Contribution of the hepatic lipase gene to the atherogenic lipoprotein phenotype in familial combined hyperlipidemia

Familial combined hyperlipidemia (FCH) is a common genetic lipid disorder with a frequency of 1-2% in the population. In addition to the hypercholesterolemia and/or hypertriglyceridemia that affected individuals exhibit, small, dense LDL particles and decreased HDL-cholesterol levels are traits frequently associated with FCH. Recently, we reported that families with FCH and families enriched for coronary artery disease (CAD) share genetic determinants for the atherogenic lipoprotein phenotype (ALP), a profile presenting with small, dense LDL particles, decreased HDL-cholesterol levels, and increased triglyceride levels. Other studies in normolipidemic populations have shown that the hepatic lipase (HL) gene is linked to HDL-cholesterol levels and that a polymorphism within the HL promoter (-514C-->T) is associated with increased HDL-cholesterol levels as well as larger, more buoyant LDL particles. In the present study, we tested whether the HL gene locus also contributes to ALP in a series of Dutch FCH families using nonparametric sibpair linkage analysis and association analysis. Evidence for linkage of LDL particle size (P < 0.019), HDL-cholesterol (P < 0.003), and triglyceride levels (P < 0.026) to the HL gene locus was observed. A genome scan in a subset of these families exhibited evidence for linkage of PPD (LOD = 2.2) and HDL-cholesterol levels (LOD = 1.2) to the HL gene locus as well. The -514C-->T promoter polymorphism was significantly associated (P < 0.0001) with higher HDL-cholesterol levels in the unrelated males of this population, but not in unrelated females. No association was observed between the polymorphism and LDL particle size or triglyceride levels. Our results provide support that ALP is a multigenic trait and suggest that the relationship between small, dense LDL particles, HDL-cholesterol, and triglyceride levels in FCH families is due, in part, to common genetic factors.     

Multilocus genetic determinants of LDL particle size in coronary artery disease families.

Recent interest in atherosclerosis has focused on the genetic determinants of low-density lipoprotein (LDL) particle size, because of (i) the association of small dense LDL particles with a three-fold increased risk for coronary artery disease (CAD) and (ii) the recent report of linkage of the trait to the LDL receptor (chromosome 19). By utilizing nonparametric quantitative sib-pair and relative-pair analysis methods in CAD families, we tested for linkage of a gene or genes controlling LDL particle sizes with the genetic loci for the major apolipoproteins and enzymes participating in lipoprotein metabolism. We confirmed evidence for linkage to the LDL receptor locus (P=.008). For six candidate gene loci, including apolipoprotein(apo)B, apoAII, apo(a), apoE-CI-CII, lipoprotein lipase, and high-density lipoprotein-binding protein, no evidence for linkage was observed by sib-pair linkage analyses (P values ranged from .24 to .81). However, in addition, we did find tentative evidence for linkage with the apoAI-CIII-AIV locus (chromosome 11) (P=.06) and significant evidence for linkage of the cholesteryl ester transfer protein locus (chromosome 16) (P=.01) and the manganese superoxide dismutase locus (chromosome 6) (P=.001), thus indicating multilocus determination of this atherogenic trait.     

New genetic variants in the apoA-I and apoC-III genes and familial combined hyperlipidemia

