From evidence to practice: development of web-based Dutch lipid reference values.

INTRODUCTION: In the Netherlands, the total number of yearly measured lipid profiles exceeds 500,000. While lipid values are strongly affected by age and sex, until recently, no up-to-date age- and sex-specific lipid reference values were available. We describe the translation of big-cohort lipid data into accessible reference values, which can be easily incorporated in daily clinical practice. METHODS: Lipid values (total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides) from all healthy adults and children in the LifeLines cohort were used to generate age- and sex-specific percentiles. A combination of RStudio, Cascading Style Sheets and HyperText Markup Language was used to interactively display the percentiles in a responsive web layout. RESULTS: After exclusion of subjects reporting cardiovascular disease or lipid-lowering therapy at baseline, 141,611 subjects were included. On the website, input fields were created for age, sex and all main plasma lipids. Upon input of these values, corresponding percentiles are calculated, and output is displayed in a table and an interactive graph for each lipid. The website has been made available in both Dutch and English and can be accessed at www.lipidtools.com . CONCLUSION: We constructed the first searchable, national lipid reference value tool with graphical display in the Netherlands to use in screening for dyslipidaemias and to reduce the underuse of lipid-lowering therapy in Dutch primary prevention. This study illustrates that data collected in big-cohort studies can be made easily accessible with modern digital techniques and preludes the digital health revolution yet to come.

Beyond the genetics of HDL: why is HDL cholesterol inversely related to cardiovascular disease?

There is unequivocal evidence that high-density lipoprotein (HDL) cholesterol levels in plasma are inversely associated with the risk of cardiovascular disease (CVD). Studies of families with inherited HDL disorders and genetic association studies in general (and patient) population samples have identified a large number of factors that control HDL cholesterol levels. However, they have not resolved why HDL cholesterol and CVD are inversely related. A growing body of evidence from nongenetic studies shows that HDL in patients at increased risk of CVD has lost its protective properties and that increasing the cholesterol content of HDL does not result in the desired effects. Hopefully, these insights can help improve strategies to successfully intervene in HDL metabolism. It is clear that there is a need to revisit the HDL hypothesis in an unbiased manner. True insights into the molecular mechanisms that regulate plasma HDL cholesterol and triglycerides or control HDL function could provide the handholds that are needed to develop treatment for, e.g., type 2 diabetes and the metabolic syndrome. Especially genome-wide association studies have provided many candidate genes for such studies. In this review we have tried to cover the main molecular studies that have been produced over the past few years. It is clear that we are only at the very start of understanding how the newly identified factors may control HDL metabolism. In addition, the most recent findings underscore the intricate relations between HDL, triglyceride, and glucose metabolism indicating that these parameters need to be studied simultaneously.

Mutations in LPL, APOC2, APOA5, GPIHBP1 and LMF1 in patients with severe hypertriglyceridaemia.

OBJECTIVES: The severe forms of hypertriglyceridaemia (HTG) are caused by mutations in genes that lead to the loss of function of lipoprotein lipase (LPL). In most patients with severe HTG (TG > 10 mmol L(-1) ), it is a challenge to define the underlying cause. We investigated the molecular basis of severe HTG in patients referred to the Lipid Clinic at the Academic Medical Center Amsterdam. METHODS: The coding regions of LPL, APOC2, APOA5 and two novel genes, lipase maturation factor 1 (LMF1) and GPI-anchored high-density lipoprotein (HDL)-binding protein 1 (GPIHBP1), were sequenced in 86 patients with type 1 and type 5 HTG and 327 controls. RESULTS: In 46 patients (54%), rare DNA sequence variants were identified, comprising variants in LPL (n = 19), APOC2 (n = 1), APOA5 (n = 2), GPIHBP1 (n = 3) and LMF1 (n = 8). In 22 patients (26%), only common variants in LPL (p.Asp36Asn, p.Asn318Ser and p.Ser474Ter) and APOA5 (p.Ser19Trp) could be identified, whereas no mutations were found in 18 patients (21%). In vitro validation revealed that the mutations in LMF1 were not associated with compromised LPL function. Consistent with this, five of the eight LMF1 variants were also found in controls and therefore cannot account for the observed phenotype. CONCLUSIONS: The prevalence of mutations in LPL was 34% and mostly restricted to patients with type 1 HTG. Mutations in GPIHBP1 (n = 3), APOC2 (n = 1) and APOA5 (n = 2) were rare but the associated clinical phenotype was severe. Routine sequencing of candidate genes in severe HTG has improved our understanding of the molecular basis of this phenotype associated with acute pancreatitis and may help to guide future individualized therapeutic strategies.

