Characterization of a novel mutation causing hepatic lipase deficiency among French Canadians
2003; Elsevier BV; Volume: 44; Issue: 8 Linguagem: Inglês
10.1194/jlr.m200479-jlr200
ISSN1539-7262
AutoresIsabelle L. Ruel, Patrick Couture, Claude Gagné, Yves Deshaies, Jacques Simard, Robert A. Hegele, Benoı̂t Lamarche,
Tópico(s)Lipid metabolism and biosynthesis
ResumoIndividuals 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 Québec-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 Québec kindred is associated with an abnormal lipoprotein-lipid profile, which may vary considerably in the presence of secondary factors such as abdominal obesity. 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 Québec-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 Québec kindred is associated with an abnormal lipoprotein-lipid profile, which may vary considerably in the presence of secondary factors such as abdominal obesity. Hepatic lipase (HL) is a 476 amino acid glycoprotein lipolytic serine hydrolase that is synthesized and secreted from hepatocytes, and anchored to the liver sinusoidal surface by heparin sulfate proteoglycans (1Doolittle M.H. Wong H. Davis R.C. Schotz M.C. Synthesis of hepatic lipase in liver and extrahepatic tissues.J. Lipid Res. 1987; 28: 1326-1334Abstract Full Text PDF PubMed Google Scholar). Evidence from clinical and animal studies supports a role for HL in the metabolism of several lipoprotein classes (2Santamarina-Fojo S. Haudenschild C. Amar M. The role of hepatic lipase in lipoprotein metabolism and atherosclerosis.Curr. Opin. Lipidol. 1998; 9: 211-219Crossref PubMed Scopus (211) Google Scholar, 3Beisiegel U. New aspects on the role of plasma lipases in lipoprotein catabolism and atherosclerosis.Atherosclerosis. 1996; 124: 1-8Abstract Full Text PDF PubMed Scopus (57) Google Scholar). Thus, HL has been reported to augment the uptake of HDL cholesterol by the liver through a reverse cholesterol transport process by participating in the reconversion of large, buoyant HDL2 to small, dense HDL3 and by modulating the phospholipid content of these particles (4Kuusi T. Saarinen P. Nikkila E.A. Evidence for the role of hepatic endothelial lipase in the metabolism of plasma high density lipoprotein2 in man.Atherosclerosis. 1980; 36: 589-593Abstract Full Text PDF PubMed Scopus (253) Google Scholar). HL is also responsible for the hydrolysis of triglycerides (TGs) and phospholipids in large, buoyant LDLs leading to the formation of small, dense, and more atherogenic LDL particles (5Zambon A. Austin M.A. Brown B.G. Hokanson J.E. Brunzell J.D. Effect of hepatic lipase on LDL in normal men and those with coronary artery disease.Arterioscler. Thromb. 1993; 13: 147-153Crossref PubMed Scopus (221) Google Scholar). Multiple lines of evidence suggest that HL participates in the conversion of VLDL remnants to LDL (6Jansen H. van Tol A. Hulsmann W.C. On the metabolic function of heparin-releasable liver lipase.Biochem. Biophys. Res. Commun. 1980; 92: 53-59Crossref PubMed Scopus (189) Google Scholar, 7Rubinstein A. Gibson J.C. Paterniti Jr., J.R. Kakis G. Little A. Ginsberg H.N. Brown W.V. Effect of heparin-induced lipolysis on the distribution of apolipoprotein E among lipoprotein subclasses. Studies with patients deficient in hepatic triglyceride lipase and lipoprotein lipase.J. Clin. Invest. 1985; 75: 710-721Crossref PubMed Scopus (57) Google Scholar) and enhances the uptake of remnant lipoproteins by functioning as a ligand between the lipoprotein and cell surface receptors (8Shafi S. Brady S.E. Bensadoun A. Havel R.J. Role of hepatic lipase in the uptake and processing of chylomicron remnants in rat liver.J. Lipid Res. 1994; 35: 709-720Abstract Full Text PDF PubMed Google Scholar). The enzyme can be released into the circulation by intravenous injection of heparin, enabling the measurement of its activity in postheparin plasma (9Huttunen J.K. Ehnholm C. Kekki M. Nikkila E.A. Post-heparin plasma lipoprotein lipase and hepatic lipase in normal subjects and in patients with hypertriglyceridaemia: correlations to sex, age and various parameters of triglyceride metabolism.Clin. Sci. Mol. Med. 1976; 50: 249-260PubMed Google Scholar). However, more specific metabolic functions of HL and its importance as a determinant of plasma lipoprotein levels have yet to be fully elucidated.The HL gene spans 35 kb of DNA, maps to chromosome 15q21, and is composed of nine exons and eight introns (10Cai S.J. Wong D.M. Chen S.H. Chan L. Structure of the human hepatic triglyceride lipase gene.Biochemistry. 