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The newly identified apolipoprotein AV (apoAV) gene is a key player in determining plasma triglyceride concentrations. Because hypertriglyceridemia is a major independent risk factor in coronary artery disease, the understanding of the regulation of the expression of this gene is of considerable importance. We presently characterize the structure, the transcription start site, and the promoter of the human apoAV gene. Since the peroxisome proliferator-activated receptor-alpha (PPARalpha) and the farnesoid X-activated receptor (FXR) have been shown to modulate the expression of genes involved in triglyceride metabolism, we evaluated the potential role of these nuclear receptors in the regulation of apoAV transcription. Bile acids and FXR induced the apoAV gene promoter activity. 5'-Deletion, mutagenesis, and gel shift analysis identified a heretofore unknown element at positions -103/-84 consisting of an inverted repeat of two consensus receptor-binding hexads separated by 8 nucleotides (IR8), which was required for the response to bile acid-activated FXR. The isolated IR8 element conferred FXR responsiveness on a heterologous promoter. On the other hand, in apoAV-expressing human hepatic Hep3B cells, transfection of PPARalpha specifically enhanced apoAV promoter activity. By deletion, site-directed mutagenesis, and binding analysis, a PPARalpha response element located 271 bp upstream of the transcription start site was identified. Finally, treatment with a specific PPARalpha activator led to a significant induction of apoAV mRNA expression in hepatocytes. The identification of apoAV as a PPARalpha target gene has major implications with respect to mechanisms whereby pharmacological PPARalpha agonists may exert their beneficial hypotriglyceridemic actions. Show less

The recently discovered APOA5 gene has been shown in humans and mice to be important in determining plasma triglyceride levels, a major cardiovascular disease risk factor. apoAV represents the first described apolipoprotein where overexpression lowers triglyceride levels. Since fibrates represent a commonly used therapy for lowering plasma triglycerides in humans, we investigated their ability to modulate APOA5 gene expression and consequently influence plasma triglyceride levels. Human primary hepatocytes treated with Wy 14,643 or fenofibrate displayed a strong induction of APOA5 mRNA. Deletion and mutagenesis analyses of the proximal APOA5 promoter firmly demonstrate the presence of a functional peroxisome proliferator-activated receptor response element. These findings demonstrate that APOA5 is a highly responsive peroxisome proliferator-activated receptor alpha target gene and support its role as a major mediator for how fibrates reduce plasma triglycerides in humans. Show less

Common, complex genetic disorders such as coronary heart disease (CHD) frequently show large population differences, contributing to health disparities. It is also well known that CHD risk factor profiles and the frequency of coronary events differ by gender. Study of premature CHD has revealed that apolipoproteins are important discriminating factors for distinguishing individuals with CHD. Recent findings indicated that apolipoprotein A-V (APOA-V) gene promoter polymorphisms are an important determinant of plasma triglycerides (TG) and lipoprotein cholesterol, and a risk factor for CHD. Variations in APOA-V may have varying impacts in different ethnic groups. The purpose of this interdisciplinary genetic research project was to determine (1) the association of the APOA-V polymorphisms with lipoprotein profiles, and (2) the gender and ethnic differences in the T-1131C promoter polymorphism of the APOA-V gene in individuals with dyslipidemia versus controls. Results indicate that the minor -1131C allele (CC homozygotes + CT heterozygotes) was associated with elevated plasma TG (p = 0.007), very low density lipoprotein (VLDL)-TG (p = 0.019), LDL-TG (p = 0.004), high-density-lipoprotein (HDL)-TG (p < 0.001), and VLDL-cholesterol (p = 0.008). We found a striking elevation in the frequency of the minor C allele in Asians (p < 0.001) compared to Europeans. We also found a significant difference in genotype frequency between men and women in Asians (p = 0.031) and Europeans (p < 0.01). Remarkably, Asian women with the C allele have a 36% increase in TG compared to Asian women homozygous for the T allele. In summary, we found significant ethnic-specific and gender-based differences in the frequency of the minor allele of the -1131 APOA-V gene promoter polymorphism. Identification of genetic variations among ethnic groups and between genders may have significant potential for a better understanding of the development of cardiovascular disease. Show less