Linkage and association between the apolipoprotein (apo) A-I/C-III/A-IV gene region on chromosome 11 and familial combined hyperlipidemia (FCHL) has been observed previously. Using sequence analysis two new allelic variants were identified, C(317) -T in intron 2 of the apoA-I gene and C(1100)-T in exon 3 of the apoC-III gene. These variants were studied in 30 FCHL probands, 159 hyperlipidemic relatives, 327 normolipidemic relatives, and 218 spouses. The allele frequencies of both variants were significantly different in probands and spouses (P < 0.002 and P < 0.001, respectively), with increased frequency of the minor alleles in the probands. The minor genotypes (TT) were associated with elevated plasma triglyceride and apoC-III. Both variants were in strong, although not complete, linkage disequilibrium with each other and with the MspI site in the promoter region of the apoA-I gene and the SstI site in the 3' untranslated region of exon 4 of the apoC-III gene. Haplotypes based on these four variants were constructed and the distributions of haplotype combinations were significantly different (P < 0.0001). Two distinct haplotypes predisposing to FCHL were found: 2-2-1-2 and 1-2-2-2 (MspI, C(317) -T; SstI, C(1100)-T). The haplotype combinations carrying one of these high risk alleles are associated with elevated lipid levels in probands and in spouses. While these effects can be attributed to the presence of the M2 and S2 minor alleles, these results suggest that the importance of specific allelic haplotypes may be greater than single genotypic effects.     

Genetic variation in lipoprotein (a) levels in families enriched for coronary artery disease is determined almost entirely by the apolipoprotein (a) gene locus.

Lipoprotein (a) (Lp[a]) is a cholesterol-rich lipoprotein resembling LDL but also containing a large polypeptide designated apolipoprotein (a) (apo[a]). Its levels are highly variable among individuals and, in a number of studies, are strongly correlated with the risk of coronary artery disease (CAD). In an effort to determine which genes control Lp(a) levels, we have studied 25 multiplex families (comprising 298 members) enriched for CAD. The apo(a) gene was genotyped among the families, using a highly informative pulse-field gel electrophoresis procedure. In addition, polymorphisms of the gene for the other major protein of Lp(a), apolipoprotein B (apoB), were examined. Quantitative sib-pair linkage analysis indicates that apo(a) is the major gene controlling Lp(a) levels in this CAD population (P = .001; 99 sib pairs), whereas the apoB gene demonstrated no significant quantitative linkage effect. We estimate that the apo(a) locus accounts for < or = 98% of variance of Lp(a) serum levels. Approximately 43% of this variation is explained by size polymorphisms within the apo(a) gene. These results indicate that the apo(a) gene is the major determinant of Lp(a) serum levels not only in the general population but also in a high-risk CAD population.     

Inherited susceptibility determines the distribution of dense low-density lipoprotein subfraction profiles in familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCH) is a heritable lipid disorder, in which dense low-density lipoprotein (LDL) subfraction profiles due to a predominance of small dense LDL particles are frequently observed. These small dense LDL particles are associated with cardiovascular disease. Using segregation analysis, we investigated to what extent these LDL subfraction profiles are genetically determined; also, the mode of inheritance was studied. Individual LDL subfraction profiles were determined by density gradient ultracentrifugation in 623 individuals of 40 well-defined Dutch FCH families. The individual LDL subfraction profile was defined as a quantitative trait by the continuous variable K, a reliable estimate of the relative contribution of each LDL subfraction to the overall profile. Variation in parameter K due to age, sex, and hormonal status was taken into account by introducing liability classes. Segregation analysis was performed by fitting a series of class D regressive models, implemented in the Statistical Analysis for Genetic Epidemiology (SAGE) program, after which genetic models were compared using log-likelihood ratio tests. Our data show that 60% of the variability of parameter K could be explained by lipid and lipoprotein levels and that a major autosomal locus, recessively inherited, with a population frequency of .42 +/- .07, and an additional polygenic component of .25 best explained the clustering of atherogenic dense LDL subfraction profiles in these FCH families. Therefore, dense LDL subfraction profiles, associated with elevated lipid levels, appear to have a genetic basis in FCH.     

Strategies for the assessment of genetic coronary artery disease risk.

In atherosclerotic diseases, genetic factors have a substantial influence on the age of onset and the frequency and severity of clinical symptoms, as well as response to therapy. In myocardial infarctions occurring at young age, genetics may be the leading causative factor. Despite such a prominent role of genetics in the pathophysiology of atherosclerosis clinical risk assessment and therapeutic decision making are still based on classical risk factors. In this paper we analyse the reasons for the current lack of predictive power of genetics-based algorithms and we speculate why future developments might open the door to a role for genetics in the clinical management of atherosclerosis.     