1989; 28: 8966-8971Crossref PubMed Scopus (89) Google Scholar, 11Ameis D. Stahnke G. Kobayashi J. McLean J. Lee G. Buscher M. Schotz M.C. Will H. Isolation and characterization of the human hepatic lipase gene.J. Biol. Chem. 1990; 265: 6552-6555Abstract Full Text PDF PubMed Google Scholar). Four missense mutations in encoding exons [R186H (12Knudsen P. Antikainen M. Uusi-Oukari M. Ehnholm S. Lahdenpera S. Bensadoun A. Funke H. Wiebusch H. Assmann G. Taskinen M.R. Ehnholm C. Heterozygous hepatic lipase deficiency, due to two missense mutations R186H and L334F, in the HL gene.Atherosclerosis. 1997; 128: 165-174Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), L334F (13Knudsen P. Antikainen M. Ehnholm S. Uusi-Oukari M. Tenkanen H. Lahdenpera S. Kahri J. Tilly-Kiesi M. Bensadoun A. Taskinen M.R. Ehnholm C. A compound heterozygote for hepatic lipase gene mutations Leu334→Phe and Thr383→Met: correlation between hepatic lipase activity and phenotypic expression.J. Lipid Res. 1996; 37: 825-834Abstract Full Text PDF PubMed Google Scholar), S267F (14Hegele R.A. Little J.A. Connelly P.W. Compound heterozygosity for mutant hepatic lipase in familial hepatic lipase deficiency.Biochem. Biophys. Res. Commun. 1991; 179: 78-84Crossref PubMed Scopus (27) Google Scholar), and T383M (15Hegele R.A. Vezina C. Moorjani S. Lupien P.J. Gagne C. Brun L.D. Little J.A. Connelly P.W. A hepatic lipase gene mutation associated with heritable lipolytic deficiency.J. Clin. Endocrinol. Metab. 1991; 72: 730-732Crossref PubMed Scopus (47) Google Scholar)] have been identified and demonstrated to be responsible for HL-deficient phenotypes. The determination of the role of HL in human lipoprotein metabolism has been facilitated by the identification of patients with HL deficiency. This rare genetic disorder, which appears to be inherited as an autosomal recessive trait, has been identified in only five families to date (15Hegele R.A. Vezina C. Moorjani S. Lupien P.J. Gagne C. Brun L.D. Little J.A. Connelly P.W. A hepatic lipase gene mutation associated with heritable lipolytic deficiency.J. Clin. Endocrinol. Metab. 1991; 72: 730-732Crossref PubMed Scopus (47) Google Scholar, 16Breckenridge W.C. Little J.A. Alaupovic P. Wang C.S. Kuksis A. Kakis G. Lindgren F. Gardiner G. Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase.Atherosclerosis. 1982; 45: 161-179Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 17Carlson L.A. Holmquist L. Nilsson-Ehle P. Deficiency of hepatic lipase activity in post-heparin plasma in familial hyper-alpha-triglyceridemia.Acta Med. Scand. 1986; 219: 435-447Crossref PubMed Google Scholar, 18Auwerx J.H. Babirak S.P. Hokanson J.E. Stahnke G. Will H. Deeb S.S. Brunzell J.D. Coexistence of abnormalities of hepatic lipase and lipoprotein lipase in a large family.Am. J. Hum. Genet. 1990; 46: 470-477PubMed Google Scholar, 19Sheriff D.S. el Fakhri M. Ghwarsha K. Libyan family with hypercholesterolemia and increased high-density lipoprotein cholesterol in plasma.Clin. Chem. 1994; 40: 2313-2316Crossref PubMed Scopus (6) Google Scholar). Affected individuals with a heterozygous state for HL mutations do not have specific lipoprotein abnormalities, and even patients with complete HL deficiency display a variable phenotype (20Hegele R.A. Little J.A. Vezina C. Maguire G.F. Tu L. Wolever T.S. Jenkins D.J. Connelly P.W. Hepatic lipase deficiency. Clinical, biochemical, and molecular genetic characteristics.Arterioscler. Thromb. 