insights into the physiologic function of HL in vivo. The metabolism of apolipoprotein B (apoB)-100 in very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) and of apoA-I and apoA-II in high-density lipoprotein (HDL) particles lipoprotein (Lp)(AI) and Lp(AI:AII) was assessed in 2 heterozygous males for compound mutations L334F/T383M or L334F/R186H, with 18% and 22% of HL activity, respectively, compared with 6 control males. Subjects were provided with a standard Western diet for a minimum of 3 weeks. At the end of the diet period, apo kinetics was assessed using a primed-constant infusion of [5,5,5-(2)H(3)] leucine. Mean plasma triglyceride (TG) and HDL cholesterol levels were 55% and 12% higher and LDL cholesterol levels 19% lower in the HL patients than control subjects. A higher proportion of apoB-100 was in the VLDL than IDL and LDL fractions of HL patients than control subjects due to a lower VLDL apoB-100 fractional catabolic rate (FCR) (4.63 v 9.38 pools/d, respectively) and higher hepatic production rate (PR) (33.24 v 10.87 mg/kg/d). Delayed FCR of IDL (2.78 and 6.31 pools/d) and LDL (0.128 and 0.205 pools/d) and lower PR of IDL (3.67 and 6.68 mg/kd/d) and LDL 4.57 and 13.07 mg/kg/d) was observed in HL patients relative to control subjects, respectively. ApoA-I FCR (0.09 and 0.13 pools/d) and PR (4.01 and 6.50 mg/kg/d) were slower in Lp(AI:AII) particles of HL patients relative to control subjects, respectively, accounting for the somewhat higher HDL cholesterol levels. HL deficiency may result in a lipoprotein pattern associated with low heart disease risk.
Title: Atorvastatin dose-dependently decreases hepatic lipase activity in type 2 diabetes: effect of sex and the LIPC promoter variant Berk P, II, Hoogerbrugge N, Stolk RP, Bootsma AH, Jansen H Ref: Diabetes Care, 26:427, 2003 : PubMed
OBJECTIVE: Hepatic lipase (HL) is involved in the metabolism of several lipoproteins and may contribute to the atherogenic lipid profile in type 2 diabetes. Little is known about the effect of cholesterol synthesis inhibitors on HL activity in relation to sex and the hepatic lipase gene, the LIPC promoter variant in type 2 diabetes. Therefore, we studied the effect of atorvastatin 10 mg (A10) and 80 mg (A80) on HL activity in 198 patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: Patients (aged 45-75 years, without manifest coronary artery disease, total cholesterol 4.0-8.0 mmol/l, and fasting triglycerides [TG] 1.5-6.0 mmol/l) were included in a double-blind, randomized, placebo-controlled trial for 30 weeks (Diabetes Atorvastatin Lipid Intervention study). RESULTS: HL activity at baseline was significantly higher in our population compared with an age-matched control group without type 2 diabetes (406 +/- 150 vs. 357 +/- 118 units/l). HL activity in men versus women (443 +/- 158 vs. 358 +/- 127 units/l), in carriers of the LIPC C/C allele versus carriers of the T/T allele (444 +/- 142 vs. 227 +/- 96 units/l), and in Caucasians versus blacks (415 +/- 150 vs. 260 +/- 127 units/l) all differed significantly (P < 0.001). Atorvastatin dose-dependently decreased HL (A10, -11%; A80, -22%; both P < 0.001). Neither sex nor the LIPC C-->T variation influenced the effect of atorvastatin on HL activity. CONCLUSIONS: Sex, LIPC promoter variant, and ethnicity significantly contribute to the baseline variance in HL activity. Atorvastatin treatment in diabetic dyslipidemia results in a significant dose-dependent decrease in HL activity, regardless of sex or the LIPC promoter variant.
Title: Hepatic lipase promoter C-514T polymorphism influences serial changes in HDL cholesterol levels since childhood: the Bogalusa Heart Study Chen W, Srinivasan SR, Boerwinkle E, Berenson GS Ref: Atherosclerosis, 169:175, 2003 : PubMed
Hepatic lipase (HL) is an important determinant of high-density lipoprotein (HDL) concentrations. A common C-to-T substitution at position -514 of the promoter region of the HL gene has been shown to be associated with HL activity and HDL cholesterol (HDL-C) levels. The current study examines the influence of this polymorphism on both levels and serial changes of HDL-C from childhood to adulthood in a community-based sample of 707 white and 291 black unrelated individuals aged 4-38 years using a repeated measures analysis. The frequency of the -514T allele was lower in whites than in blacks (0.228 vs. 0.545, P<0.001). After adjusting for age and BMI, the genotype effect on longitudinal profiles of HDL-C levels was significant (P=0.003) in white males with values in the order of T/T>T/C>C/C. Although a similar trend was seen, the genotype effect was not significant in white females and blacks. Further, the slopes of the age trajectories of HDL-C were similar in three genotype groups in blacks and whites. A sex-genotype interaction effect (P=0.043) on longitudinal profiles of HDL-C levels was found in whites, but not in blacks. White males showed a stronger genotype effect (3.6 mg/dl, P=0.003) than white females (0.5 mg/dl, P=0.601). Thus, the -514T variant of the HL gene is consistently associated with higher levels of HDL-C longitudinally since childhood, but not with rate of change over time. These results suggest that the HL gene may play an important role in the regulation of HDL-C levels from childhood to adulthood, especially in white males.
This study explored the genetic basis of the combination of extreme blood levels of HDL-C and LDL-C, a well-studied endophenotype for CVD, which has several attractive features as a target for genetic analysis: (1) the trait is moderately heritable; (2) non-genetic risk factors account for a significant but still limited portion of the phenotypic variance; (3) it is known to be moderated by a number of gene products. We exhaustively surveyed 11 candidate genes for allelic variation in a random population-based sample characterized for known CVD risk factors and blood lipid profiles. With the goal of generating specific etiological hypotheses, we compared two groups of subjects with extreme lipid phenotypes, from the same source population, using a case-control design. Cases (n=186) were subjects, within the total sample of 1708 people, who scored in the upper tertile of LDL-C and the lowest tertile of HDL-C, while controls (n=185) scored in the lowest tertile of LDL-C and the upper tertile of HDL-C. We used logistic regression and a four-tiered, systematic model building strategy with internal cross-validation and bootstrapping to investigate the relationships between the trait and 275 genetic variants in the presence of 10 non-genetic risk factors. Our results implicate a subset of nine genetic variants, spanning seven candidate genes, together with five environmental risk factors, in the etiology of extreme lipoprotein phenotypes. We propose a model involving these 14 genetic and non-genetic risk factors for evaluation in future independent studies.