Plasma levels of lecithin:cholesterol acyltransferase and risk of future coronary artery disease in apparently healthy men and women: a prospective case-control analysis nested in the EPIC-Norfolk population study.

LCAT plays a key role in the maturation of HDL, as evidenced by low HDL-cholesterol levels in carriers of deleterious mutations in LCAT. However, the role of LCAT in atherosclerosis is unclear. We set out to study this in a prospective study. Plasma LCAT levels, which strongly correlate with LCAT activity, were measured in baseline nonfasting samples of 933 apparently healthy men and women who developed coronary artery disease (CAD) and 1,852 matched controls who remained free of CAD during 6 year follow-up. LCAT levels did not differ between cases and controls but were higher in women than men. Stratification into LCAT quartiles revealed a positive association with plasma LDL-cholesterol and triglyceride levels in the unexpected absence of an association with HDL-cholesterol. In mixed-gender analyses, the odds ratio (OR) for future CAD in the highest LCAT quartile versus the lowest was 1.00 [confidence interval (CI): 0.76-1.29, P for linearity = 0.902], although opposite trends were observed in men and women. In fact, high LCAT levels were associated with an increased CAD risk in women (unadjusted OR 1.45, CI: 0.94-2.22, P for linearity = 0.036). In contrast to our studies in carriers of LCAT mutations, the current data show that low LCAT plasma levels are not associated with increased atherosclerosis in the general population.

Effects of six APOA5 variants, identified in patients with severe hypertriglyceridemia, on in vitro lipoprotein lipase activity and receptor binding.

OBJECTIVE: The purpose of this study was to identify rare APOA5 variants in 130 severe hypertriglyceridemic patients by sequencing, and to test their functionality, since no patient recall was possible. METHODS AND RESULTS: We studied the impact in vitro on LPL activity and receptor binding of 3 novel heterozygous variants, apoAV-E255G, -G271C, and -H321L, together with the previously reported -G185C, -Q139X, -Q148X, and a novel construct -Delta139 to 147. Using VLDL as a TG-source, compared to wild type, apoAV-G255, -L321 and -C185 showed reduced LPL activation (-25% [P=0.005], -36% [P<0.0001], and -23% [P=0.02]), respectively). ApoAV-C271, -X139, -X148, and Delta139 to 147 had little affect on LPL activity, but apoAV-X139, -X148, and -C271 showed no binding to LDL-family receptors, LR8 or LRP1. Although the G271C proband carried no LPL and APOC2 mutations, the H321L carrier was heterozygous for LPL P207L. The E255G carrier was homozygous for LPL W86G, yet only experienced severe hypertriglyceridemia when pregnant. CONCLUSIONS: The in vitro determined function of these apoAV variants only partly explains the high TG levels seen in carriers. Their occurrence in the homozygous state, coinheritance of LPL variants or common APOA5 TG-raising variant in trans, appears to be essential for their phenotypic expression.

Reduced leucocyte cholesteryl ester transfer protein expression in acute coronary syndromes.