1993; 13: 720-728Crossref PubMed Scopus (213) Google Scholar). HL-deficient subjects may present with features characteristic of Type III hyperlipoproteinemia, including hypercholesterolemia, hypertriglyceridemia, and β-VLDL, and some have premature cardiovascular disease (20Hegele R.A. Little J.A. Vezina C. Maguire G.F. Tu L. Wolever T.S. Jenkins D.J. Connelly P.W. Hepatic lipase deficiency. Clinical, biochemical, and molecular genetic characteristics.Arterioscler. Thromb. 1993; 13: 720-728Crossref PubMed Scopus (213) Google Scholar, 21Breckenridge W.C. Little J.A. Alaupovic P. Wang C.S. Kuksis A. Kakis G. Lindgren F. Gardiner G. Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase.Atherosclerosis. 1982; 45: 161-179Abstract Full Text PDF PubMed Scopus (231) Google Scholar, 22Connelly P.W. Maguire G.F. Lee M. Little J.A. Plasma lipoproteins in familial hepatic lipase deficiency.Arteriosclerosis. 1990; 10: 40-48Crossref PubMed Scopus (96) Google Scholar, 23Connelly P.W. Ranganathan S. Maguire G.F. Lee M. Myher J.J. Kottke B.A. Kuksis A. Little J.A. The beta very low density lipoprotein present in hepatic lipase deficiency competitively inhibits low density lipoprotein binding to fibroblasts and stimulates fibroblast acyl-CoA:cholesterol acyltransferase.J. Biol. Chem. 1988; 263: 14184-14188Abstract Full Text PDF PubMed Google Scholar). One of the most consistent findings in the lipoprotein phenotype of HL-deficient subjects is an elevation of HDL2 cholesterol (21Breckenridge W.C. Little J.A. Alaupovic P. Wang C.S. Kuksis A. Kakis G. Lindgren F. Gardiner G. Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase.Atherosclerosis. 1982; 45: 161-179Abstract Full Text PDF PubMed Scopus (231) Google Scholar, 24Carlson L.A. Holmquist L. Nilsson-Ehle P. Deficiency of hepatic lipase activity in post-heparin plasma in familial hyper-alpha-triglyceridemia.Acta Med. Scand. 1986; 219: 435-447Crossref PubMed Scopus (90) Google Scholar, 25Little J.A. Connelly P.W. Familial hepatic lipase deficiency.Adv. Exp. Med. Biol. 1986; 201: 253-260PubMed Google Scholar) and a marked TG enrichment of LDL and HDL particles (20Hegele R.A. Little J.A. Vezina C. Maguire G.F. Tu L. Wolever T.S. Jenkins D.J. Connelly P.W. Hepatic lipase deficiency. Clinical, biochemical, and molecular genetic characteristics.Arterioscler. Thromb. 1993; 13: 720-728Crossref PubMed Scopus (213) Google Scholar, 22Connelly P.W. Maguire G.F. Lee M. Little J.A. Plasma lipoproteins in familial hepatic lipase deficiency.Arteriosclerosis. 1990; 10: 40-48Crossref PubMed Scopus (96) Google Scholar, 24Carlson L.A. Holmquist L. Nilsson-Ehle P. Deficiency of hepatic lipase activity in post-heparin plasma in familial hyper-alpha-triglyceridemia.Acta Med. Scand. 1986; 219: 435-447Crossref PubMed Scopus (90) Google Scholar).In the present study, we describe the underlying molecular defects in the HL gene of three patients from the Québec-based Hepatic Lipase Deficiency (QHLD) kindred presenting with very low to undetectable postheparin plasma HL activity. Hegele et al. previously reported the presence of the T383M mutation in the HL gene in one of two alleles among the HL-deficient subjects from the QHLD kindred (15Hegele R.A. Vezina C. Moorjani S. Lupien P.J. Gagne C. Brun L.D. Little J.A. Connelly P.W. A hepatic lipase gene mutation associated with heritable lipolytic deficiency.J. Clin. Endocrinol. Metab. 1991; 72: 730-732Crossref PubMed Scopus (47) Google Scholar). In vitro expression studies have confirmed that the T383M mutant protein retains partial activity but is poorly secreted (26Durstenfeld A. Ben-Zeev O. Reue K. Stahnke G. Doolittle M.H. Molecular characterization of human hepatic lipase deficiency. In vitro expression of two naturally occurring mutations.Arterioscler. Thromb. 