Individuals with hepatic lipase (HL) deficiency are often characterized by elevated levels of triglycerides (TGs) and cholesterol. The aim of the present study was to characterize the molecular defect leading to severe HL deficiency in a Quebec-based kindred. In the proband and two of her brothers, the very low to undetectable HL activity resulted from compound heterozygosity for two rare HL gene mutations, a previously unknown missense mutation in exon 5 designated A174T and the previously reported T383M mutation in exon 8 of the HL gene. The mutation at codon 174 resulted in the substitution of alanine for threonine, a polar amino acid, in a highly conserved nonpolar region of the protein involved in the catalytic activity of the enzyme. The severe HL deficiency among the three related compound heterozygotes was associated with a marked TG enrichment of LDL and HDL particles. The two men with severe HL deficiency also presented with abdominal obesity, which appeared to amplify the impact of HL deficiency on plasma TG-rich lipoprotein levels. Our results demonstrated that HL deficiency in this Quebec kindred is associated with an abnormal lipoprotein-lipid profile, which may vary considerably in the presence of secondary factors such as abdominal obesity.
Low density lipoprotein (LDL) particle size is a genetically influenced trait associated with coronary heart disease (CHD). This study investigates the effects of genetic variation in plasma factors with important roles in lipoprotein metabolism on LDL heterogeneity. Common variants in the cholesteryl ester transfer protein (CETP-629C/A), lipoprotein lipase (LPL S447X), hepatic lipase (HL-480C/T) and apolipoprotein E (apoE e2/e3/e4) genes were studied in relation to LDL particle size distribution in 377 healthy, middle-aged men. A high-resolution polyacrylamide gradient gel electrophoresis technique was used to measure plasma concentrations of four LDL subfractions. The CETP-629A and LPL 447X alleles were associated with moderately increased LDL peak particle size. In contrast, the apoE e4 allele was associated with a marked reduction in LDL peak particle size and an increased relative proportion and plasma concentration of small, dense LDL. An interaction between the HL-480C/T and apo E polymorphisms contributed significantly to increased plasma concentration of small, dense LDL (LDL-III) in HL-480T carriers. In summary, the investigated polymorphisms were associated with diverse effects on the LDL particle size distribution, consistent with respect to protein function and proposed association with CHD risk. The observed associations were further modulated by gene-gene and gene-environment interactions.
Title: Interaction effect of Serine447Stop variant of the lipoprotein lipase gene and C-514T variant of the hepatic lipase gene on serum triglyceride levels in young adults: the Bogalusa Heart Study Xin X, Srinivasan SR, Chen W, Boerwinkle E, Berenson GS Ref: Metabolism, 52:1337, 2003 : PubMed
The opposing effects of lipoprotein lipase (LPL) Serin447Stop (S447X) polymorphism and hepatic lipase (HL) C-514T polymorphism on serum triglyceride (TG) levels have been known. However, little is known about the interaction effect of these 2 functional gene variants on serum triglyceride levels. This aspect was examined in a community-based sample of 902 whites and 389 blacks aged 18 to 41 years, using a repeated measures analysis in a mixed model. The frequency of the LPL X447 allele was higher in whites than blacks (16% v 11%, P <.05); whereas the frequency of HL T-514 allele was higher in blacks than whites (77% v 40%, P <.001). The combined genotype distribution was also different between whites and blacks (P <.001). Although the frequency of carriers of both variants was similar in whites and blacks (7% v 8%), more whites carried the LPL X447 allele only (9% v 3%), and more blacks carried the HL T-514 allele only (70% v 33%). Mean levels of TG adjusted for age, sex, and body mass index (BMI) in carriers versus noncarriers of the LPL X447 allele were lower by 13.5% (P <.0001) in whites, 15.8% (P <.01) in blacks and 16.0% (P <.0001) in the total sample. No such phenotypic effect was noted with respect to HL T-514 allele either in blacks or whites, although the mean level in carriers was marginally (P =.08) higher in the total sample. The interaction effect of LPL and HL variants on TG levels was significant in the total sample (P =.016) and marginal in whites (P =.079). In the total sample, the decrease of TG in carriers versus noncarriers of the LPL X447 was 1.8-fold greater in carriers versus noncarriers of the HL T-514 allele (13.6 mg/dL v 7.4 mg/dL, P =.016). Whites tended to show a similar trend (16.8 mg/dL v 6.1 mg/dL, P =.079). Blacks also showed a similar, but nonsignificant, trend (10.4 mg/dL v 8.6 mg/dL, P =.45). These results by showing modulation of association between S447X variant of the LPL gene and serum TG by C-514T variant of the HL gene underscore the importance of gene-gene interactions in the assessment of genetic effects on complex traits.
Title: Contribution of hepatic lipase, lipoprotein lipase, and cholesteryl ester transfer protein to LDL and HDL heterogeneity in healthy women Carr MC, Ayyobi AF, Murdoch SJ, Deeb SS, Brunzell JD Ref: Arterioscler Thromb Vasc Biol, 22:667, 2002 : PubMed
Hepatic lipase (HL) and cholesteryl ester transfer protein (CETP) have been independently associated with low density lipoprotein (LDL) and high density lipoprotein (HDL) size in different cohorts. These studies have been conducted mainly in men and in subjects with dyslipidemia. Ours is a comprehensive study of the proposed biochemical determinants (lipoprotein lipase, HL, CETP, and triglycerides) and genetic determinants (HL gene [LIPC] and Taq1B) of small dense LDL (sdLDL) and HDL subspecies in a large cohort of 120 normolipidemic, nondiabetic, premenopausal women. HL (P<0.001) and lipoprotein lipase activities (P=0.006) were independently associated with LDL buoyancy, whereas CETP (P=0.76) and triglycerides (P=0.06) were not. The women with more sdLDL had higher HL activity (P=0.007), lower HDL2 cholesterol (P<0.001), and lower frequency of the HL (LIPC) T allele (P=0.034) than did the women with buoyant LDL. The LIPC variant was associated with HL activity (P<0.001), HDL2 cholesterol (P=0.034), and LDL buoyancy (P=0.03), whereas the Taq1B polymorphism in the CETP gene was associated with CETP mass (P=0.002) and HDL3 cholesterol (P=0.039). These results suggest that HL activity and HL gene promoter polymorphism play a significant role in determining LDL and HDL heterogeneity in healthy women without hypertriglyceridemia. Thus, HL is an important determinant of sdLDL and HDL2 cholesterol in normal physiological states as well as in the pathogenesis of various disease processes.