OBJECTIVE: Cholesterol ester transfer protein (CETP) plays an important role in HDL cholesterol metabolism. Leucocytes, including monocyte-derived macrophages in the arterial wall synthesize and secrete CETP, but its role in atherosclerosis is unclear. The aim of the current study was to investigate the effect of acute coronary syndromes (ACS) on leucocyte CETP expression. RESEARCH DESIGN: Peripheral blood mononuclear cells (PBMCs) were freshly isolated from hospitalized ACS patients displaying Braunwald class IIIB unstable angina pectoris (UAP) on admission (t = 0) and at 180 days post inclusion (t = 180) for analysis of CETP expression. In addition, to prove the potential correlation between leucocyte CETP and ACS the effect of acute myocardial infarction on leucocyte CETP expression was studied in CETP transgenic mice. RESULTS: Upon admission, UAP patients displayed approximately 3-6 fold (P < 0.01) lower CETP mRNA and nearly absent CETP protein expression in PBMCs, as compared to healthy age-/sex-matched controls. Interestingly, CETP mRNA and protein levels were significantly elevated in PBMCs isolated from UAP patients (both stabilized and refractory) at t = 180 as compared to t = 0 (P < 0.01), which was correlated with a reduced inflammatory status after medical treatment. In agreement with the data obtained in UAP patients, markedly down-regulated leucocyte CETP mRNA expression was observed after coronary artery ligation in CETP transgenic mice, which also correlated with increased serum amyloid A levels. CONCLUSIONS: We are the first to report that episodes of UAP in humans and myocardial infarction in CETP transgenic mice are associated with reduced leucocyte CETP expression. We propose that the impairment in leucocyte CETP production is associated with an enhanced inflammatory status, which could be clinically relevant for the pathogenesis of ACS.

Pediatric lipid reference values in the general population: The Dutch lifelines cohort study.

BACKGROUND: Atherosclerosis starts in childhood and its progression is influenced by lifelong low-density lipoprotein cholesterol (LDL-c) exposure, the so-called cholesterol burden. Early identification of children and adolescents with severely elevated LDL-c is thus of major clinical significance. This is especially true for children with familial hypercholesterolemia (FH), a frequent but undertreated genetic disorder. To identify children with possible FH, insight in the distribution of lipid levels in children is a prerequisite. OBJECTIVE: To provide health care professionals with contemporary age- and gender-based pediatric reference values for lipid and lipoprotein levels to help the identification of children with dyslipidemia, especially FH. METHODS: Lifelines is a large prospective population-based Dutch cohort study. Children from 8 till 18 years of age were included and fasting lipid levels were measured. Smoothed reference curves and percentiles (5th, 10th, 25th, 50th, 75th, 90th, and 95th) were generated using the Generalized Additive Models for Location, Scale and Shape package in the statistical software R. RESULTS: A total of 8071 children (3823 boys and 4248 girls) were included. In the total cohort we noted marked dynamic changes in lipid and lipoprotein levels over age, which were in part gender specific. Our data highlight a high and unexpected prevalence of severely elevated LDL-c (>190 mg/dL) in both boys and girls. CONCLUSION: Our cross-sectional data provide contemporary reference ranges for plasma lipids that can assist physicians in identifying children at increased risk of premature atherosclerosis, especially FH.

A vascular bed-specific pathway.

The endothelial nitric oxide synthase (eNOS) gene is induced by a variety of extracellular signals under both in vitro and in vivo conditions. To gain insight into the mechanisms underlying environmental regulation of eNos expression, transgenic mice were generated with the 1,600-bp 5' flanking region of the human eNos promoter coupled to the coding region of the LacZ gene. In multiple independent lines of mice, transgene expression was detected within the endothelium of the brain, heart, skeletal muscle, and aorta. beta-galactosidase activity was consistently absent in the vascular beds of the liver, kidney, and spleen. In stable transfection assays of murine endothelial progenitor cells, the 1,600-bp promoter region was selectively induced by conditioned media from cardiac myocytes, skeletal myocytes, and brain astrocytes. Cardiac myocyte-mediated induction was partly abrogated by neutralizing anti-platelet-derived growth factor (PDGF) antibodies. In addition, promoter activity was upregulated by PDGF-AB. Analysis of promoter deletions revealed that a PDGF response element lies between -744 and -1,600 relative to the start site of transcription, whereas a PDGF-independent cardiac myocyte response element is present within the first 166 bp of the 5' flanking region. Taken together, these results suggest that the eNos gene is regulated in the cardiac endothelium by both a PDGF-dependent and PDGF-independent microvascular bed-specific signaling pathway.

An intronic mutation in a lariat branchpoint sequence is a direct cause of an inherited human disorder (fish-eye disease).