1994; 14: 381-385Crossref PubMed Google Scholar). We report a previously unknown missense mutation in exon 5 of the HL gene due to a G→A base change resulting in the change of an alanine for a threonine in codon 174 of the mature protein. HL deficiency among three patients from the QHLD kindred resulted from compound heterozygosity for the two missense mutations A174T and T383M in the HL gene. We also describe the effects of severe HL deficiency on lipoprotein phenotype.METHODSSubjectsThe proband, now a healthy 44-year-old French Canadian female, was referred to our clinic in the early 1980s for investigation of a moderate hyperlipidemia; she was 22 years old at the time. Physical examination was entirely normal. Thyroid, hepatic, and renal functions, as well as fasting blood glucose and urinalyses were normal. Plasma lipid and lipoprotein profile showed unusual abnormalities, including hyperalphalipoproteinemia with an HDL cholesterol value of 2.5 mmol/l, which was observed in spite of a concomitant hypertriglyceridemia of 4.1 mmol/l. Agarose gel electrophoresis of the VLDL fraction isolated by ultracentrifugation revealed a β-VLDL band. Analysis of the apolipoprotein (apo)E phenotype indicated that the patient was a carrier of the apoE3/3 genotype, thus ruling out the diagnosis of dysbetalipoproteinemia associated with the apoE2/2 genotype. It was then hypothesized that this patient could be affected with HL deficiency.In the early 1990s, measurement of postheparin plasma lipase activities was performed and confirmed the diagnosis of severe HL deficiency. The analyses revealed undetectable HL activity with normal lipoprotein lipase (LPL) activity. First-degree relatives were then investigated: the proband had two sisters and four brothers. Three of the seven first-degree relatives, including the proband, were found to have undetectable HL activity; and sequencing of the HL gene, however, revealed the presence of the T838M missense mutation in one allele only (15Hegele R.A. Vezina C. Moorjani S. Lupien P.J. Gagne C. Brun L.D. Little J.A. Connelly P.W. A hepatic lipase gene mutation associated with heritable lipolytic deficiency.J. Clin. Endocrinol. Metab. 1991; 72: 730-732Crossref PubMed Scopus (47) Google Scholar). We have recently revisited the family to better understand the molecular defect responsible for HL deficiency in this kindred. The lipid-lipoprotein phenotype was characterized in the first-degree relatives (one of the proband's brothers refused to participate in the study) as well as in six individuals on the proband's paternal side (Fig. 1). The family members were generally healthy with no cases of Type 2 diabetes or previous history of coronary heart disease (CHD). One compound heterozygous male (1Doolittle M.H. Wong H. Davis R.C. Schotz M.C. Synthesis of hepatic lipase in liver and extrahepatic tissues.J. Lipid Res. 1987; 28: 1326-1334Abstract Full Text PDF PubMed Google Scholar-5Zambon A. Austin M.A. Brown B.G. Hokanson J.E. Brunzell J.D. Effect of hepatic lipase on LDL in normal men and those with coronary artery disease.Arterioscler. Thromb. 1993; 13: 147-153Crossref PubMed Scopus (221) Google Scholar) was treated for hypothyroidism. Subject 1-8 was taking oral contraceptives, Subject 2-1 received conjugated estrogens alone, and Subjects 2-4 and 2-5 received conjugated estrogens with medroxyprogesterone. Two of the participants also received HMG-CoA reductase inhibitors (2-2 and 2-3), one received a β-blocker (2Santamarina-Fojo S. Haudenschild C. Amar M. The role of hepatic lipase in lipoprotein metabolism and atherosclerosis.Curr. Opin. Lipidol. 1998; 9: 211-219Crossref PubMed Scopus (211) Google Scholar), and finally, Patients 2-1 and 2-2 were taking an angiotensin II receptor inhibitor for essential arterial hypertension. None of the family members were smokers at the time of investigation. All participants gave their written consent to participate in this study, which received the approval from the local ethics committees. Unrelated men were recruited at the Lipid Research Center to serve as control subjects.Lipid and lipoprotein analysesBlood was drawn in tubes containing 0.15% EDTA after a 12 h fast, and plasma was isolated by centrifugation (1,500 g