ATP-binding Cassette Sub-family G Member 1 Research Articles

Common Variants in Cholesterol Synthesis– and Transport–Related Genes Associate with Circulating Cholesterol Responses to Intakes of Conventional Dairy Products in Healthy Individuals

Background: Dairy intake has been associated with varying impacts on circulating cholesterol concentrations across nutritional epidemiology and intervention studies, with findings attributed mainly to differences in the nature of dairy products consumed or study designs. The contribution of the genomic architecture to such observations has yet to be revealed.Objective: We assessed the impact of multiple common genetic variations in cholesterol-related genes on responses of serum cholesterol to the recommended amount of dairy product intake in Canada.Methods: In a multicenter, randomized crossover design, 101 normolipidemic adults (n = 29 men and 72 women), with a mean ± SD age of 41.7 ± 16.7 y and a body mass index (BMI, in kg/m2) of 25.9 ± 4.3 consumed 3 servings/d of dairy [375 mL 1% milk-fat (MF) milk, 175 g 1.5% MF yogurt, and 30 g of 34% MF cheese] or energy-matched control products (juice, cashews, and cookies) provided within a prudent background diet for 4 wk each, separated by a 4- to 8-wk washout period. Serum lipid variables were determined by standard enzymatic methods by using an autoanalyzer. Candidate single nucleotide polymorphisms were assessed by TaqMan genotyping assay.Results: The responsiveness of serum total cholesterol (TC) and LDL cholesterol to the dairy compared with the control diet was associated with individuals’ genotypes. The cholesterol transport gene ATP-binding cassette subfamily G, member 5 (ABCG5) rs6720173-GG homozygotes had higher concentrations of TC (+0.18 mmol/L; P = 0.0118) and LDL cholesterol (+0.17 mmol/L; P = 0.0056) relative to C-allele carriers (−0.07 and −0.06 mmol/L, respectively). The bile acid synthesis gene cholesterol 7α-hydroxylase (CYP7A1) rs3808607-G-allele carriers had higher TC (+0.20 to +0.28 mmol/L; P = 0.0026) and LDL cholesterol (+0.19 mmol/L for GT genotype; P = 0.0260) relative to TT homozygotes (−0.11 and −0.03 mmol/L, respectively). In addition, the cholesterol synthesis gene 7-dehydrocholesterol reductase (DHCR7) rs760241-A-allele carriers had higher LDL cholesterol (+0.26 mmol/L; P = 0.0399) relative to GG homozygotes (+0.06 mmol/L).Conclusion: Genetic variations in ABCG5, CYP7A1, and DHCR7 may contribute to differing responses of serum cholesterol to dairy intake among healthy adults. This trial was registered at clinicaltrials.gov as NCT01444326.

CYP7A1-rs3808607and APOEisoform associate with LDL cholesterol lowering after plant sterol consumption in a randomized clinical trial