The first step in the splicing of an intron from nuclear precursors of mRNA results in the formation of a lariat structure. A distinct intronic nucleotide sequence, known as the branchpoint region, plays a central role in this process. We here describe a point mutation in such a sequence. Three sisters were shown to suffer from fish-eye disease (FED), a disorder which is caused by mutations in the gene coding for lecithin:cholesterol acyltransferase (LCAT). Sequencing of the LCAT gene of all three probands revealed compound heterozygosity for a missense mutation in exon 4 which is reported to underlie the FED phenotype, and a point mutation located in intron 4 (IVS4:T-22C). By performing in vitro expression of LCAT minigenes and reverse transcriptase PCR on mRNA isolated from leukocytes of the patient, this gene defect was shown to cause a null allele as the result of complete intron retention. In conclusion, we demonstrated that a point mutation in a lariat branchpoint consensus sequence causes a null allele in a patient with FED. In addition, our finding illustrates the importance of this sequence for normal human mRNA processing. Finally, this report provides a widely applicable strategy which ensures fast and effective screening for intronic defects that underlie differential gene expression.

A unique genetic and biochemical presentation of fish-eye disease.

This paper describes a novel genetic defect which causes fish-eye disease in four homozygous probands and its biochemical presentation in 34 heterozygous siblings. The male index patient presented with premature coronary artery disease, corneal opacification, HDL deficiency, and a near total loss of plasma lecithin:cholesterol acyltransferase (LCAT) activity. Sequencing of the LCAT gene revealed homozygosity for a novel missense mutation resulting in an Asp131 - Asn (N131D) substitution. Heterozygotes showed a highly significant reduction of HDL-cholesterol and apolipoprotein A-I levels as compared with controls which was associated with a specific decrease of LpA-I:A-II particles. Functional assessment of this mutation revealed loss of specific activity of recombinant LCAT(N131D) against proteoliposomes. Unlike other mutations causing fish-eye disease, recombinant LCAT(N131D) also showed a 75% reduction in specific activity against LDL. These unique biochemical characteristics reveal the heterogeneity of phenotypic expression of LCAT gene defects within a range specified by complete loss of LCAT activity and the specific loss of activity against HDL. The impact of this mutation on HDL levels and HDL subclass distribution may be related to the premature coronary artery disease observed in the male probands.

Cholesteryl ester transfer protein TaqIB variant, high-density lipoprotein cholesterol levels, cardiovascular risk, and efficacy of pravastatin treatment: individual patient meta-analysis of 13,677 subjects.

BACKGROUND: Several studies have reported that the cholesteryl ester transfer protein (CETP) TaqIB gene polymorphism is associated with HDL cholesterol (HDL-C) levels and the risk of coronary artery disease (CAD), but the results are inconsistent. In addition, an interaction has been implicated between this genetic variant and pravastatin treatment, but this has not been confirmed. METHODS AND RESULTS: A meta-analysis was performed on individual patient data from 7 large, population-based studies (each >500 individuals) and 3 randomized, placebo-controlled, pravastatin trials. Linear and logistic regression models were used to assess the relation between TaqIB genotype and HDL-C levels and CAD risk. After adjustment for study, age, sex, smoking, body mass index (BMI), diabetes, LDL-C, use of alcohol, and prevalence of CAD, TaqIB genotype exhibited a highly significant association with HDL-C levels, such that B2B2 individuals had 0.11 mmol/L (0.10 to 0.12, P<0.0001) higher HDL-C levels than did B1B1 individuals. Second, after adjustment for study, sex, age, smoking, BMI, diabetes, systolic blood pressure, LDL-C, and use of alcohol, TaqIB genotype was significantly associated with the risk of CAD (odds ratio=0.78 [0.66 to 0.93]) in B2B2 individuals compared with B1B1 individuals (P for linearity=0.008). Additional adjustment for HDL-C levels rendered a loss of statistical significance (P=0.4). Last, no pharmacogenetic interaction between TaqIB genotype and pravastatin treatment could be demonstrated. CONCLUSIONS: The CETP TaqIB variant is firmly associated with HDL-C plasma levels and as a result, with the risk of CAD. Importantly, this CETP variant does not influence the response to pravastatin therapy.