BACKGROUND: A common polymorphism in the hepatic lipase (HL) gene promoter, -514C/T, affecting enzyme activity, has been associated with alterations in plasma lipoprotein levels. However a relationship with coronary heart disease (CHD) is less well documented. DESIGN AND METHODS: We studied HL -514 C/T in 562 Caucasian CHD patients aged under 50 years and in 642 Caucasian community recruited subjects without historical evidence of CHD. RESULTS: Male CHD subjects (n = 490) had a 41% carrier rate for the C to T substitution, compared with 33% in corresponding controls (n = 330), [OR = 1.42 (95% CI:1.06-1.90), P < 0.02], T allele frequencies being 0.231 and 0.177 respectively [OR = 1.39 (1.08-1.78), P < 0.01]. In male CHD subjects, the T allele was associated with higher HDL-cholesterol (HDL-C) (CC: 0.95 +/- 0.24 (SD); CT: 1.04 +/- 0.41; TT: 1.01 +/- 0.20 mmol/l, P = 0.02, ANOVA) but the trend was not significant in females. In male CHD patients the T allele was more frequently encountered in those with high (> 4.5 mmol/l) than in those with low triglycerides [68% vs. 39%, OR = 3.13 (1.54-6.67), P = 0.001]. In community control subjects, the T allele was associated with a trend to higher HDL-C levels, the significance varying between subgroups while, in males, serum total and LDL-cholesterol were significantly lower in T homozygotes than in the other two genotypes (LDL-C: 2.73 +/- 0.63 vs. 3.56 +/- 0.95 mmol/l; P = 0.01). During the course of this study, a previously unreported promoter region polymorphism was found exclusively on -514C chromosomes (-592A/G, A allele frequency 0.108, 95% CI 0.09 - 0.126). It can lead to mistyping of C as T alleles in C/T heterozygotes, resulting in overestimation of -514 T homozygotes. CONCLUSIONS: The T allele of the hepatic lipase -514 C/T polymorphism is associated with changes in plasma lipids. The superficially paradoxical predisposition to CHD in males is attributable to impairment of TG rich lipoprotein metabolism and reverse cholesterol transport.
Title: Genetic and environmental determinants of plasma high density lipoprotein cholesterol and apolipoprotein AI concentrations in healthy middle-aged men Talmud PJ, Hawe E, Robertson K, Miller GJ, Miller NE, Humphries SE Ref: Ann Hum Genet, 66:111, 2002 : PubMed
The effects of common variants of cholesteryl ester transfer protein (CETP) (TaqIB), hepatic lipase (HL) (-514C>T), lipoprotein lipase (LPL) (S447X) and lecithin cholesterol acyl transferase (LCAT) (S208T) on the determination of high density lipoprotein cholesterol (HDL-C) and apolipoprotein AI (apoAI) levels were examined in 2773 healthy middle-aged men participating in the second Northwick Park Heart Study. The extent of gene:gene, gene:smoking and gene:alcohol interactions were determined. For HDL-C levels, only CETP genotype was associated with significant effects (p&0.0001), with the B2 allele being associated with higher levels in both smokers and non-smokers. This interaction was significant at the lowest tertile of TG, suggesting that TG levels were rate limiting. As previously reported, CETP, LPL and HL genotypes were all associated with significant effects on apoAI levels (all p&0.01), with carriers of the rare alleles having higher levels and with no evidence of heterogeneity of effects in smokers and non-smokers. LCAT genotype was not associated with significant effects on either trait. There was no significant interaction between any of the genotypes and alcohol consumption on either HDL-C or apoAI levels. All genotypic effects were additive for HDL-C and apoAI. Environmental and TG levels explained more than 20% and 5.5% of the variance in HDL-C and apoAI, respectively. The novel aspect of this finding is that genetic variation at these loci explained in total only 2.5% of the variance in HDL-C and 1.89% of the variance in apoAI levels. Thus despite the key roles played by these enzymes in HDL metabolism, variation at these loci, at least as detected by these common genotypes, contributes minimally to the variance in HDL-C and apoAI levels in healthy men, highlighting the polygenic and multifactorial control of HDL-C.
Title: Hepatic lipase promoter activity is reduced by the C-480T and G-216A substitutions present in the common LIPC gene variant, and is increased by Upstream Stimulatory Factor Botma GJ, Verhoeven AJ, Jansen H Ref: Atherosclerosis, 154:625, 2001 : PubMed
The common -216G-->A and -480C-->T substitutions in the promoter region of the human hepatic lipase (LIPC) gene show high allelic association, and are correlated with decreased hepatic lipase activity and increased high-density lipoprotein cholesterol levels. To test the functionality of these substitutions, CAT-reporter assays were performed in HepG2 cells. LIPC (-650/+48) but not (-650/+61) promoter constructs showed transcriptional activity. LIPC (-650/+48) constructs with both -216A and -480T exhibited significantly lower promoter activity (-45%) than the wild-type form. Activities of -289/+48 constructs were not significantly affected by the -216G-->A substitution. The -480C/T site lies within a binding region for Upstream Stimulatory Factor (USF). Gel-shift assays showed that the binding affinity of USF protein for HL specific oligonucleotides was decreased four-fold by the -480C-->T substitution. However, promoter activity of the -650/+48 constructs was not significantly affected by the -480C-->T substitution alone. Co-transfection of HepG2 cells with USF(43) cDNA yielded a similar dose-dependent increase in activity of all -650/+48 constructs; the absolute difference in promoter activity increased but the relative difference between the variant promoter forms was maintained. Our studies demonstrate that the common LIPC promoter variation is functional, which explains the association of the -480T allele with a lower hepatic lipase activity in man.
BACKGROUND: Impaired coronary flow reserve (CFR) can be used to indicate vascular dysfunction before the appearance of angiographic lesions. The hepatic lipase (HL) gene has a functional promoter polymorphism at position C-480T, which affects transcription and leads to high activity (C/C) and low activity (C/T, T/T) genotypes. These genotypes modulate HL activity, but their role in coronary artery disease is controversial and the effect on coronary function has not been studied. We investigated whether HL genotypes are associated with coronary artery function in healthy young men. MATERIALS AND METHODS: We studied 49 healthy, mildly hypercholesterolemic men (aged 35 +/- 4 years). Myocardial blood flow was measured at rest and during adenosine induced hyperaemia with positron emission tomography using [15O] H2O. HL genotype was determined by PCR and Nla III enzyme digestion. RESULTS: Resting myocardial blood flow was not statistically different in subjects with high and low activity HL genotypes. However, CFR (the ratio of adenosine flow to resting flow) was 24% higher (4.62 +/- 1.52 vs. 3.73 +/- 1.08 mL g-1 min-1, P = 0.024) in men with the high activity genotype (n = 26) than in those with low activity (n = 23). In multivariate analysis, the HL genotype remained a significant predictor of CFR (P = 0.038) after adjusting for age, body mass index, serum lipids and smoking. CONCLUSIONS: The findings of our preliminary study suggest that the C-480T polymorphism of the HL gene may modify coronary reactivity and reflect differences in the early pathogenesis of coronary dysfunction in these healthy young men. If the association between HL polymorphism and impaired CFR is also present in subjects with other dyslipoproteinemias, the HL polymorphism could be a new risk factor for cardiovascular disease.