Postprandial triglyceride levels in familial combined hyperlipidemia. The role of apolipoprotein E and lipoprotein lipase polymorphisms

The effect of apolipoprotein E genotype and polymorphisms of lipoprotein lipase gene on plasma postprandial triglyceride levels in familial combined hyperlipidemic subjects and their relatives have not been sufficiently studied. This study included sixteen familial combined hyperlipidemic parents (G1): age: 52 +/- 9 years with total-cholesterol: 7.2 +/- 1.7 mmol/L, fasting triglycerides: 2.8 +/- 1.4 mmol/L and sixteen children (G2) (twelve were normolipidemic): of age: 22 +/- 5 years with total-cholesterol: 5.2 +/- 1.1 mmol/L, fasting triglycerides: 2.06 +/- 1.8 mmol/L and twelve normolipidemic, healthy controls. Blood samples were taken fasting and 2, 4, 6, 8, 10 hr postprandially after the standard fat rich test meal. We determined lipid parameters, apolipoprotein E and lipoprotein lipase HindIII and PvuII polymorphisms as well. The 6-hr critical postprandial triglyceride values were abnormal in both G1: 5.88 +/- 2.7 mmol/L and G2: 3.53 +/- 2.7 mmol/L (p <0.001), respectively, and differed significantly (p <0.001) from each other. The subjects of familial combined hyperlipidemic families with E4 allele in both generations exhibited significantly (p <0.001) higher and extended postprandial lipemia. We did not find significant effects of lipoprotein lipase HindIII or PvuII polymorphisms on the fasting lipid values alone, however in normolipidemic subjects from the same families the homozygosity of HindIII variation was associated with higher triglyceride postprandial peak (p <0.01). The main findings of our study are that i.) normolipidemic G2 subjects in familial combined hyperlipidemic families have already abnormal postprandial status, and ii.) the 6 h postprandial triglyceride values were correlated with fasting triglyceride levels, which showed association with the apolipoprotein E4 allele.     

The effect of hepatic lipase on coronary artery disease in humans is influenced by the underlying lipoprotein phenotype

Increased or decreased hepatic lipase (HL) activity has been associated with coronary artery disease (CAD). This is consistent with the findings that gene variants that influence HL activity were associated with increased CAD risk in some population studies but not in others. In this review, we will explain the conditions that influence the effects of HL on CAD. Increased HL is associated with smaller and denser LDL (sdLDL) and HDL (HDL₃) particles, while decreased HL is associated with larger and more buoyant LDL and HDL particles. The effect of HL activity on CAD risk is dependent on the underlying lipoprotein phenotype or disorder. Central obesity with hypertriglyceridemia (HTG) is associated with high HL activity that leads to the formation of sdLDL that is pro-atherogenic. In the absence of HTG, where large buoyant cholesteryl ester-enriched LDL is prominent, elevation of HL does not raise the risk for CAD. In HTG patients, drug therapy that decreases HL activity selectively decreases sdLDL particles, an anti-atherogenic effect. Drug therapy that raises HDL₂ cholesterol has not decreased the risk for CAD. In trials where inhibition of cholesterol ester transfer protein (CETP) or HL occurs, the increase in HDL₂ most likely is due to inhibition of catabolism of HDL₂ and impairment of reverse cholesterol transport (RCT). In patients with isolated hypercholesterolemia, but with normal triglyceride levels and big-buoyant LDL particles, an increase in HL activity is beneficial; possibly because it increases RCT. Drugs that lower HL activity might decrease the risk for CAD only in hypertriglyceridemic patients with sdLDL by selectively clearing sdLDL particles from plasma, which would override the potentially pro-atherogenic effect on RCT. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Remnant-like lipoprotein particles as risk factors for coronary artery disease in elderly patients.