T-->G or T-->A mutation introduced in the branchpoint consensus sequence of intron 4 of lecithin:cholesterol acyltransferase (LCAT) gene: intron retention causing LCAT deficiency.

Previous mutations associated with lecithin:cholesteryl acyltransferase (LCAT) deficiency syndromes have been identified in the coding regions of the LCAT gene. However, recently, an intron mutation was found in a family in which three sisters presented with fish-eye disease (FED). The probands were shown to be heterozygotes for a mutation in intron 4. The respective T-->C nucleotide substitution, 22 bases upstream of the 3'-splice site, causes a null allele as the result of complete intron retention. Since the natural mutation occurs in a putative branchpoint consensus sequence of the intron, it was hypothesized that the point mutation may disrupt the splicing of the pre-mRNA. To further study the functional significance of the above thymine residue in the branchpoint sequence, we introduced other nucleotides at this position, i.e., LCAT Int-4 MUT-1 (T-->G) and LCAT Int-4 MUT-2 (T-->A). After stable transfection of the mutated pNUT-LCAT minigenes into BHK cells, we could detect neither LCAT activity nor LCAT protein in the culture medium of the pNUT-LCAT Int-4 MUT-1 and pNUT-LCAT Int-4 MUT-2 cell lines, as was previously described for the natural mutation. To determine the effects of the introduced mutations on pre-mRNA splicing, total RNA from transfected BHK cells was used for RT-PCR analysis. All BHK cell lines were shown to transcribe the integrated LCAT minigenes. However, the sizes of these LCAT messengers indicated that intron 4 was retained in the pNUT-LCAT Int-4 MUT-1 and pNUT-LCAT Int-4 MUT-2 cell lines. Subsequent sequence analysis of the RT-PCR products demonstrated that the unspliced intronic sequences contained the introduced mutations. In conclusion, the observed retention of intron 4 of the LCAT gene is the result of the specific loss of a thymine residue two bases upstream of the branchpoint adenosine residue in the putative branchpoint consensus sequence. The results confirm that a single base change in the branchpoint consensus sequence of an intron can cause human disease although this sequence is poorly conserved in mammals.

Increasing high-density lipoprotein cholesterol through cholesteryl Ester transfer protein inhibition: a next step in the fight against cardiovascular disease?

Over the past decades, lowering of LDL-cholesterol (LDL-c) levels has been established as the foundation for preventing atherosclerotic disease. It is, however, widely accepted that additional risk reduction has to come from modifying other risk factors than LDL-c. In this context, increasing HDL-cholesterol (HDL-c) levels by pharmacological inhibition of the cholesteryl ester transfer protein (CETP) is currently under intense investigation. Two small-molecule compounds, JTT-705 and Torcetrapib, have been shown to effectively increase HDL-c levels in humans, without inducing clinically significant side effects when used as monotherapy or combined with statins. Whether this approach will translate into a reduction in risk of atherosclerotic disease has not yet been established. Data from studies focusing on genetic CETP deficiency as well as those studying the relationship between CETP plasma levels and risk of atherosclerosis do not provide clear answers. Several long-term clinical studies addressing this crucial issue have recently been initiated, results of which will follow within the next few years. This review focuses on CETP, its role in human lipid metabolism and its relation to atherosclerotic disease. Furthermore, it summarizes the currently available data regarding pharmacological CETP inhibition. Finally, it will highlight a number of issues basic to the considerations of whether CETP inhibition will fulfill its promises.

Gene therapy for genetic lipoprotein lipase deficiency: from promise to practice.

Lipoprotein lipase (LPL) deficiency is a rare, hereditary disorder of lipoprotein metabolism characterised by severely increased triglyceride levels, and associated with an increased risk for pancreatitis. Since no adequate treatment modality is available for this disorder, we set out to develop an LPL gene therapy protocol. This paper focuses on the clinical presentation of LPL deficiency, summarises the preclinical investigations in animal models and describes the rationale to evaluate gene therapy for this monogenetic disorder of lipid metabolism in humans.