We have determined the genotypes of two common polymorphisms in the lipoprotein lipase (S447X) and hepatic lipase (-480C/T) genes in a cohort of 285 representative selected Czech probands (131 male and 154 female), examined in 1988 and reinvestigated in 1996. The genotype distributions of both polymorphisms were in Hardy-Weinberg equilibrium and did not differ between male and female subjects. The rare allele frequency of the lipoprotein lipase polymorphism did not differ significantly from the other European populations. Compared to the German populations, the frequency of the hepatic lipase -480T allele was significantly higher in the Czech group (20% vs. 36%, p<0.0001). There were no significant associations between the lipoprotein lipase gene variants and lipid parameters measured either in 1988, or in 1996 or with changes of lipid parameters over the 8-year period. The carriers of the T-480 allele of the hepatic lipase polymorphism were found to have higher HDL cholesterol levels (p=0.02). However, this difference was confined to female subjects only. The male carriers of the -480T allele had higher concentrations of total cholesterol (p=0.03) as compared to CC-480 subjects. Both associations were observed in 1996 only. In the Slavic Czech population, a common polymorphism in the hepatic lipase gene (-480C/T), but not in the lipoprotein lipase gene (S447X), is a significant determinant of plasma HDL cholesterol in females and plasma total cholesterol in males and indicates the importance of gender-associated effects in the genetic determinations of plasma lipids.
Title: Effects of hepatic lipase gene promoter nucleotide variations on serum HDL cholesterol concentration in the general Japanese population Inazu A, Nishimura Y, Terada Y, Mabuchi H Ref: J Hum Genet, 46:172, 2001 : PubMed
Genetic factors may play a major role in determining serum high-density lipoprotein (HDL) cholesterol (HDL-C) levels in the general population. Cholesteryl ester transfer protein (CETP) is a strong genetic factor as a determinant of HDL-C levels in Japanese, whereas hepatic lipase (HL) plays a predominant role in Caucasian populations. We investigated the effects of HL gene promoter polymorphisms on HDL-C levels in a general population of Japanese men (n = 299). An HL promoter polymorphism of -514C/T explained a considerable variance of HDL-C (2.9%), as compared with CETP mutations of D442G and IVS14 + 1G > A (3.6% and 1.9%). HL promoter variation of the -514C/C genotype, reported to have high HL activity, had significant effects on decreasing HDL-C levels (-3.8mg/dl), but -514T allele carriers had a weak effect on increasing HDL-C levels. The frequency of the -514T allele was three times higher (0.50) in the Japanese than in Caucasian populations (0.15-0.19). Thus, the higher frequency of the HL -514T allele, along with CETP gene mutations, could explain about 9% of phenotypic variability of HDL-C. These genetic attributes may be among the many factors that contribute to the relatively higher serum HDL-C levels in Japanese subjects.
Title: Association of the C-514T polymorphism in the hepatic lipase gene with variations in lipoprotein subclass profiles: The Framingham Offspring Study Couture P, Otvos JD, Cupples LA, Lahoz C, Wilson PW, Schaefer EJ, Ordovas JM Ref: Arterioscler Thromb Vasc Biol, 20:815, 2000 : PubMed
Hepatic lipase is involved in the metabolism of several lipoproteins and has a key role in reverse cholesterol transport. A common C-to-T substitution at position -514 of the hepatic lipase promoter has been associated with variations in plasma high density lipoprotein cholesterol (HDL-C) levels and hepatic lipase activity. The aim of the current study was to investigate the association of this polymorphism to lipoprotein levels in a population-based sample of 1314 male and 1353 female Framingham Offspring Study participants. In men and women, carriers of the -514T allele had higher HDL-C and apolipoprotein A-I (apoAI) concentrations compared with noncarriers. The higher HDL-C levels associated with the -514T allele was due to an increase in the HDL(2)-C subfraction, and this association was stronger in women compared with men (P=0.0043 versus 0.0517). To gain further understanding about the metabolic basis of these effects, HDL and low density lipoprotein (LDL) subclass profiles were measured by using automated nuclear magnetic resonance spectroscopy and gradient gel electrophoresis, respectively. The association of the -514T allele with higher HDL-C levels seen in men and women was primarily due to significant increases in the large HDL subfractions (size range 8.8 to 13.0 nm). In contrast, there was no relationship between the hepatic lipase polymorphism at position -514 and the LDL particle size distribution after adjustment for familial relationships, age, body mass index, smoking, alcohol intake, use of beta-blockers, apoE genotype, and menopausal status and estrogen therapy in women. Moreover, multiple regression analyses suggested that the C-514T polymorphism contributed significantly to the variability of HDL particle size in men and women (P<0.04). Thus, our results show that the C-514T polymorphism in the hepatic lipase gene is associated with significant variations in the lipoprotein profile in men and women.
Title: The C-514T polymorphism in the human hepatic lipase gene promoter diminishes its activity Deeb SS, Peng R Ref: J Lipid Res, 41:155, 2000 : PubMed
Four common polymorphisms in the promoter of the hepatic lipase gene (LIPC) that are in almost complete linkage disequilibrium have been described in different ethnic groups. The T and A alleles at positions -514 and -250, respectively, were observed to be associated with decreased hepatic lipase (HL) activity and increased triglyceride content of HDL and LDL particles. We investigated whether these polymorphisms have any effect on transcriptional activity of the proximal promoter (-639 to +29) in transient transfection assays. We found that the promoter with T at position -514 had approximately 30% lower activity than the one with C at the same position (P < 0.0005) regardless of the genotype at position -250. In conclusion, these data indicate that the -514T allele may contribute significantly to decreased HL activity and the resultant increase in plasma levels of triglyceride-rich HDL(2) and large buoyant LDL particles. In addition, this promoter variant may explain the finding that its presence attenuates the increase in HL activity with increasing intra-abdominal fat in women.
Familial combined hyperlipidemia (FCHL) is a heritable lipid disorder characterized by multiple lipoprotein phenotypes within a single family. Previously, we have shown an increased incidence of mutations in the LPL gene which was associated with elevated levels of very low density lipoprotein (VLDL) and decreased levels of high density lipoprotein among the families studied. Now, we report the results of our study on the hepatic lipase gene. We found the HL V73M variant to be present in four FCHL families. By means of a pedigree-based maximum log-likelihood method we analyzed the effect of this variant on the lipid levels in these families. Carriers of the HL V73M variant revealed significantly higher levels of total cholesterol (P < 0.01) and apoB (P <0.01). These findings show that the HL V73M mutant explains another part of the variability in the phenotype observed among FCHL family members, compared with mutations in the LPL gene. Family analysis shows that in these FCHL families, carriers of mutations in the LPL or HL genes have an increased risk for FCHL compared with their non-carrier relatives.