Although remnant-like lipoprotein particles (RLPs) are known to be atherogenic, the relationship between serum RLP-cholesterol (RLP-C) level and coronary artery disease (CAD) has not as yet been evaluated. This clinical study was aimed at investigating the pathological significance of serum RLP-C among several coronary risk factors with a clear focus on elderly patients. We took fasting venous blood samples to determine lipid profiles including RLP-C from 188 patients with angiographically identified CAD and 68 control patients. Overall analysis showed that the RLP-C/HDL-C ratio was higher in both single-vessel CAD group (n = 67; p < 0.01) and multi-vessel CAD group (n = 121; p < 0.001) compared to controls. Further, multiple logistic regression analysis indicated that the diabetes, HDL-C and the RLP-C/HDL-C ratio could discriminate CAD patients from controls. In patients younger than 65 years, diabetes, HDL-C, LDL-C and the LDL-C/HDL-C ratio as well as the RLP-C/HDL-C ratio could discriminate CAD. In patients 65 aged years or older, however, diabetes, triglyceride and RLP-C as well as the RLP-C/HDL-C ratio could discriminate CAD, whereas LDL-C and the LDL-C/HDL-C ratio could not. These results led us to believe that the contribution of a given risk factor to the development of CAD in elderly patients may be different from that in younger patients. In elderly patients, RLP-C rather than LDL-C was strongly associated with the development of CAD. Accordingly, serum RLP-C levels may serve as a convenient and reliable index for assessing CAD.     

Molecular mechanisms,prevalence, and molecular methods for familial combined hyperlipidemia disease: A review

Familial combined hyperlipidemia (FCHL) is the most common genetic dyslipidemia disorder which is accompanied by increasing of triglyceride and cholesterol. This disorder is a complex genetic disease although it also has monogenic forms. The familial form has several criteria for diagnosis that can be distinguished of nonfamilial position. It has been shown that a variety of internal and external risk factors are involved in the pathogenesis of FCHL. Environmental factors and the genetic background also play an important role in the FCHL pathogenesis. Many mechanisms and pathways are involved in lipid metabolism (ie, dysfunctional adipose tissue, hepatic fat and very low-density lipoprotein overproduction, triglyceride-rich lipoproteins, and clearance of low-density lipoprotein particles) that could lead to FCHL. Individuals with a positive family history like those who have a positive family history of cardiovascular diseases are more predispositions for this disorder. To date several methods have been used to identify the genetic background of the FCHL. In the current review, we summarized the prevalence and the molecular mechanisms involved in the FCHL disease. Moreover, we highlighted the used molecular methods for determining the genes involved in the FCHL.     

Mapping the genetic determinants of pathogenicity and plaque phenotype in swine vesicular disease virus

A series of recombinant viruses were constructed using infectious cDNA clones of the virulent J1'73 (large plaque phenotype) and the avirulent H/3'76 (small plaque phenotype) strains of swine vesicular disease virus to identify the genetic determinants of pathogenicity and plaque phenotype. Both traits could be mapped to the region between nucleotides (nt) 2233 and 3368 corresponding to the C terminus of VP3, the whole of VP1, and the N terminus of 2A. In this region, there are eight nucleotide differences leading to amino acid changes between the J1'73 and the H/3'76 strains. Site-directed mutagenesis of individual nucleotides from the virulent to the avirulent genotype and vice versa indicated that A at nt 2832, encoding glycine at VP1-132, and G at nt 3355, encoding arginine at 2APRO-20, correlated with a large-plaque phenotype and virulence in pigs, irrespective of the origin of the remainder of the genome. Of these two sites, 2APRO-20 appeared to be the dominant determinant for the large-plaque phenotype but further studies are required to elucidate their relative importance for virulence in pigs.     