Adherence to guidelines to prevent cardiovascular diseases: The LifeLines cohort study.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death worldwide. While there is indisputable evidence that statin treatment reduces the burden of CVD, undertreatment remains a concern for primary and secondary prevention. The aim of this study was to assess the use of lipid-lowering drugs (LLD) among 70,292 individuals in the Netherlands as a proxy of adherence to the national guideline for prevention and treatment of CVD. METHODS: LifeLines is a population-based prospective cohort study in the three Northern provinces of the Netherlands. At baseline, all participants completed questionnaires, and underwent a physical examination and lab testing. The national guidelines were used to assess how many participants were eligible for LLD prescription and we analysed how many indeed reported LLD use. RESULTS: For primary prevention, 77% (2515 of 3268) of those eligible for LLD treatment did not report using these drugs, while for secondary prevention this was 31% (403 of 1302). Patients with diabetes mellitus were treated best (67%) for primary prevention. Notably, of the patients with stroke, only 47% (182 of 386) reported LLD treatment. CONCLUSION: Despite clear guidelines and multiple national initiatives to improve CVD risk management, adherence to guidelines for the treatment of CVD in the Netherlands remains a major challenge. This study calls out for improving public awareness of CVD and to improve primary and secondary prevention to prevent unnecessary CVD-related morbidity and mortality.

Targeting the Hprt locus in mice reveals differential regulation of Tie2 gene expression in the endothelium.

To study the in vivo expression of the murine Tie2 gene, we have targeted the hypoxanthine phosphoribosyltransferase (Hprt) gene locus to generate two single-copy transgenic mice: T1, containing the 2,100-bp Tie2 promoter upstream from the beta-galactosidase (LacZ) gene, and T5, which also included an enhancing element originating from the first intron of the Tie2 gene. Comparing T1 and T5 embryos at day E10.5 revealed differential endothelial cell-specific expression of LacZ, whereas colocalization analyses showed that the expression was confined to endothelial cells. Moderate reporter gene activity was observed in the brain and kidney of T1 adults, whereas extensive LacZ gene expression was seen in the vasculature of most organs of the T5 adults. This study demonstrates the feasibility of targeting the Hprt locus with endothelial cell-specific sequences to analyze the spatial-temporal expression of transgenes. Of particular importance is the observation that the analysis of a single transgene copy in a defined locus allows for an accurate and rapid comparison of transcriptional activity among regulatory DNA sequences.

Targeting of human eNOS promoter to the Hprt locus of mice leads to tissue-restricted transgene expression.

Phenotypic heterogeneity of the endothelium arises from cell type-specific differences in gene expression. An understanding of the mechanisms that underlie differential gene expression would provide important insight into the molecular basis of vascular diversity. In standard transgenic assays, multiple copies of heterologous DNA cassettes are randomly integrated into the mouse genome, resulting in significant line-to-line variation in expression. To overcome these limitations, we have targeted a single copy of a transgene that contains 1,600 bp of the human endothelial nitric oxide synthase (eNOS) promoter coupled to the LacZ reporter gene to the X-linked hypoxanthine phosphoribosyltransferase (Hprt) locus of mice by homologous recombination. The transgene was inserted in either of the orientations relative to that of the Hprt gene. In mice derived from multiple embryonic stem (ES) cell clones, the expression pattern was limited to a subset of endothelial cells, cardiomyocytes, and vascular smooth muscle cells. These findings suggest that Hprt locus targeting is a feasible tool for studying endothelial cell-restricted gene regulation.

Lipid-free apolipoprotein (apo) A-I is converted into alpha-migrating high density lipoproteins by lipoprotein-depleted plasma of normolipidemic donors and apo A-I-deficient patients but not of Tangier disease patients.