at 4°C, 15 min). The lipid content of plasma and lipoprotein subfractions isolated by sequential ultracentrifugations was determined by enzymatic methods with a Technicon RA-500 analyzer (Bayer Corporation, Tarrytown, NY) according to standardized procedures that have been described previously (27Couillard C. Despres J.P. Lamarche B. Bergeron J. Gagnon J. Leon A.S. Rao D.C. Skinner J.S. Wilmore J.H. Bouchard C. Effects of endurance exercise training on plasma HDL cholesterol levels depend on levels of triglycerides: evidence from men of the Health, Risk Factors, Exercise Training and Genetics (HERITAGE) Family Study.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1226-1232Crossref PubMed Scopus (236) Google Scholar). Plasma apoB and apoA-I levels were measured by nephelometry (Dade Behring, Newark, DE). The apoE genotype was determined using the procedure described by Hixson and Vernier (28Hixson J.E. Vernier D.T. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI.J. Lipid Res. 1990; 31: 545-548Abstract Full Text PDF PubMed Google Scholar). Nondenaturing 2–16% and 4–30% polyacrylamide gradient gel electrophoreses were used to determine LDL and HDL particle sizes respectively, as previously described (29Lamarche B. St-Pierre A.C. Ruel I.L. Cantin B. Dagenais G.R. Despres J.P. A prospective, population-based study of low density lipoprotein particle size as a risk factor for ischemic heart disease in men.Can. J. Cardiol. 2001; 17: 859-865PubMed Google Scholar, 30Pérusse M. Pascot A. Després J.P. Couillard C. Lamarche B. A new method for HDL particle sizing by polyacrylamide gradient gel electrophoresis using whole plasma.J. Lipid Res. 2001; 42: 1331-1334Abstract Full Text Full Text PDF PubMed Google Scholar).Lipolytic enzyme activity determinationsLPL and HL activities were measured in subjects after a 12 h fast, 10 min after an intravenous injection of heparin (60 IU/kg body weight). LPL and HL activities were determined in postheparin plasma after preincubation with SDS, as previously described by Watson et al. (31Watson T.D. Tan C.E. McConnell M. Clegg S.K. Squires L.F. Packard C.J. Measurement and physiological significance of lipoprotein and hepatic lipase activities in preheparin plasma.Clin. Chem. 1995; 41: 405-412Crossref PubMed Scopus (35) Google Scholar). The two lipolytic enzyme activities were expressed as micromoles of free fatty acids released per milliliter of plasma per hour. The coefficient of variation for the analysis was 4.8%.Amplification of the exons of the HL gene by PCRDNA was extracted from leukocytes as described earlier (32Sambrook J. Fritsh E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York1989Google Scholar). PCR primers, homologous to intron sequences flanking the exons of the HL gene and containing 18–23 nucleotides, were designed based on published HL gene structure (10Cai S.J. Wong D.M. Chen S.H. Chan L. Structure of the human hepatic triglyceride lipase gene.Biochemistry. 1989; 28: 8966-8971Crossref PubMed Scopus (89) Google Scholar). Sequences of amplification primers are available on request. The PCR was carried out using an automated 9600 GeneAmp (PE Applied Biosystems, Foster City, CA) in a total volume of 50 μl after 35 cycles (30 s at 95°C, 30 s at 55°C, and 90 s at 72°C).DNA sequencingThe nucleotide sequence of both strands of the PCR products was determined by the dideoxy nucleotide chain termination method (33Sanger F. Nicklen S. Coulson A.R. DNA sequencing with chain-terminating inhibitors.Proc. Natl. Acad. Sci. USA. 1977; 74: 5463-5467Crossref PubMed Scopus (52357) Google Scholar) using a T7 sequencing kit from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). After the identification of the novel A174T missense mutation in exon 5 of the HL gene in the proband, its allele frequency was determined in 50 unrelated normal subjects by a complete sequencing of exon 5 of the HL gene. All family members of the QHLD kindred were then genotyped for the A174T mutation with digestion of the PCR-amplified exon 5 with SacII. The T383M mutation in the HL gene was detected by standard PCR followed by digestion with the enzyme NlaIII, as previously described (15Hegele R.A. Vezina C. Moorjani S. Lupien P.J. Gagne C. Brun L.D. Little J.A. Connelly P.W. A hepatic lipase gene mutation associated with heritable lipolytic deficiency.J. Clin. Endocrinol. Metab. 