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Lipoprotein lipase (LPL) plays a pivotal role in lipid metabolism by hydrolyzing triglyceride (TG)-rich lipoprotein particles. Abnormalities in normal LPL function are associated with the risk of coronary artery disease (CAD). A number of genetic variants have been identified in the LPL gene that affects different functions of the LPL protein. A common HindIII polymorphism in intron 8 (T/G) of the LPL gene has been found to be associated with altered plasma TG and HDL-cholesterol, and CAD risk in several studies, but its functional significance is unknown. It has been shown that certain intronic sequence contain regulatory elements that are important for transcription and translational regulation of a gene. In this study we tested the hypothesis that this polymorphism affects the binding site of a transcription factor that regulates the transcription of LPL gene. Electrophoretic mobility shift assays (EMSAs) revealed that the HindIII site binds to a transcription factor and that the mutant allele has lower binding affinity than the wild type allele. Transcription assays containing the entire intron 8 sequence along with full-length human LPL promoter were carried out in COS-1 and human vascular smooth muscle cells. The mutant allele was associated with significantly decreased luciferase expression level compared to the wild type allele in both the muscle (3.394+/-0.022 vs. 4.184+/-0.028; P=4.7 x 10(-6)) and COS-1 (11.603+/-0.409 vs. 14.373+/-1.096; P<0.0001) cells. In conclusion, this study demonstrates for the first time that the polymorphic HindIII site in the LPL gene is functional because it affects the binding of a transcription factor and it also has an impact on LPL expression.

Genetic variation in the apolipoprotein E (APOE) gene is a significant determinant of variation in plasma cholesterol levels and it also affects the risk of coronary artery disease (CAD). We examined the association of the APOE polymorphism with CAD severity in women from the NHLBI-sponsored Women's Ischemia Syndrome Evaluation (WISE) study. Quantitative coronary angiography was used to classify subjects as having normal/minimal CAD (<20% stenosis), mild CAD (20-49% stenosis) and significant CAD (>or=50% stenosis). The women with or=50% stenosis were further stratified according to the number of vessel disease they have (one, two, or three). In white subjects, the frequency of APOE*4 carriers (3/4 and 4/4 genotypes) was significantly higher in the combined mild/significant CAD group (>or=20% stenosis) compared with the normal/minimal CAD group (<20% stenosis) (31.3 vs. 19.2%; P=0.025) with an adjusted OR of 2.40 (95% CI: 1.47-3.93; P=0.0005). Furthermore, the APOE*4 allele was found to be significantly associated with the increased vessel disease number (chi(2)=8.04; P=0.0046). This association of the APOE*4 allele with CAD severity was present only in women with family history of CAD. APOE polymorphism also showed significant associations with increasing plasma total cholesterol (P=0.01) and low-density lipoprotein (LDL)-cholesterol (P<0.001) in whites. These data support the hypothesis that the APOE*4 allele is an independent risk factor not only for the presence of CAD and hyperlipidemia, but also for the angiographic severity of CAD in white women with a family history of disease.

Apolipoprotein D (APOD, gene; apoD, protein) is a plasma high-density lipoprotein (HDL)-associated glycoprotein, with a putative role in the cholesterol (CHOL) transport pathway. An apoD protein polymorphism has been previously reported by us. The cathodically shifted pattern seen on isoelectric focusing gels, controlled by the APOD*2 allele, was found to be unique to populations of African ancestry. To characterize the molecular basis of the protein polymorphism and to identify new mutations, we used a combination of SSCP, DHPLC and DNA sequencing techniques to screen the entire coding region of the APOD gene. We identified three distinct missense mutations, including Phe36Val, Tyr108Cys, and Thr158Lys with frequencies ranging from 2.1 to 2.8% in 722 African blacks from Nigeria. In addition, a common 8 bp deletion polymorphism was observed in intron 1 with a carrier frequency of 30.1%. The missense mutation, Thr158Lys correlated with the APOD*2 allele of the protein polymorphism. None of the 454 Caucasians screened for these polymorphisms showed any variation. We also determined the effect of these polymorphisms on plasma lipid levels in the African black population by generalized linear model (GLM). The Val36 allele was associated with significantly decreased HDL3-C (P=0.027) and apoA-I (P=0.030) levels among females. The Lys158 allele was associated with significantly increased Lp(a) (P=0.018) and triglyceride (P=0.017) levels, among females and males, respectively. In addition, males heterozygous for both intron 1 and codon 108 polymorphisms showed significantly increased HDL-C (P=0.011), HDL3-C (P=0.041), HDL2-C (P=0.009), apoA-I (P=0.005) and decreased LDL-C (P=0.025) levels. The results of our study show that the APOD gene harbors several polymorphisms, which are unique to African populations. Further study of these polymorphisms may help to characterize the role of apoD in lipid metabolism, and in cardiovascular disease among African populations.