The -1131T>C polymorphism in the newly identified apolipoprotein A5 (APOA5) gene has been associated with elevated plasma triglycerides. We determined its incidence in 915 patients attending a lipid outpatient clinic. The frequency of the C allele was significantly higher in patients with triglycerides above the 90th percentile and patients with type III hyperlipidemia compared to those with hypercholesterolemia. The C allele was associated with increased plasma triglycerides and decreased plasma HDL cholesterol, conditions associated with an increased risk of coronary heart disease. The effects on plasma lipids were only observed in overweight (BMI>25) patients and were greater in patients who were also carriers of a least one epsilon4 allele in the APOE gene. Thus additional genetic and/or metabolic factors are required in order for the triglyceride raising and HDL lowering effect of the -1131T>C polymorphism in APOA5 to be expressed. Show less

The apolipoprotein A5 gene (APOA5 ) has been shown to play an important role in determining plasma triglyceride concentrations in humans. We describe here a novel variant, c.553G>T, in the apolipoprotein A5 gene that is associated with hypertriglyceridemia. In contrast to some other polymorphisms, which occur in non-coding regions of the gene, this variant occurs within the coding region and causes the change of amino acid sequence (a substitution of a cysteine for a glycine residue). The minor allele frequencies were 0.042 and 0.27 (P<0.001) for control and hypertriglyceridemic patients, respectively. The serum triglyceride level was significantly different among the genotypic groups (G/G 92.5+/-37.8 mg/dl, G/T 106.6+/-34.8 mg/dl, T/T 183.0 mg/dl, P=0.014) in control subjects. Multiple logistic regression revealed individuals carrying the minor allele had age, gender and BMI (body mass index)-adjusted odds ratio of 11.73 (95% confidence interval of 6.617-20.793; P<0.0001) for developing hypertriglyceridemia in comparison to individuals without that allele. These findings suggest the possible use of c.553G>T polymorphisms in APOA5 as prognostic indicators for hypertriglyceridemia susceptibility in Chinese. Show less

Apolipoprotein A-V (apoA-V), the newest member of the plasma apolipoprotein family, was recently discovered by comparison of the mouse and human genomes. Studies in rodents and population surveys of human apoA-V polymorphisms have noted a strong effect of apoA-V on plasma triglyceride levels. Toward the elucidation of the biologic function of apoA-V, we used spectroscopic and surface chemistry techniques to probe its structure and interfacial activity. Computer-assisted sequence analysis of apoA-V predicts that it is very hydrophobic, contains a significant amount of alpha-helical secondary structure, and probably is composed of discrete structural regions with varying degrees of lipid affinity. Fluorescence spectroscopy of recombinant human apoA-V provided evidence of tertiary folding, and light scattering studies indicated that apoA-V transforms dimyristoylphosphatidylcholine vesicles into discoidal complexes with an efficiency similar to that of apoA-I. Surface chemistry techniques revealed that apoA-V displays high affinity, low elasticity, and slow binding kinetics at hydrophobic interfaces, properties we propose may retard triglyceride-rich particle assembly. Metabolic labeling and immunofluorescence studies of COS-1 cells transfected with human apoA-V demonstrated that apoA-V is poorly secreted, remains associated with the endoplasmic reticulum, and does not traffic to the Golgi. Given that overexpression of the apoA-V gene lowers plasma triglycerides in mice, these data together suggest that apoA-V may function intracellularly to modulate hepatic VLDL synthesis and/or secretion. Show less