A major goal in human genetics is to understand the role of common genetic variants in susceptibility to common diseases. This will require characterizing the nature of gene variation in human populations, assembling an extensive catalogue of single-nucleotide polymorphisms (SNPs) in candidate genes and performing association studies for particular diseases. At present, our knowledge of human gene variation remains rudimentary. Here we describe a systematic survey of SNPs in the coding regions of human genes. We identified SNPs in 106 genes relevant to cardiovascular disease, endocrinology and neuropsychiatry by screening an average of 114 independent alleles using 2 independent screening methods. To ensure high accuracy, all reported SNPs were confirmed by DNA sequencing. We identified 560 SNPs, including 392 coding-region SNPs (cSNPs) divided roughly equally between those causing synonymous and non-synonymous changes. We observed different rates of polymorphism among classes of sites within genes (non-coding, degenerate and non-degenerate) as well as between genes. The cSNPs most likely to influence disease, those that alter the amino acid sequence of the encoded protein, are found at a lower rate and with lower allele frequencies than silent substitutions. This likely reflects selection acting against deleterious alleles during human evolution. The lower allele frequency of missense cSNPs has implications for the compilation of a comprehensive catalogue, as well as for the subsequent application to disease association.
Title: A hepatic lipase gene promoter polymorphism attenuates the increase in hepatic lipase activity with increasing intra-abdominal fat in women Carr MC, Hokanson JE, Deeb SS, Purnell JQ, Mitchell ES, Brunzell JD Ref: Arterioscler Thromb Vasc Biol, 19:2701, 1999 : PubMed
High hepatic lipase (HL) activity is associated with an atherogenic lipoprotein profile of small, dense LDL particles and lower HDL(2)-C. Intra-abdominal fat (IAF) is positively associated with HL activity. A hepatic lipase gene (LIPC) promoter variant (G-->A(-250)) is associated with lower HL activity, higher HDL(2)-C, and less dense LDL particles. To determine whether the LIPC promoter polymorphism acts independently of IAF to regulate HL, 57 healthy, premenopausal women were studied. The LIPC promoter A allele was associated with significantly lower HL activity (GA/AA=104+/-34 versus GG=145+/-57 nmoles x mL(-1) x min(-1), P=0.009). IAF was positively correlated with HL activity (r=0.431, P<0.001). Multivariate analysis revealed a strong relationship between both the LIPC promoter genotype (P=0. 001) and IAF (P<0.001) with HL activity. The relationship between IAF and HL activity for carriers and noncarriers of the A allele was curvilinear with the carriers having a lower apparent maximum level of plasma HL activity compared with noncarriers (138 versus 218 nmoles x mL(-1) x min(-1), P<0.001). In addition, the LIPC A allele was associated with a significantly higher HDL(2)-C (GA/AA=16+/-7 versus GG=11+/-5 mg/dL, P=0.003). We conclude that the LIPC promoter A allele attenuates the increase in HL activity due to IAF in premenopausal women.
Title: The role of hepatic lipase in lipoprotein metabolism Connelly PW Ref: Clinica Chimica Acta, 286:243, 1999 : PubMed
Hepatic lipase (HL) is one of two major lipases released from the vascular bed by intravenous injection of heparin. HL hydrolyzes phospholipids and triglycerides of plasma lipoproteins and is a member of a lipase superfamily that includes lipoprotein lipase and pancreatic lipase. The enzyme can be divided into an NH2-terminal domain containing the catalytic site joined by a short spanning region to a smaller COOH-terminal domain. The NH2-terminal portion contains an active site serine in a pentapeptide consensus sequence, Gly-Xaa-Ser-Xaa-Gly, as part of a classic Ser-Asp-His catalytic triad, and a putative hinged loop structure covering the active site. The COOH-terminal domain contains a putative lipoprotein-binding site. The heparin-binding sites may be distributed throughout the molecule, with the characteristic elution pattern from heparin-sepharose determined by the COOH-terminal domain. Of the three N-linked glycosylation sites, Asn-56 is required for efficient secretion and enzymatic activity. HL is hypothesized to directly couple HDL lipid metabolism to tissue/cellular lipid metabolism. The potential significance of the HL pathway is that it provides the hepatocyte with a mechanism for the uptake of a subset of phospholipids enriched in unsaturated fatty acids and may allow the uptake of cholesteryl ester, free cholesterol and phospholipid without catabolism of HDL apolipoproteins. HL can hydrolyze triglyceride and phospholipid in all lipoproteins, but is predominant in the conversion of intermediate density lipoproteins to LDL and the conversion of post-prandial triglyceride-rich HDL into the post-absorptive triglyceride-poor HDL. It has been suggested that enzymatically inactive HL can play a role in hepatic lipoprotein uptake forming a 'bridge' by binding to the lipoprotein and to the cell surface. This raises the interesting possibility that production and secretion of mutant inactive HL could promote clearance of VLDL remnants. We have described a rare family with HL deficiency. Affected patients are compound heterozygotes for a mutation of Ser267Phe that causes an inactive enzyme and a mutation of Thr383Met that results in impaired secretion of HL and reduced specific activity. Human HL deficiency in the context of a second factor causing hyperlipidemia is strongly associated with premature coronary artery disease.
The promoter sequence variant -480T in the hepatic lipase gene (LIPC) has been shown to be significantly associated with low post-heparin hepatic lipase activity. Some studies have also found that the -480T variant is associated with elevation in plasma HDL cholesterol. We tested for associations of LIPC -480T with plasma lipoprotein traits in samples taken from three distinct Canadian populations: 657 Alberta Hutterites, 328 Ontario Oji-Cree and 210 Keewatin Inuit. Plasma HL activity was not available for analyses. The LIPC -480T allele frequencies in these three groups, respectively, were 0.219, 0.527 and 0.383, and the prevalence of LIPC -480T/T homozygotes was, respectively, 0.042, 0.274 and 0.167. No significant association was found between LIPC -480T and plasma HDL cholesterol or apolipoprotein AI concentration, after adjusting for covariates including gender and body mass index. There was no consistent relationship between the population mean plasma HDL cholesterol concentration and the population LIPC -480T frequency. Our findings are consistent with the idea that the common promoter variation in LIPC, 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.