The benefits of plant sterols (PSs) for cholesterol lowering are hampered by large heterogeneity across individuals, potentially because of genetic polymorphisms. We investigated the impact of candidate genetic variations on cholesterol response to PSs in a trial that recruited individuals with high or low endogenous cholesterol synthesis, estimated by lathosterol to cholesterol (L:C) ratio. Mildly hypercholesterolemic adults preselected as possessing either high endogenous cholesterol synthesis (n = 24; mean ± SEM: L:C ratio = 2.03 ± 0.39 μmol/mmol) or low endogenous cholesterol synthesis (n = 39; mean ± SEM: L:C ratio = 0.99 ± 0.28 μmol/mmol) consumed 2 g PS/d or a placebo for 28 d by using a dual-center, single-blind, randomized crossover design. Cholesterol synthesis and change in cholesterol absorption were measured with stable isotopic tracers. Candidate single-nucleotide polymorphisms and apolipoprotein E (APOE) isoform were assessed by TaqMan genotyping assay. The cholesterol fractional synthesis rate was higher (P < 0.001) in participants with high endogenous cholesterol synthesis (mean ± SEM: placebo: 9.16% ± 0.47%; PSs: 9.74% ± 0.47%) than in participants with low endogenous cholesterol synthesis (mean ± SEM placebo: 5.72% ± 0.43%; PS: 7.10% ± 0.43%). Low-density lipoprotein (LDL) cholesterol lowering in response to PSs was associated with individuals' genotypes. Cholesterol 7 alpha-hydroxylase (CYP7A1-rs3808607) T/T homozygotes showed no LDL cholesterol lowering (mean ± SEM: -0.05 ± 0.07 mmol/L, P = 0.9999, n = 20), whereas the presence of the G-allele associated with LDL cholesterol response in a dose-dependent fashion (mean ± SEM G/T: -0.22 ± 0.06 mmol/L, P = 0.0006, n = 35; G/G: -0.46 ± 0.12 mmol/L, P = 0.0009, n = 8). Similarly, APOE ɛ3 carriers (mean ± SEM: -0.13 ± 0.05 mmol/L, P = 0.0370, n = 40) responded less than APOE ɛ4 carriers (mean ± SEM: -0.31 ± 0.07 mmol/L, P < 0.0001, n = 23). Moreover, genoset CYP7A1-rs3808607 T/T/APOE ɛ3 was associated with nonresponsiveness (mean ± SEM: +0.09 ± 0.08 mmol/L, P = 0.9999, n = 14). rs5882 in cholesteryl ester transfer protein (CETP) and rs4148217 in ATP-binding cassette subfamily G member 8 (ABCG8) did not associate with LDL cholesterol lowering. Cholesterol absorption decreased as a result of PS consumption, but this decrease was not related to circulating LDL cholesterol concentrations, cholesterol synthesis phenotype, or genotypes. CYP7A1-rs3808607 and APOE isoform are associated with the extent of reduction in circulating LDL cholesterol in response to PS consumption and could serve as potential predictive genetic markers to identify individuals who would derive maximum LDL cholesterol lowering with PS consumption. The trial was registered at clinicaltrials.gov as NCT01131832.

ABCG5/8 variants are associated with susceptibility to coronary heart disease

ATP-binding cassette sub-family G member 5 (ABCG5) and ABCG8 are members of an ATP-binding cassette transporter superfamily. ABCG5 and ABCG8 variants affected serum levels of cholesterol and were considered as risk factors for coronary heart disease (CHD). The present control study analyzed ABCG5 and ABCG8 variants in a population for association with the risk of CHD. A total of 417 CHD patients and 267 controls were recruited for genotyping of four single nucleotide polymorphisms (SNPs; i.e. i7892T>C in ABCG5 and Tyr54CysA>G, Thr400LysC>A and 5U145A>C in ABCG8) using quantitative PCR high-resolution melting (qPCR-HRM). Serum lipid levels were measured using an automatic biochemical analyzer. The association of ABCG5/8 variants with lipid levels was analyzed using a Chi-square test. The impact of candidate ABCG5/8 SNPs on CHD was evaluated in a dominant genetic model with stepwise multiple regression analysis. Subgroup analyses were performed with regard to these SNPs, tobacco smoking status, alcohol consumption and gender. Genotypic and allelic frequencies of ABCG8 Thr400LysC>A were significantly different (P<0.05) between CHD patients and controls. CC homozygotes of the ABCG8 Thr400LysC>A SNP had greater triglyceride levels than CA/AA carriers with CHD. Logistic analysis revealed CHD risk was significantly higher in CC homozygotes of ABCG8 Thr400LysC>A than in carriers of the A allele (adjusted P=0.048; OR=2.034; 95% CI=0.983-4.207). Furthermore, there was a significant gene-tobacco smoking interaction. CC homozygotes of ABCG8 Thr400LysC>A SNP had significantly higher triglyceride concentrations (P=0.012) and an increased risk of CHD than tobacco smoking carriers of the A allele. The data from the current study suggested that ABCG8 Thr400LysC>A SNP genetic variants modulated plasma triglyceride levels and thereby affected CHD risk in the population studied. The genetic variant of ABCG8 also contributed to CHD risk through interaction with tobacco smoking.