A new apolipoprotein gene, APOA5, was recently discovered near the APOA1/C3/A4 gene cluster. Transgenic mice overexpressing the homologous gene, apoa5, showed reduced plasma triglyceride levels, while knockout mice had greatly increased triglycerides, suggesting that human genetic variants affecting expression of the protein product, APOAV, might affect triglyceride levels. Polymorphisms in the APOA5 gene were indeed found to be associated with triglyceride levels in men, though not in women. We sought to confirm the association of the APOA5-1131T>C polymorphism with triglyceride levels in 167 Chinese men chosen for having either high (>/=1.7 mm, n = 82) or low (Show less

The newly recognised apolipoprotein (apo) AV gene (APOAV) has been linked to fasting plasma triglyceride (TG) concentrations with some polymorphisms associated with elevated fasting TGs. Since fasting plasma TGs are mainly determined by the hepatic production of TG-rich particles (very low density lipoprotein; VLDL), and fasting TGs are the major determinants of postprandial lipaemia, we have evaluated the effects of an APOAV polymorphism on postprandial triglyceridaemia, which is largely determined by the intestinal production and clearance of chylomicrons. For this purpose, diurnal capillary triglyceridaemia (reflecting postprandial lipaemia) was determined in a cohort of 88 healthy volunteers (48 males and 40 females) in relation with a -1131T>C variant in the promoter of APOAV. Thirteen of these subjects (7 males and 6 females) were carriers of the -1131C allele, which has been associated with higher fasting plasma TG levels. The carriers had higher fasting capillary TG concentrations, although plasma TGs were not significantly different from non-carriers in this cohort. Surprisingly, total diurnal triglyceridaemia calculated as the area under the capillary TG curve was similar in carriers compared to non-carriers but after correction for fasting capillary TG levels, incremental diurnal triglyceridaemia was significantly lower in carriers (1.74 (5.27) mmol/h/l) than in non-carriers (4.91 (4.90) mmol/h/l; p = 0.036). The same trends were found for both males and females when analysed separately. Since dietary intake, which is a major determinant of incremental diurnal triglyceridaemia, did not differ between the two groups, we believe that these differences are at least partly explained by the APOAV. In summary, the APOAV assessed by means of the -1131T>C variant seemed to have a paradoxical effect on postprandial lipaemia when compared to fasting TG levels. Show less

The availability of the human genome sequence and the recently completed draft sequences of two major mammalian model species, the mouse (Mus musculus) and the rat (Rattus norvegicus), allow researchers to apply novel approaches for gene identification and characterization, using methods of comparative and functional genomics. Recently, a new gene coding for apolipoprotein A-V was identified in the vicinity of APOA-I/C-III/A-IV cluster on human chromosome 11q23 by comparative sequencing method. In a relatively short time, compelling evidence accumulated for the substantial role of APOA-V in lipid metabolism. Studies in knock-out and transgenic mice revealed that its expression pattern correlates negatively with triglyceride levels. This observation was verified in human population studies in variety of ethnic and age groups. Several single nucleotide polymorphisms were described and particular SNP alleles and haplotypes in the APO A-V gene region were shown to be associated with dyslipidemia. The discovery and characterization of the APO A-V demonstrates current possibilities of the integrative approaches in biology, boosted by the available bioinformatic tools. Show less

Recent discovery and characterization of APOAV suggests a role in metabolism of triglyceride (TG)-rich lipoproteins. Previously, variation at the APOAV locus was shown to modestly influence plasma TGs in normolipidemic samples. The aims of this study were to assess the effects of a polymorphism in APOAV (T-1131C) in terms of its frequency among three dyslipidemic populations and a control population, differences of allele frequency across available ethnic groups, and associations with specific lipoprotein TG and cholesterol compartments. We found a striking elevation in the frequency of the rare allele in a Chinese population (P = 0.0002) compared with Hispanic and European populations. The rare allele of the polymorphism was associated with elevated plasma TG (P = 0.012), VLDL cholesterol (P = 0.0007), and VLDL TG (P = 0.012), LDL TG (P = 0.003), and HDL TG (P = 0.016). Linear regression models predict that possession of the rare allele elevates plasma TG by 21 mg/dl (P = 0.009) and VLDL cholesterol by 8 mg/dl (P = 0.0001), and reduces HDL cholesterol by 2 mg/dl (P = 0.017). The association of the polymorphism with altered lipoprotein profiles was observed in combined hyperlipidemia, hypoalphalipoproteinemia, and hyperalphalipoproteinemia, and in controls. These findings indicate that APOAV is an important determinant of plasma TG and lipoprotein cholesterol, and is potentially a risk factor for cardiovascular disease. Show less