Title: The T allele of the hepatic lipase promoter variant C-480T is associated with increased fasting lipids and HDL and increased preprandial and postprandial LpCIII:B : European Atherosclerosis Research Study (EARS) II Jansen H, Chu G, Ehnholm C, Dallongeville J, Nicaud V, Talmud PJ Ref: Arterioscler Thromb Vasc Biol, 19:303, 1999 : PubMed
The common C-480T transition in the hepatic lipase (HL) promoter has been shown to be associated with lower HL activity and increased high density lipoprotein (HDL) cholesterol. We examined the frequency and lipid associations of this HL polymorphism in 385 healthy, young (18- to 28-year-old) men whose fathers had had a premature myocardial infarction (designated cases) and 405 age-matched controls. These individuals were participants in the European Atherosclerosis Research Study II postprandial trial, who had been recruited from 11 European countries in 4 regions (the Baltic; United Kingdom; and central and southern Europe). Overall, the frequency of the T allele was 0.207 in controls and 0.244 in cases (P=0.08). The T allele was associated with higher fasting plasma total cholesterol (P<0.01), triglycerides (P<0.01), and HDL cholesterol (P<0.01). The strongest association was found with apolipoprotein (apo) A-I concentration, which was 10% higher in individuals homozygous for the T allele compared with those homozygous for the C allele (P<0.001). This polymorphism had no effect on the rise in plasma triglyceride levels after a fatty meal. However, before and after the fat load was ingested, levels of particles containing both apoC-III and apoB (LpC-III:B) were higher in carriers of the T allele, with homozygotes having 23% and 27% higher levels preprandially and postprandially, respectively, than those homozygous for the C allele (P<0.05). Thus, our results demonstrate that the C-480T polymorphism in the HL promoter is associated with alterations in plasma lipids and lipoproteins and the accumulation of atherogenic LpC-III:B particles.
Title: Common variants in the promoter of the hepatic lipase gene are associated with lower levels of hepatic lipase activity, buoyant LDL, and higher HDL2 cholesterol Zambon A, Deeb SS, Hokanson JE, Brown BG, Brunzell JD Ref: Arterioscler Thromb Vasc Biol, 18:1723, 1998 : PubMed
Increased hepatic lipase (HL) activity is associated with small, dense, low density lipoprotein (LDL) and low high density lipoprotein2 (HDL2) cholesterol (-C) levels. A polymorphism in the promoter region of the HL gene (LIPC) is associated with HDL-C levels. To test whether this association is mediated by differences in HL activity between different LIPC promoter genotypes, the LIPC promoter polymorphism at position -250 (G-->A), HL activity, LDL buoyancy, and HDL-C levels were studied in white normolipidemic men and men with coronary artery disease (CAD). The less common A allele (frequency=0.21 and 0.25 in normal and CAD subjects, respectively) was associated with lower HL activity (P<0.005 by ANOVA) and buoyant LDL particles (P</=0.01) in both groups. Normal and CAD subjects heterozygous for the A allele had lower HL activity (by 24% and 29%, respectively) and significantly more buoyant LDL particles. Homozygosity for this allele (AA) was associated with an even lower HL activity in normal (-26%) and CAD (-46%) subjects. The A allele was associated with higher HDL2-C in CAD patients (P=0.007); heterozygotes and homozygotes for the A allele had a 92% and a 140% higher HDL2-C level (P<0.01) than did GG individuals. In a small number of normolipidemic subjects, the same trend in HDL2-C was seen. In a univariate analysis, the LIPC genotype accounted for 20% to 32% of the variance in HL levels among normal subjects and CAD patients, respectively. After adjustment for HL, the association between LIPC genotype and LDL buoyancy was no longer significant, suggesting that the effect of LIPC genotype on LDL buoyancy is mediated by its effects on HL activity. The LIPC A allele was more frequent in Japanese-Americans and African-Americans than in whites. In summary, these results suggest that variants in the LIPC promoter may significantly contribute to the variance in levels of HL activity and consequently, to the prevalence of the atherogenic small, dense, LDL particles and low HDL2-C levels.
We studied the molecular basis of low hepatic lipase (HL) activity in normolipidemic male patients with angiographically documented coronary artery disease (CAD). In 18 subjects with a lowered HL activity (< 225 mU/mL), all nine exons of the HL gene and part of the promoter region (nucleotides -524 to +7) were sequenced. No structural mutations in the coding part of the HL gene were found, but 50% of the subjects showed a C-to-T substitution at nucleotide -480. Screening for the base substitution in 782 patients yielded an allele frequency of 0.213 (297 heterozygotes, 18 homozygotes). In a group of 316 nonsymptomatic control subjects, the allele frequency was 0.189, which is significantly less than in the CAD patients (P = .035). In the CAD patients, the C-to-T substitution was associated with a lowered lipase activity (heterozygotes -15%, homozygotes -20%). The patients were divided into quartiles on the basis of HL activity. Sixty percent (allele frequency 0.32) of the patients in the lowest quartile (HL activity < 306 mU/mL) had the gene variant against 27% (allele frequency 0.14) in the highest quartile (HL activity > 466 mU/mL). In the noncarriers, but not in the carriers, HL activity was related with plasma insulin, being increased at higher insulin concentration. Homozygous carriers had a significantly higher HDL cholesterol level-than noncarriers (1.13 +/- 0.28 mmol/L versus 0.92 +/- 0.22 mmol/L, P < .02). Our results show that a C-to-T substitution at -480 of the HL promoter is associated with a lowered HL activity. The base substitution, or a closely linked gene variation, may contribute to the variation in HL activity and affect plasma lipoprotein metabolism.
Hepatic lipase (HL) is an endothelial enzyme involved in the metabolism of intermediate density lipoproteins (IDL) and high density lipoproteins (HDL) in plasma. In a Finnish pedigree consisting of 18 members belonging to three generations two missense mutations RI86H and L334F in exons 5 and 7 of the HL gene co-segregated with low post-heparin HL activity. Haplotype analysis of the HL gene in family members revealed a high degree of genetic variation and demonstrated that the two missense mutations reside on the same chromosome. In vitro site-directed mutagenesis and expression of the cDNA constructs in COS-1 cells revealed that the R186H mutation leads to a protein that is not secreted while the L334F mutation results in the production of a HL protein that is secreted but has only about 30% of wild type HL activity. Carriers of the mutated HL gene exhibited clearly reduced HL activity and mass in post-heparin plasma. Probably due to their heterozygous carrier status they had only moderate elevation of total triglycerides, IDL, and LDL-triglycerides. The LDL-particles were enriched in triglycerides and depleted of cholesterol. Also their HDL2- and HDL3-particles were enriched in triglycerides.