1991; 72: 730-732Crossref PubMed Scopus (47) Google Scholar).StatisticsPlasma, HDL, and LDL-TG levels were log10-transformed in order to reduce the skewness of their distribution, although untransformed values are shown in tables. Age, BMI, waist girth, and all lipid parameters in the different HL mutation groups, and in control subjects were compared using a general linear model with the Duncan posthoc test to locate subgroup differences. The statistical analyses were performed with the SAS software package (SAS Institute).RESULTSIn order to establish the molecular defect underlying HL deficiency in the proband, we sequenced all nine coding exon and introns exon boundaries of the HL gene from PCR-amplified DNA. Sequencing of the PCR products revealed a variable site in exon 5 of the HL gene: the proband presented a normal G base on one allele as well as an abnormal base on the other allele, an A, at the first position of codon 174 in the sequencing gel. This novel single nucleotide mutation resulting in the substitution of alanine for threonine at amino acid 174 was identified in the proband and her two younger brothers (Fig. 1). The T383M missense mutation in exon 8 was found in the proband, her mother, and three brothers. The results obtained in the first-degree relatives suggest that the father had the A174T mutation. He had a myocardial infarction at age 49 but died at the age of 55 from an oropharyngeal cancer. The allele frequency of the A174T variant was determined by direct sequencing of exon 5 in 50 unrelated normal subjects. Each HL gene variant site was di-allelic (Table 1). None of the control subjects were carriers of the A174T mutation in the HL gene. Gene variants of previously reported codons 193 and 202 in exon 5 were present in the controls with a frequency of 35% and 49%, respectively.TABLE 1Hepatic lipase gene variants in exon 5 and their frequencies in 50 control subjectsVariantExonBaseCodonDNA ChangeAmino Acid ChangeGenotype FrequencyMinor Allele FrequencyA174T5593174G→AaNucleotide specifying minor allele for each site indicated in italic type.Ala→ThrGG100%0%GA0%AA0%N193S5651193A→GAsn→SerAA36%35%AG58%GG6%T202T5679202C→GThr→ThrCC24%49%CG54%GG22%a Nucleotide specifying minor allele for each site indicated in italic type. Open table in a new tab The proband and all first-degree relatives of the proband were E3 homozygotes. The three subjects carrying the A174T and T383M mutations were found to have extremely low to undetectable HL activity, with normal LPL activity. For the purpose of the present study, we arbitrarily refer to these compound heterozygotes as patients with complete HL deficiency. As shown in Fig. 1, the two compound heterozygous males for the A174T and T383M mutations (1-5 and 1-6) were hypertriglyceridemic and exhibited abdominal obesity. Compared with her two brothers with complete HL deficiency, the complete HL-deficient proband female (1Doolittle M.H. Wong H. Davis R.C. Schotz M.C. Synthesis of hepatic lipase in liver and extrahepatic tissues.J. Lipid Res. 1987; 28: 1326-1334Abstract Full Text PDF PubMed Google Scholar) presented elevated HDL cholesterol levels, mostly due to a greater proportion of HDL2 cholesterol compared with HDL3 cholesterol levels. The two completely HL-deficient men were also characterized by small LDL particles (252.7 and 250.2 Å for Subjects 1-5 and 1-6, respectively), which was not found in the proband, who had very large LDL particles (265.8 Å, not shown).The mean concentrations of cholesterol and TG in plasma and lipoprotein fractions of the complete and partial HL-deficient, unaffected family members, and unrelated control subjects