Singapore comprises three ethnic groups: Chinese (76.7%), Malays (14%), and Asian-Indians (7.9%). Overall, Singaporeans experience coronary heart disease rates similar to those found in the United States. However, there is a dramatic interethnic gradient, with Asian-Indians having significantly higher risk than Chinese and Malays. These differences are associated with HDL cholesterol levels and cannot be solely explained by environmental exposure, and may be driven by genetic factors. The gene encoding apolipoprotein A-V (APOA5) has been located on chromosome 11, and it is emerging as an important candidate gene for lipoprotein metabolism. We investigated associations between APOA5 polymorphisms and plasma lipids in 3,971 Singaporeans to establish whether they accounted for some of the ethnic differences in plasma lipids. We found significant associations between the minor alleles at each of four common polymorphisms and higher plasma triglycerides (TGs) across ethnic groups. Haplotype analyses showed significant associations with TGs, explaining 6.9%, 5.2%, and 2.7% of the TG variance in Malays, Asian-Indians, and Chinese, respectively. Conversely, we observed significant inverse associations between the minor alleles and HDL cholesterol concentrations for Chinese and Malays. These data suggest that APOA5 plays a role in the ethnic differences observed for plasma TG and HDL cholesterol concentrations. Show less

The recently identified apolipoprotein A5 gene (APOA5) has been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. We previously identified an APOA5 haplotype (designated APOA5*2) that is present in approximately 16% of Caucasians and is associated with increased plasma triglyceride concentrations. In this report we describe another APOA5 haplotype (APOA5*3) containing the rare allele of the single nucleotide polymorphism c.56C>G that changes serine to tryptophan at codon 19 and is independently associated with high plasma triglyceride levels in three different populations. In a sample of 264 Caucasian men and women with plasma triglyceride concentrations above the 90th percentile or below the 10th percentile, the APOA5*3 haplotype was more than three-fold more common in the group with high plasma triglyceride levels. In a second independently ascertained sample of Caucasian men and women (n=419) who were studied while consuming their self-selected diets as well as after high-carbohydrate diets and high-fat diets, the APOA5*3 haplotype was associated with increased plasma triglyceride levels on all three dietary regimens. In a third population comprising 2660 randomly selected individuals, the APOA5*3 haplotype was found in 12% of Caucasians, 14% of African-Americans and 28% of Hispanics and was associated with increased plasma triglyceride levels in both men and women in each ethnic group. These findings establish that the APOA5 locus contributes significantly to inter-individual variation in plasma triglyceride levels in humans. Together, the APOA5*2 and APOA5*3 haplotypes are found in 25-50% of African-Americans, Hispanics and Caucasians and support the contribution of common human variation to quantitative phenotypes in the general population. Show less

Mice lacking ApoA-V, a novel HDL-associated apolipoprotein identified by our group and independently by Pennacchio et al. [Science 294 (2001) 169], were recently shown to be hypertriglyceridemic. To study the role of ApoA-V in triglyceride homeostasis, we compared lipid profiles in mice expressing normal and highly elevated levels of ApoA-V. For this purpose, adenoviral vectors expressing sense or antisense ApoA-V cDNA were constructed. Treatment of mice with sense adenoviral constructs resulted in circa 20-fold higher serum ApoA-V levels compared with mice injected with either PBS or antisense adenoviral constructs. ApoA-V overexpressing mice had markedly decreased (-70%) serum triglyceride levels caused primarily by lowered triglyceride content of the VLDL fraction. Furthermore, in these mice cholesterol levels were found to be lowered in all lipoprotein fractions with the largest mass decrease in the HDL fraction. This resulted in a 40% drop of serum cholesterol content. These findings suggest a role of ApoA-V in regulating levels of circulating triglycerides and cholesterol. Show less