Title: Compound heterozygosity for mutant hepatic lipase in familial hepatic lipase deficiency Hegele RA, Little JA, Connelly PW Ref: Biochemical & Biophysical Research Communications, 179:78, 1991 : PubMed
In a kindred with three hyperlipidemic subjects who had premature atherosclerosis and complete deficiency of hepatic lipase activity, we had previously identified a novel structural hepatic lipase gene variant. We now report the identification of three more hepatic lipase gene mutations in this family and demonstrate that compound heterozygosity for two hepatic lipase mutations (designated S267F and T383M) underlies hepatic lipase deficiency.
Absent hepatic lipase (HL) activity results in dyslipidemia and premature atherosclerosis. DNA sequencing of the HL gene from subjects with heritable HL deficiency identified a new C to T substitution within exon 8 that in the mature enzyme caused a threonine to methionine change at position 383 (T383M). With a rapid DNA detection method we observed that all 6 individuals with complete HL deficiency from 2 families had the T383M mutation. None of 50 random unrelated unaffected subjects had this mutation. We propose that T383M is specific to families with heritable HL deficiency. Furthermore, structural variation at the HL gene, possibly in combination with other factors, appears to be etiologic in HL deficiency.
Overlapping bacterial phage and cosmid genomic clones were isolated spanning an area of approximately 60 kilobases that contains the human hepatic lipase (HL) gene. It is composed of 9 exons spanning approximately 35 kilobases of DNA. The entire coding regions, the 5'-flanking sequences, and the exon-intron junctions were sequenced. The intron positions correspond to those of human lipoprotein lipase and canine pancreatic lipase, supporting the concept that these genes constitute a dispersed gene family of lipases and have evolved by duplication of a common ancestral gene. A region of the HL gene, which displays a significant homology with various other lipolytic enzymes and contains the putative catalytic site serine residue of HL, was encoded by exon 4. A major transcription start site of the human HL gene was located by primer extension analysis, 43 nucleotides upstream of the translation initiation codon. Two possible promoter elements were located 25 and 63 nucleotides upstream of the transcription initiation site: a "TATA" box-like sequence, TAATA, and a sequence found in the promoter region of many liver-specific genes, AGGTTAATTATTAAT. In addition, sequences homologous to glucocorticoid and cAMP-responsive elements were identified in the 5'-nontranscribed region.
A large family is reported with familial hepatic triglyceride lipase (HTGL) deficiency and with the coexistence of reduced lipoprotein lipase (LPL) similar to the heterozygote state of LPL deficiency. The proband was initially detected because of hypertriglyceridemia and chylomicronemia. He was later demonstrated to have beta-VLDL despite an apo E3/E3 phenotype and the lack of stigmata of type III hyperlipoproteinemia. The proband had no HTGL activity in postheparin plasma. Two of his half-sisters had very low HTGL activity (39 and 31 nmol free fatty acids/min/ml; normal adult female greater than 44). His son and daughters had decreased HTGL activity (normal male and preadolescent female greater than 102), which would be expected in obligate heterozygotes for HTGL deficiency. Low HTGL activity was associated with LDL particles which were larger and more buoyant. Several family members, including the proband, had reduced LPL activity and mass less than that circumscribed by the 95% confidence-interval ellipse for normal subjects and had hyperlipidemia similar to that described in heterozygote relatives of patients with LPL deficiency. All the sibs with hyperlipidemia had a reduced LPL activity and mass, while subjects with isolated reduced HTGL (with normal LPL activity) had normal lipid phenotypes. Analysis of genomic DNA from these subjects by restriction-enzyme digestion revealed no major abnormalities in the structure of either the HTGL or the LPL gene. Compound heterozygotes for HTGL and LPL deficiency show lipoprotein physiological characteristics typical for HTGL deficiency, while their variable lipid phenotype is typical for LPL deficiency.
Title: Combined lipase deficiency in the mouse. Evidence of impaired lipase processing and secretion Davis RC, Ben-Zeev O, Martin D, Doolittle MH Ref: Journal of Biological Chemistry, 265:17960, 1990 : PubMed
Newborn combined lipase-deficient (cld) mice have severe hypertriglyceridemia associated with a marked decrease of lipoprotein lipase (LPL) and hepatic lipase (HL) activities. Since the cld mutation and lipase genes reside on separate chromosomes, combined lipase deficiency cannot result from defects occurring within the LPL or HL structural genes. To elucidate the biochemical basis of this trans-acting defect, cld mice were compared to unaffected littermates for changes in lipase mRNA levels, rates of synthesis, and posttranslational processing and secretion. LPL and HL mRNA levels in cld liver and LPL in cld heart were comparable to controls; corresponding lipase synthetic rates were modestly decreased by about 30%. However, these reduced synthetic rates were not lipase-specific, since the rates of apolipoprotein (apo) A-I and apoA-II synthesis in cld liver were similarly decreased. Despite LPL synthetic rates that were 70% of controls, LPL mass in cld postheparin plasma was markedly reduced to only 7% of control values, suggesting that the majority of LPL is not secreted but remains intracellular. Consistent with a lipase secretory defect, neither the LPL nor HL oligomannosyl forms were converted to their respective complex forms in cld tissues, indicating that the lipases had failed to move from the endoplasmic reticulum/cis-Golgi to the medial/trans-Golgi network. In addition, the majority of intracellular LPL was catalytically inactive, since LPL specific activity (units/mg LPL protein) in cld heart, kidney, and brain was reduced 80-97%. In contrast to the severe impairment of lipase posttranslational processing and secretion, cld mouse plasma contained normal levels of another secretory N-linked glycoprotein, adipsin, with its oligosaccharide chains fully processed to the complex form. Thus, the cld mutation appears not to globally disrupt the secretion of all N-linked glycoproteins, but rather selectively impairs LPL and HL at points essential to their normal intracellular transport and secretion.
Title: Structure of the human hepatic triglyceride lipase gene Cai SJ, Wong DM, Chen SH, Chan L Ref: Biochemistry, 28:8966, 1989 : PubMed
The structure of the human hepatic triglyceride lipase gene was determined from multiple cosmid clones. All the exons, exon-intron junctions, and 845 bp of the 5' and 254 bp of the 3' flanking DNA were sequenced. Comparison of the exon sequences to three previously published cDNA sequences revealed differences in the sequence of the codons for residues 133, 193, 202, and 234 that may represent sequence polymorphisms. By primer extension, hepatic lipase mRNA initiates at an adenine 77 bases upstream of the translation initiation site. The hepatic lipase gene spans over 60 kb containing 9 exons and 8 introns, the latter being all located within the region encoding the mature protein. The exons are all of average size (118-234 bp). Exon 1 encodes the signal peptide, exon 4, a region that binds to the lipoprotein substrate, and exon 5, an evolutionarily highly conserved region of potential catalytic function, and exons 6 and 9 encode sequences rich in basic amino acids thought to be important in anchoring the enzyme to the endothelial surface by interacting with acidic domains of the surface glycosaminoglycans. The human lipoprotein lipase gene has been recently reported to have an identical exon-intron organization containing the analogous structural domains [Deeb & Peng (1989) Biochemistry 28, 4131-4135]. Our observations strongly support the common evolutionary origin of these two lipolytic enzymes.