Human apolipoprotein C-IV (apoC-IV, protein; APOC4, gene) is a new member of the APO E/C1/C2 gene cluster. In transgenic mice, human apoC-IV is predominantly associated with very low-density lipoprotein (VLDL) and thus may play an important role in lipid metabolism. To our knowledge, the extent and nature of APOC4 genetic variation and its role in lipid metabolism are unknown. In this study we have assessed the presence of genetic variation in all three exons of APOC4 and their flanking intronic sequence by SSCP and DNA sequencing. A total of five point mutations were observed, including two in the non-coding part of exon 1 (A609G and G620A), two in exon 2 (codons 36 and 52) and one in exon 3 (codon 96). The three mutations in exons 2 and 3 predict amino acid substitutions, Leu36Pro, Gly52Asp, and Leu96Arg. The frequencies of the variant alleles were: 0.010 for 609G, 0.039 for 620A, 0.502 for Pro36, 0.003 for Asp52 and 0.357 for Arg96. Significant pairwise linkage disequilibrium was observed between five of the ten APOC4 pairs, including nt. 620/codon 36, nt. 620/codon 96, codon 36/codon 52, codon 36/codon 96 and codon 52/codon 96. A general linear model analysis reveled a significant association of the Leu36Pro and the Leu96Arg polymorphisms with triglyceride levels in women. This is consistent with the proposed role of apoC-IV in triglyceride metabolism. The characterization of APOC4 genetic variation may lead to the identification of a specific role of apoC-IV in lipid metabolism or in other physiologic pathways.

The low density lipoprotein (LDL) receptor-related protein (LRP) is a cell receptor that has close structural homology to the LDL and very low density lipoprotein receptors and thus is believed to play an important role in lipid metabolism. This study was carried out to evaluate the distribution of a known tetranucleotide repeat polymorphism in the LRP gene and its association with serum lipoprotein-lipid and apolipoprotein levels in four large samples comprising Hispanics (n=373) and non-Hispanic Whites (n=522) from the U.S. and Nigerian Blacks from Sokoto (n=390) and Benin (n=800). A total of four alleles, designated 83, 87, 91 and 95 bp, were observed. The 83 bp allele was observed at 0.4-1.1% in the two U.S. populations but was completely absent in African Blacks. Sokoto Blacks had significantly different frequencies of the 87 and 91 bp alleles compared to Hispanics (P=0.008) and non-Hispanic Whites (P=0.024). The frequency of the 91 bp allele was also significantly higher in Benin Blacks compared to Hispanics (P=0.026) and non-Hispanic Whites (P=0.054). The analysis of the relationship between the LRP polymorphism and serum lipid traits yielded some significant race and gender specific significant association for lipoprotein(a) in non-Hispanic White males (P=0.02); HDL2-cholesterol in Hispanic females (P=0.03) and apolipoprotein B in Benin males (P=0.04). We also observed an interaction between the LRP polymorphism and menopausal status for Lp(a) in Hispanic females (P=0.014). However, considering multiple comparisons were performed, these associations could be due to chance. Our data indicate that although the LRP tetranucleotide polymorphism exhibits inter-racial differences in its distribution, it does not appear to have a significant role in affecting serum lipid traits.

Recently several but not all studies have implicated the codon 192 polymorphism in the paraoxonase 1 (PON1) gene with the risk of coronary heart disease (CHD). These findings suggest that this polymorphism is not functional but rather may be in linkage disequilibrium with a functional mutation in the PON1 or a nearby gene. In this investigation, we have evaluated the role of another common polymorphism in the PON1 gene at codon 55 with the risk of CHD in a biracial sample of Asian Indians and Chinese. We observed a significant inter-racial variability in the allelic distribution as the frequency of the less common allele, codon 55/L, was significantly higher in Indians than Chinese (0.202 versus 0.036; P < 0.0001). However, despite this inter-racial difference the codon 55 polymorphism was neither associated with CHD risk nor with plasma lipoprotein-lipids variation in both racial groups. We also used two site haplotype data (codons 55 and 192) to assess the combined contribution of the two polymorphisms to the risk of CHD. There was a strong linkage disequilibrium between the two polymorphic sites in both racial groups (P < 0.0001). While the haplotype data revealed no association with CHD in Chinese, the frequency of the BL haplotype was significantly higher (0.430 versus 0.311; P = 0.004) and the frequency of the AL haplotype was significantly lower (0.368 versus 0.483; P = 0.006) in Indian patients than controls. Since the B allele of the codon 192 polymorphism was shown to be an independent risk factor for CHD in Indians in our previous study, the positive association of the BL haplotype with CHD appears to be mediated by the B allele with no independent contribution from the codon 55 polymorphism.