Plasma of patients with Tangier disease (TD) is devoid of alpha-LpA-I (apolipoprotein A-I-containing lipoprotein), which in normolipidemic plasma constitutes the majority of high density lipoprotein (HDL). The residual amounts of apolipoprotein A-I (apo A-I) in TD plasma have electrophoretic prebeta1-LpA-I mobility. We have previously demonstrated that TD plasma does not convert prebeta1-LpA-I into alpha-LpA-I. In this study we found that plasmas of normolipidemic controls, apo A-I-deficient patients and patients with fish-eye disease, but not plasmas of six TD patients, convert biotinylated lipid-free apo A-I into alpha-LpA-I. Supplementation of plasma with free oleic acid or fatty acid free albumin neither inhibited conversion activity in normal plasmas nor reconstituted it in TD plasma. In normal plasma the conversion activity was assessed in HDL and in the lipoprotein-free fraction. The latter fraction, however, generated larger particles only in the presence of exogenous phospholipid vesicles. To obtain particles with alpha-mobility, these vesicles had to contain phosphatidylinositol and/or cholesterol. Lipoprotein-depleted TD plasma did not convert lipid-free apo A-I into alpha-LpA-I even in the presence of exogenous vesicles with phospholipids or cholesterol. Taken together we conclude that disturbed transfer of glycerophospholipds onto apo A-I or prebeta1-LpA-I prevents maturation of HDL and thereby possibly causes deficiency of HDL cholesterol in patients with TD. Moreover, the lack of alpha-LpA-I in TD plasma together with its failure to convert exogenous apo A-I into an alpha-migrating particle provide specific tests for the diagnosis of TD.

Parental history of myocardial infarction: lipid traits, gene polymorphisms and lifestyle.

To investigate the relationship between parental history of myocardial infarction (MI), lipid traits and gene polymorphisms involved in lipid metabolism, we examined Dutch men and women, who were selected from a large population-based study. Subjects whose father (n=112), mother (n=115) or both parents (n=115) suffered from a premature MI presented with significantly higher apolipoprotein B (apo B) levels than subjects without a parental history (n=114). Genetic analyses revealed that the apo E4 isoform and the D9N mutation of lipoprotein lipase (LPL) were more frequent among subjects with a parental history (P< or =0.05). A similar trend was found for the LPL N291S mutation. In contrast, the LPL S447X mutation and polymorphisms at the cholesteryl ester transfer protein (TaqIB) and apo CIII (SstI) loci proved to be noninformative. Body mass index and lifestyle could not explain differences in apo B levels between parental history groups. In contrast, the apo E polymorphism and the LPL D9N mutation accounted for some, but not all, of the higher apo B levels in subjects with a parental history. Therefore, other genetic or lifestyle-related factors must be responsible for the increased levels of apo B in individuals with a family history of myocardial infarction.

Heterozygosity for ABCA1 gene mutations: effects on enzymes, apolipoproteins and lipoprotein particle size.

A cohort of 13 female and 14 male heterozygotes for ATP binding cassette A1 (ABCA1) gene defects was directly compared with 13 and 14 unaffected female and male family members of almost exact same age. The activities of three proteins that play key roles in HDL metabolism were measured in addition to extensive lipid and (apo) lipoprotein subfraction analysis. Compared to controls, LCAT activity was reduced by 15% in affected subjects (P < 0.001) while PLTP activity was unaffected. Interestingly, CETP activity was elevated by 50% in the heterozygote siblings of one kindred but was unaffected in heterozygotes of the three other families. With respect to lipids, the heterozygotes had normal total cholesterol (TC), and LDL-cholesterol concentrations but presented with a trend towards increased triglyceride levels (13%; P = 0.08). HDL metabolism, by contrast, was severely affected as illustrated by 40% reductions in HDL-cholesterol (P < 0.001) with concomitant reductions in apoAI (25%; P < 0.001) levels and in lipoprotein subfraction LpAI (28%; P < 0.001), LpAI:AII (24%; P=0.014), and LpCIII:nonB (34%; P < 0.001) concentrations. We furthermore observed reduced average HDL particle size (5%; P = 0.004; 16% in female and 3.6% in male) and reduced plasma apoCIII concentration (15%; P = 0.006) while apoAII, apoAIV, apoE and apoB levels were unchanged. In conclusion, heterozygosity for ABCA1 defects was associated with reduced LCAT activity in absence of effects on PLTP activity. Of special interest was our finding that the effects of compromised ABCA1 function on HDL were more pronounced in women than in men.