Title: Human hepatic lipase. Cloned cDNA sequence, restriction fragment length polymorphisms, chromosomal localization, and evolutionary relationships with lipoprotein lipase and pancreatic lipase Datta S, Luo CC, Li WH, VanTuinen P, Ledbetter DH, Brown MA, Chen SH, Liu SW, Chan L Ref: Journal of Biological Chemistry, 263:1107, 1988 : PubMed
Human hepatic lipase is an important enzyme in high density lipoprotein (HDL) metabolism, being implicated in the conversion of HDL2 to HDL3. Three human hepatic lipase cDNA clones were identified in two lambda gt11 libraries from human liver. The cDNA-derived amino acid sequence predicts a protein of 476 amino acid residues, preceded by a 23-residue signal peptide. Four potential N-glycosylation sites are identified, two of which are conserved in rat hepatic lipase. On alignment with human, mouse, and bovine lipoprotein lipase, the same two sites were also conserved in lipoprotein lipase in all three species. Stringent conservation of the cysteine residues was also evident. Comparative analysis of amino acid sequences shows that hepatic lipase evolves at a rapid rate, 2.07 x 10(-9) substitutions/site/year, about four times that in lipoprotein lipase and half that in pancreatic lipase. Further, hepatic lipase and pancreatic lipase appear to be evolutionarily closer to each other than either of them is to lipoprotein lipase. Southern blot analysis revealed high frequency restriction fragment length polymorphisms of the hepatic lipase gene for the enzymes HindIII and MspI. these polymorphisms will be useful for haplotype and linkage analysis of the hepatic lipase gene. Using cloned human hepatic lipase cDNA as a hybridization probe, we performed Southern blot analysis of a panel of 13 human-rodent somatic cell hybrids. Concordance analysis of the various hybrid clones indicates that the hepatic lipase gene is located on the long arm of human chromosome 15. Analysis of hybrids containing different translocations of chromosome 15 localized the gene to the region 15q15----q22.
Hepatic triglyceride lipase (H-TGL) was isolated from human postheparin plasma by column chromatography on heparin-Sepharose and phenyl-Sepharose and immunoaffinity chromatography with monoclonal antibodies. The purified enzyme had an apparent molecular weight of 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an amino-terminal sequence of Leu-Gly-Gln-Ser-Leu-Lys-Pro-Glu. Partial amino acid sequences of seven cyanogen bromide peptides were obtained. A human hepatoma cDNA library was screened with synthetic oligonucleotides derived from the partial protein sequence. The cloned H-TGL cDNA of 1569 nucleotides predicts a mature protein of 477 amino acids plus a leader sequence of 22 amino acids. Blot hybridization analysis of poly(A)+ mRNA with a putative H-TGL cDNA clone gave a single hybridizing band of 1.7 kilobases. The protein contains four consensus N-glycosylation sequences based on the cDNA sequence. Comparison of the enzyme sequence with that of other lipases reveals highly conserved sequences in regions of putative lipid and heparin binding. The carboxyl terminus of H-TGL contains a highly basic sequence which is not reported to be present in rat H-TGL or other members of the lipase gene family.
We have used cDNA probes for lipoprotein lipase and hepatic lipase to determine the chromosomal and subchromosomal locations of the human genes for these lipolytic enzymes. Southern blot analysis of genomic DNA from 17 independent mouse-human somatic cell hybrids demonstrated the presence of the gene for human lipoprotein lipase on chromosome 8, whereas the gene for hepatic lipase was on chromosome 15. Regional mapping of the genes by in situ hybridization to human chromosomes indicated that the lipoprotein lipase gene (LPL) resides in the p22 region of chromosome 8, while hepatic lipase gene (HL) resides in the q21 region of chromosome 15. We previously reported, on the basis of nucleotide and amino acid homologies, that these genes are members of a gene family of lipases, and, thus, the present findings indicate that the members of this family are dispersed. The results are also of significance with respect to disorders involving deficiencies of the enzymes. In particular, they suggest that certain rare combined deficiencies of both enzymes do not involve mutations of the structural loci.
Title: Deficiency of hepatic lipase activity in post-heparin plasma in familial hyper-alpha-triglyceridemia Carlson LA, Holmquist L, Nilsson-Ehle P Ref: Acta Med Scand, 219:435, 1986 : PubMed
Hyper-alpha-triglyceridemia is a rare dyslipoproteinemia characterized by a pronounced increase in the concentration of triglycerides in the plasma high density lipoprotein (HDL) fraction. One case with this condition, an apparently healthy 61-year-old man, has been studied. Additional lipoprotein abnormalities were present, such as abnormally cholesterol-rich very low density lipoproteins (VLDL) with retarded electrophoretic mobility (beta-VLDL) and triglyceride enrichment of low density lipoproteins (LDL). The patient's plasma concentration of apolipoproteins A-I, A-II and B were normal and those of C-I, C-II, C-III and E were elevated. No abnormal forms of the soluble apolipoproteins of VLDL and high density lipoproteins (HDL) were found after analysis by isoelectric focusing. Lecithin:cholesterol acyltransferase activities, plasma cholesterol esterification rates and lipid transfer protein activities were normal. Post-heparin plasma activity of hepatic lipase was virtually absent and that of lipoprotein lipase was reduced by 50%. In plasma of this patient, HDL was almost exclusively present as large triglyceride-rich particles corresponding in size to particles of the HDL2 density fraction. The only brother of the patient also had hyper-alpha-triglyceridemia together with the other lipoprotein abnormalities described for the index case and deficiency of postheparin plasma activity of hepatic lipase. The findings presented below support the hypothesis that one primary function of hepatic lipase is associated with degradation of plasma HDL2. Deficiency of this enzyme activity thus causes accumulation of HDL2 in plasma leading to hyper-alpha-triglyceridemia. The results further suggest that the abnormal chemical and electrophoretic properties of VLDL and LDL in plasma from the patient, reminiscent of type III hyperlipoproteinemia, are secondary to the lack of the action of hepatic lipase on the HDL particles.
Title: Familial hepatic lipase deficiency Little JA, Connelly PW Ref: Advances in Experimental Medicine & Biology, 201:253, 1986 : PubMed