Apolipoprotein A-IV (apo A-IV, protein; apo A4, gene) is a major constituent of triglyceride-rich and high-density lipoprotein particles and may, therefore, play an important role in lipid metabolism. We studied the distribution of two apo A4 polymorphisms at codons 347 (alleles A and T) and 360 (alleles 1 and 2) in relation to plasma lipoprotein-lipid and apolipoprotein levels in 176 non-fasting male blood donors from New Delhi, Northern India. The frequencies of the T allele at codon 347 and the 2 allele at codon 360 were 0.12 and 0.03 respectively. Carriers of the T allele (AT and TT genotypes) had significantly lower plasma total cholesterol (P = 0.04) and low density lipoprotein (LDL)-cholesterol (P = 0.02) levels than individuals homozygous for the A allele (AA genotype). The codon 347 polymorphism explained 2.2 and 2.6% of the phenotypic variation in total cholesterol and LDL-cholesterol, respectively. The 2 allele at codon 360 was associated with marginally reduced plasma LDL-cholesterol (P = 0.09) and increased triglyceride (P = 0.05) levels compared to the 1 allele. To further elucidate the combined effects of the two polymorphism we constructed two-site haplotypes. The haplotype data showed a stronger influence and explained 3.0 and 5.2% of the phenotypic variation in total cholesterol and LDL-cholesterol, respectively. The two uncommon haplotypes, T1 and A2, were associated with 24.2 and 23.5 mg/dl lower total cholesterol and 22.5 and 42.0 mg/dl lower LDL-cholesterol levels, respectively. The accentuated effect of apo A4 polymorphisms on non-fasting plasma cholesterol suggest that apo A-IV may play an important role in regulating the postprandial metabolism of lipoproteins.

A common MspI polymorphism (G/A) in the promoter region of the APOA1 gene (-75 bp) has been shown to be associated with plasma apo A-I and HDL-C variation in several, but not all, studies. Recently another MspI polymorphic site (+/-) in the 5'region of APOA1 (+83 bp) has been identified which may also be relevant to HDL metabolism. This study was undertaken to elucidate the individual and combined effects of these two polymorphisms on plasma apo A-I and HDL-C levels in a cohort of 534 normoglycemic US Whites from the San Luis Valley, Colorado. Both polymorphisms were in strong linkage disequilibrium (P < 0.005); of the expected four haplotypes (G+, G-, A+, A-) the A- was not observed in this sample. Single site RFLP analysis revealed an independent and significant effect associated with each polymorphism on plasma apo A-I variation but not on HDL-C variation. Further analyses showed that the genotype effects of both polymorphisms were confined to non-smokers only. Haplotype analysis, combining both RFLPs, was more informative as this explained almost twice the amount of phenotypic variation in plasma apo A-I compared to single RFLP analysis in non-smokers. Compared to the most common haplotype (G+), the A+ and G- haplotypes were associated with increased plasma apo A-I levels by 6.7 mg/dl and 22.0 mg/dl, respectively in non-smoking men, and by 4.6 mg/dl and 15.1 mg/dl in non-smoking women, respectively. These data indicate that haplotype analysis in this region may be important to elucidate the functional significance of the APOA1 gene in HDL metabolism.

Genetic polymorphism at the apolipoprotein E locus (APOE) has been shown to have a significant impact on quantitative risk factors for coronary artery disease (CAD) in diverse populations. However, despite the recognition that atherosclerosis begins in childhood and that genetic factors are related to the initial stages of atherosclerosis, prior studies were carried out mostly on adults and little attention has been paid to genetic risk factors for CAD in children. We have examined the impact of APOE polymorphism on quantitative risk factors for CAD (apoAI, apoB, TC, LDL-C, HDL-C and TG) in a sample of 647 African American and 573 White 9-10-year-old girls who were enrolled in the National Heart, Lung and Blood Institute Growth and Healthy Study. The frequencies of the APOE*2, APOE*3 and APOE*4 alleles were 0.09, 0.76 and 0.15 in Whites and 0.11, 0.70 and 0.19 in African Americans, respectively. The APOE*2 allele was significantly associated with lower mean levels of LDL-C and apoB and the APOE*4 allele with higher levels of LDL-C and apoB in both racial groups. Variation in maturation stage, body fat and fat patterning, as assessed by skin fold measures and waist/hip ratio, accounted for a significant proportion of the variation in quantitative CAD risk factors.