The purpose of this study was to assess the influence of single nucleotide polymorphism 3 (SNP3) of the apolipoprotein A-V ( APOA5) gene on the serum triglyceride (TG) level in Japanese schoolchildren. To determine the frequency of the genotype, we analyzed 552 schoolchildren. The frequencies of the T/T, T/C and C/C genotypes of the APOA5 gene were 225 (40.8%), 263 (47.6%) and 64 (11.6%), respectively. The serum TG level was significantly different among the genotypic groups after adjustments for age, gender and obesity index (T/T 71.6+/-34.8 mg/dl, T/C 80.7+/-36.1 mg/dl, C/C 94.4+/-69.4 mg/dl, P<0.0001). The odds ratio (95% confidence interval) for hypertriglyceridemia of the C allele was 2.4 (1.0-6.2). Our data suggested that the T/C promoter region polymorphism of the APOA5 gene appears to be a genetic risk factor for hypertriglyceridemia in Japanese children. Show less

The newly identified apoprotein AV (apoAV) gene was suggested to have a significant effect on triglyceride (TG) metabolism in Caucasians. We studied the genetic effect of this gene on serum TG in a Japanese population. Participants (481 male and 412 female) were recruited at a health examination. A T/C single nucleotide polymorphism called SNP3 in the 5'-region of the apoAV gene was genotyped as described previously. The frequency of the C allele was much greater in Japanese than in Caucasians (0.34 vs. 0.08). The serum TG level in subjects with the TT genotype was significantly lower than the level in those with TC/CC (1.10, 1.25 and 1.21 mmol/l for TT, TC and CC, respectively, P=0.0003 by ANOVA), while there were no significant differences either in the serum total cholesterol or the low- and high-density lipoprotein cholesterol levels among the three genotypes. Multiple regression analysis indicated that SNP3 had a significant independent effect on the serum TG level in Japanese (P<0.0001). This result indicates that polymorphism in the apoAV gene influence serum TG in populations of different ethnicities. Show less

Comparison of genomic DNA sequences from human and mouse revealed a new apolipoprotein (APO) gene (APOAV) located proximal to the well-characterized APOAI/CIII/AIV gene cluster on human 11q23. Mice expressing a human APOAV transgene showed a decrease in plasma triglyceride concentrations to one-third of those in control mice; conversely, knockout mice lacking Apoav had four times as much plasma triglycerides as controls. In humans, single nucleotide polymorphisms (SNPs) across the APOAV locus were found to be significantly associated with plasma triglyceride levels in two independent studies. These findings indicate that APOAV is an important determinant of plasma triglyceride levels, a major risk factor for coronary artery disease. Show less

Liver regeneration in response to various forms of liver injury is a complex process, which ultimately results in restoration of the original liver mass and function. Because the underlying mechanisms that initiate this response are still incompletely defined, this study was aimed to identify novel factors. Liver genes that were up-regulated 6 h after 70% hepatectomy (PHx) in the rat were selected by cDNA subtractive hybridization. Besides known genes associated with cell proliferation, several novel genes were isolated. The novel gene that was most up-regulated was further studied. Its mRNA showed a liver-specific expression and encoded a protein comprising 367 amino acids. The mouse and human cDNA analogues were also isolated and appeared to be highly homologous. The human gene analogue was located at an apolipoprotein gene cluster on chromosome 11q23. The protein encoded by this gene had appreciable homology with apolipoproteins A-I and A-IV. Maximal expression of the gene in the rat liver and its gene product in rat plasma was observed 6 h after PHx. The protein was present in plasma fractions containing high density lipoprotein particles. Therefore, we have identified a novel apolipoprotein, designated apolipoprotein A-V, that is associated with an early phase of liver regeneration. Show less