A common genetic mechanism determines plasma apolipoprotein B levels and dense LDL subfraction distribution in familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCH) is a common lipid disorder characterized by elevations of plasma cholesterol and/or triglyceride in first-degree relatives. A predominance of small, dense LDL particles and elevated apolipoprotein B (apoB) levels is commonly found in members of FCH families. Many studies have investigated the genetic mechanisms determining individuals' lipid levels, in FCH families. Previously, we demonstrated a major gene effect on LDL particle size and codominant Mendelian inheritance involved in determination of apoB levels in a sample of 40 well-defined FCH families. An elevation of apoB levels is associated metabolically with a predominance of small, dense LDL particles in FCH. To establish whether a common gene regulates both traits, we conducted a bivariate genetic analysis to test the hypothesis of a common genetic mechanism. In this study, we found that 66% of the total phenotypic correlation is due to shared genetic components. Further bivariate segregation analysis suggested that both traits share a common major gene plus individual polygenic components. This common major gene explains 37% of the variance of adjusted LDL particle size and 23% of the variance of adjusted apoB levels. Our study suggests that a major gene that has pleiotropic effects on LDL particle size and apoB levels may be the gene underlying FCH in the families we studied.     

Relation of angiographically defined coronary artery disease to plasma lipoprotein subfractions and apolipoproteins.

The relation of coronary artery disease to plasma lipoproteins was examined in 104 men aged 35-65 years undergoing coronary angiography for suspected myocardial ischaemia. A score reflecting the number, degree, and length of stenoses in seven major coronary arteries was assigned to each angiogram. Lipid concentrations in lipoprotein subfractions were measured after preparative ultracentrifugation; plasma apolipoprotein concentrations were measured by electroimmunoassay. Men with high coronary scores tended to have lower plasma high-density lipoprotein (HDL) cholesterol concentrations and higher low-density lipoprotein (density 1.019-1.063 g/ml) cholesterol concentrations than subjects of similar age with low coronary scores (p approximately equal to 0.1). The strongest relation, however, was with the cholesterol concentration in the HDL2 subfraction (density 1.063-1.125 g/ml) of HDL, which averaged 44% lower in the severely affected patients (p less than 0.005). No associations were found between the coronary score and HDL3 cholesterol, the cholesterol content of lipoproteins of density less than 1.019 g/ml, plasma triglyceride, or the concentrations of apolipoproteins AI, AII, and E. The high coronary scores associated with low HDL2 concentrations reflected an increase in the number of both partial and complete stenoses distributed throughout the coronary tree. In contrast the sizes of the lesions and the proportion producing complete occlusion were unrelated to HDL2.     

Association of two genetic variations of lipoprotein lipase, S447X and Hind III, with coronary artery disease and hypertriglyceridemia

This study was performed to assess the effect of the S447X and Hind III lipoprotein lipase gene polymorphisms on development of coronary artery disease and hypertriglyceridemia. The study included 132 patients and 98 healthy control subjects of Croatian descent. The lipoprotein lipase S447X polymorphism was associated with coronary artery disease and hypertriglyceridemia, as indicated by the lower frequency of S447 allele in the patient group (p = 0.005) and odds ratio (O.R = 0.40, p = 0.006). The patient and control groups also showed a significant difference in the distribution of Hind III/S447X genotype combinations (p = 0.013). There were no significant associations with lipid parameters for any genotype or genotype combination in the patient group. Frequencies of the S447X polymorphism and S447X/Hind III combinations differed between the CAD/TG and control group, thus these polymorphisms may be associated with CAD and hypertriglyceridemia.     

A genome scan for familial combined hyperlipidemia reveals evidence of linkage with a locus on chromosome 11.