The distributions of plasma total cholesterol, apolipoproteins A-I and B and lipoprotein(a) levels as well as genetic typings of apolipoprotein(a) and apolipoprotein E were determined in a randomly selected sample of American Black women (mean age 22.2 +/- 6.5 years) . Mean plasma levels of cholesterol, apolipoprotein A-I, apolipoprotein B and lipoprotein(a) were 184.5 +/- 3.0 mg/dl, 138.0 +/- 3.1 mg/dl, 79.5 +/- 1.8 mg/dl and 24.5 +/- 1.5 mg/dl, respectively. Plasma lipoprotein (a) levels correlated significantly with apolipoprotein B and cholesterol. The contribution of apolipoprotein (a) and apolipoprotein E polymorphisms in affecting these quantitative traits was evaluated. The apolipoprotein(a) locus was extremely polymorphic with 27 alleles, while the 3 common alleles were observed in the apolipoprotein E gene. The frequencies of the APOE*2, APOE* and APOE*4 alleles were 0.094, 0.674 and 0.232, respectively. An inverse relationship was observed between the size of apolipoprotein(a) isoforms and lipoprotein(a) levels (r = 0.37; P = 0.0001). The apolipoprotein E polymorphism revealed a significant genotypic effect on apolipoprotein B (P = 0.0008) and cholesterol (P= 0.005) levels; these concentrations were lower in the APOE 2-3 genotype and higher in the 3-4 and 4-4 genotypes compared with the common 3-3 genotype. The apolipoprotein E polymorphism explained 15.8% and 6.3% of the phenotypic variance in apolipoprotein B and cholesterol levels, respectively. This study demonstrates that genetics play an important role in determining quantitative risk factors for coronary heart disease among American Black women.

The extent of apolipoprotein E (apo E) polymorphism and its effect on eight quantitative risk factors for coronary heart disease (total cholesterol; low density lipoprotein (LDL) cholesterol; total high density lipoprotein and its subfractions, HDL2 and HDL3; triglycerides; fasting glucose and fasting insulin) has been determined in 238 randomly selected Hispanics (120 males and 118 females) and 201 non-Hispanic whites (NHWs) (105 males and 96 females) from the San Luis Valley, Colorado. The frequencies for the E * 2, E * 3 and E * 4 alleles were 0.048, 0.853 and 0.099, respectively, in Hispanics and 0.080, 0.783 and 0.137, respectively, in NHWs. Relatively low frequency of the E * 2 and E * 4 alleles in Hispanics compared with NHWs is consistent with the genetic and anthropologic data that Hispanics have substantial Amerindian admixture. The impact of apo E polymorphism on each quantitative trait was estimated after adjusting for concomitant variables including age, cigarette smoking and body mass index in both genders and pre- or post-menopause status in females. The distribution of eight quantitative traits was analyzed among three common apo E phenotypes, 3-2, 3-3 and 4-3. In Hispanics, significant variability among apo E phenotypes was observed for total cholesterol (P = 0.001) in females only and the apo E polymorphism accounts for 12.4% variation in total cholesterol and 15.2% variation in LDL-cholesterol. In NHWs, significant mean differences among apo E phenotypes were observed for total cholesterol in both males (P = 0.007) and females (P = 0.0004). In NHW males and females, the apo E polymorphism explained 9.2% and 12.4%, respectively, of the variation in total cholesterol, and 15.1% and 6.6%, respectively, of the variation in LDL-cholesterol. In NHWs, borderline significance levels were also noted for phenotype specific differences in HDL2-cholesterol in males (P = 0.04) and females (P = 0.05), for total HDL cholesterol in females (P = 0.02) and HDL3-cholesterol in females (P = 0.06). While the estimated effects of the apo E polymorphism on quantitative traits differ somewhat between Hispanics and non-Hispanic whites, this probably reflects the overall difference in frequencies of the less common alleles in the Hispanics rather than a biological difference in the effects of these alleles on lipid metabolism.

Apolipoprotein A-IV (apo A-IV) is a major component of several lipoprotein particles and may, therefore, play an important role in lipid metabolism. Genetic polymorphism of apo A-IV has been reported in humans and several other animal species. The presence of two common alleles, apo A-IV * 1 and apo A-IV * 2 has been documented in several human populations. In this investigation, we have determined apo A-IV polymorphism by isoelectric focusing-immunoblotting in 82 non-insulin-dependent diabetic and 204 control non-Hispanic Whites from the San Luis Valley, Colorado. We have also estimated the impact of apo A-IV polymorphism on eight quantitative traits: total cholesterol, HDL-cholesterol, HDL3 and HDL2-cholesterol, LDL-cholesterol, triglycerides, fasting glucose and fasting insulin. No statistically significant difference was seen in apo A-IV allele frequencies between the control and diabetics, and these frequencies were comparable with those reported for U.S. White and European populations. Among controls, the impact of the apo A-IV polymorphism was significant on LDL-cholesterol (P = 0.04) in females and on fasting insulin levels (P = 0.06) in males. In diabetics, the effect was significant on insulin (P = 0.03) levels in males only. Furthermore, our data suggest that when making comparison of lipid profiles between the controls and diabetics the presence or absence of common apo A-IV phenotypes should be taken into account as these appear to effect these comparisons.