are shown in Table 2. Carriers of the A174T/T383M combination presented an altered lipoprotein-lipid profile compared with control subjects, while partial HL-deficient patients, i.e., patients presenting with only one of the two mutations in the HL gene, tended to have an intermediate lipoprotein-lipid profile. The three complete HL-deficient patients were characterized by a marked hypertriglyceridemia (5.5 ± 4.0 mmol/l) versus partial HL-deficient patients and control subjects (A174T: 1.45 ± 0.37 mmol/l; T383M: 1.79 ± 0.61 mmol/l; and controls: 1.48 ± 0.58 mmol/l; P = 0.007). They also presented a marked 3- to 4-fold TG enrichment of LDL and HDL particles. In addition, gradient gel electrophoresis confirmed that the three patients with severe HL deficiency had large HDL particles (106.7 ± 2.3 vs. 83.0 ± 0.8 Å in noncarriers; P < 0.0001) representing the HDL2 subclass. These subjects also presented phospholipid-enriched HDL particles compared with the control subjects (1.54 ± 0.30 vs. 1.15 ± 0.18, P = 0.02). Partial HL-deficient patients presented low HL activity compared with noncarriers (A174T: 4.8 ± 1.0 and T383M: 3.7 ± 1.5 μmol/ml/h vs. 14.0 ± 7.2 μmol/ml/h for noncarriers; P = 0.0006). In all subjects, the postheparin plasma LPL activities were within normal range. Finally, partial HL-deficient patients carrying the A174T mutation presented a lipoprotein-lipid profile similar to that of those carrying the T383M mutation in the HL gene.TABLE 2Comparison of plasma lipid and lipoprotein values between complete and partial hepatic lipase-deficient subjects, unaffected family members, and unrelated control subjectsHL MutationA174T/T383M (n = 3)A174T (n = 3)T383M (n = 2)Noncarriers (n = 11)P aP values for all variables (except for age) were obtained after an adjustment for age and gender.Age (yrs)38 ± 448 ± 1657 ± 2246 ± 140.49BMI (kg/m2)29.5 ± 3.427.1 ± 1.226.5 ± 1.127.3 ± 4.30.84Waist girth (cm)96.0 ± 15.790.0 ± 11.592.5 ± 4.993.5 ± 11.70.77Cholesterol (mmol/l) Plasma7.2 ± 0.86.5 ± 0.55.6 ± 0.1bSignificantly different compared with complete HL-deficient subjects.5.3 ± 0.9bSignificantly different compared with complete HL-deficient subjects.0.02 LDL3.1 ± 1.04.5 ± 0.93.4 ± 0.43.5 ± 0.80.32 HDL1.55 ± 0.671.57 ± 0.261.45 ± 0.381.31 ± 0.290.50Triglycerides (mmol/l) Plasma5.5 ± 4.01.45 ± 0.37bSignificantly different compared with complete HL-deficient subjects.1.79 ± 0.61bSignificantly different compared with complete HL-deficient subjects.1.48 ± 0.58bSignificantly different compared with complete HL-deficient subjects.0.01 LDL1.26 ± 0.390.41 ± 0.05bSignificantly different compared with complete HL-deficient subjects.0.35 ± 0.01bSignificantly different compared with complete HL-deficient subjects.0.24 ± 0.08bSignificantly different compared with complete HL-deficient subjects.,cSignificantly different compared with partial HL-deficient subjects (A174T).,dSignificantly different compared with partial HL-deficient subjects (T383M).<0.001 HDL0.78 ± 0.200.32 ± 0.09bSignificantly different compared with complete HL-deficient subjects.0.37 ± 0.13bSignificantly different compared with complete HL-deficient subjects.0.23 ± 0.04bSignificantly different compared with complete HL-deficient subjects.,cSignificantly different compared with partial HL-deficient subjects (A174T).,dSignificantly different compared with partial HL-deficient subjects (T383M).<0.001Phospholipids (mmol/l) Plasma4.16 ± 0.393.38 ± 0.13bSignificantly different compared with complete HL-deficient subjects.3.10 ± 0.38bSignificantly different compared with complete HL-deficient subjects.2.81 ± 0.32bSignificantly different compared with complete HL-deficient subjects.<0.001 LDL1.44 ± 0.321.71 ± 0.131.35 ± 0.081.25 ± 0.260.09 HDL1.54 ± 0.301.38 ± 0.141.33 ± 0.171.15 ± 0.18bSignificantly different compared with complete HL-deficient subjects.0.02Apolipoprotein B (g/l)1.48 ± 0.301.32 ± 0.101.06 ± 0.02bSignificantly different compared with complete HL-deficient subjects.1.04 ± 0.23bSignifican
Referência(s)