Familial combined hyperlipidemia (FCHL) is a common familial lipid disorder characterized by a variable pattern of elevated levels of plasma cholesterol and/or triglycerides. It is present in 10%-20% of patients with premature coronary heart disease. The genetic etiology of the disease, including the number of genes involved and the magnitude of their effects, is unknown. Using a subset of 35 Dutch families ascertained for FCHL, we screened the genome, with a panel of 399 genetic markers, for chromosomal regions linked to genes contributing to FCHL. The results were analyzed by use of parametric-linkage methods in a two-stage study design. Four loci, on chromosomes 2p, 11p, 16q, and 19q, exhibited suggestive evidence for linkage with FCHL (LOD scores of 1.3-2.6). Markers within each of these regions were then examined in the original sample and in additional Dutch families with FCHL. The locus on chromosome 2 failed to show evidence for linkage, and the loci on chromosome 16q and 19q yielded only equivocal or suggestive evidence for linkage. However, one locus, near marker D11S1324 on the short arm of human chromosome 11, continued to show evidence for linkage with FCHL, in the second stage of this design. This region does not contain any strong candidate genes. These results provide evidence for a candidate chromosomal region for FCHL and support the concept that FCHL is complex and heterogeneous.     

A genomewide scan for early-onset coronary artery disease in 438 families: the GENECARD Study

A family history of coronary artery disease (CAD), especially when the disease occurs at a young age, is a potent risk factor for CAD. DNA collection in families in which two or more siblings are affected at an early age allows identification of genetic factors for CAD by linkage analysis. We performed a genomewide scan in 1,168 individuals from 438 families, including 493 affected sibling pairs with documented onset of CAD before 51 years of age in men and before 56 years of age in women. We prospectively defined three phenotypic subsets of families: (1) acute coronary syndrome in two or more siblings; (2) absence of type 2 diabetes in all affected siblings; and (3) atherogenic dyslipidemia in any one sibling. Genotypes were analyzed for 395 microsatellite markers. Regions were defined as providing evidence for linkage if they provided parametric two-point LOD scores >1.5, together with nonparametric multipoint LOD scores >1.0. Regions on chromosomes 3q13 (multipoint LOD = 3.3; empirical P value <.001) and 5q31 (multipoint LOD = 1.4; empirical P value <.081) met these criteria in the entire data set, and regions on chromosomes 1q25, 3q13, 7p14, and 19p13 met these criteria in one or more of the subsets. Two regions, 3q13 and 1q25, met the criteria for genomewide significance. We have identified a region on chromosome 3q13 that is linked to early-onset CAD, as well as additional regions of interest that will require further analysis. These data provide initial areas of the human genome where further investigation may reveal susceptibility genes for early-onset CAD.     

Classical determinants of coronary artery disease as predictors of complexity of coronary lesions,assessed with the SYNTAX score

Background

We need new biomarkers that can predict cardiovascular disease to improve both diagnosis and therapeutic strategies. The CIRCULATING CELLS study was designed to study the role of several cellular mediators of atherosclerosis as biomarkers of coronary artery disease (CAD). An objective and reproducible method for the quantification of CAD extension is required to establish relationships with these potential biomarkers. We sought to analyse the correlation of the SYNTAX score with known CAD risk factors to test it as a valid marker of CAD extension.

Methods and results

A subgroup of 279 patients (67.4% males) were included in our analysis. Main exclusion criteria were a history of previous percutaneous coronary intervention or surgical revascularisation that prevent an accurate assessment of the SS. Diabetes mellitus, smoking, renal insufficiency, body mass index and a history of CAD and myocardial infarction were all positively and strongly associated with a higher SYNTAX score after adjustment for the non-modifiable biological factors (age and sex). In the multivariate model, age and male sex, along with smoking and renal insufficiency, remain statistical significantly associated with the SYNTAX score.

Conclusion

In a selected cohort of revascularisation-naive patients with CAD undergoing coronary angiography, non-modifiable cardiovascular risk factors such as advanced age, male sex, as well as smoking and renal failure were independently associated with CAD complexity assessed by the SYNTAX score. The SYNTAX score may be a valid marker of CAD extension to establish relationships with potential novel biomarkers of coronary atherosclerosis.