Abstract 9314: Clinical Impact of Heterozygous Carrier of ATP-binding Cassette Sub-family G Member 5 Gene on Asymptomatic Normolipidemic Patients: Evidence From Familial Gene Analysis

Background: Sitosterolemia is a rare recessive form of inherited disease characterized by increased levels of plant sterols such as sitosterol, the cause of which is ATP-binding cassette (ABC) sub-family G member 5 (ABCG5) or ABCG8 gene mutations. However, few data exist regarding the clinical features and significance of heterozygous sitosterolemic carrier due to the rarity of this disease. The aim of our study was to determine the clinical impact of heterozygous mutation carriers of ABCG5 or ABCG8 gene. Methods and Results: We performed genetic analyses of ABCG5 and ABCG8 genes for the 15 family members of patients with homozygous sitosterolemia from different 3 families, and investigated their serum sitosterol levels as well as their lipid levels. We identified 12 heterozygous carriers (male=6, mean age=40) which exhibit single mutation in ABCG5 gene among these families (c.130C&gt;T, c.1306G&gt;A, c.1813_1817delCTTTT) as well as 3 normal family members (male=1, mean age=48). Although there were no significant differences in TC, TG, HDL-C, and LDL-C levels between heterozygous carriers and normal family members, serum sitosterol level of heterozygous carriers was significantly higher than that of normal family members (10.8±4.3 vs 2.7±0.5 μg/ml, p&lt;0.05). Interestingly, heterozygous carriers showed neither xanthomas nor any coronary artery diseases. Conclusions: These results demonstrate that heterozygous ABCG5 gene mutation carriers which are somewhat difficult to be identified by the conventional methods may exist among the common normolipidemic patients without significant clinical manifestations. We suggest that the functional analysis of ABCG5 or ABCG8 may facilitate identifying hyper sitosterolemic carriers.

Genetic variations involved in interindividual variability in carotenoid status

As shown in most clinical studies dedicated to carotenoids, there is a huge interindividual variability in absorption, and blood and tissue responses, of dietary carotenoids. The recent discovery that several proteins are involved in carotenoid metabolism in humans has prompted a possible explanation for this phenomenon: genetic variants in genes encoding for these proteins may affect their expression or activity, and in turn carotenoid metabolism and carotenoid status. The proteins clearly identified so far are (i) the carotene oxygenases β,β-carotene-15,15'-monooxygenase (BCMO1) and β,β-carotene-9',10'-oxygenase (BCDO2), which are involved in carotenoid cleavage, (ii) scavenger receptor class B type I (SR-BI), cluster determinant 36 (CD36), and Niemann Pick C1-like 1 (NPC1L1), which are involved in carotenoid uptake by cells, and (iii) glutathione S-transferase Pi 1 (GSTP1) and human retinal lutein-binding protein (HR-LBP), which are involved in the transport of xanthophylls in the retina. Other proteins, such as ATP-binding cassette subfamily G member 5 (ABCG5) and the fatty acid-binding proteins (FABPs) are also apparently involved although firmer evidence is still required. A genome-wide association study, as well as several candidate gene association studies, has shown that groups of subjects bearing different alleles in single nucleotide polymorphisms located in or near several of the above-mentioned genes display different blood and/or tissue concentrations of carotenoids. Further studies are needed to identify all the proteins involved in carotenoid metabolism and assess whether other types of genetic variation, e.g. copy number variants and epigenetic modifications, can modulate carotenoid status. One potential application of such research could be personalized dietary guidelines for carotenoids according to individual genetic characteristics.

Endometrial Transporters and Cytochrome P450 Family Member in Endometriosis

Purpose To investigate expression of molecular transporters and CYP4B1 in endometrium from women with and without endometriosis. Methods Expression of genes encoding proteins: ATP-binding cassette sub-family G member 1 (ABCG1), aquaporin 3 (AQP3), solute carrier family 16, member 6 (monocarboxylic acid transporter 7) (SLC16A6), and transmembrane emp24 protein transport domain containing 6 (TMED6), as well as a member of the cytochrome P450 family 4 subfamily B, polypeptide 1 (CYP4B1) were assessed by quantitative (Q)RT-PCR in proliferative (PE) and early secretory (ESE) endometrium from 27 normoovulatory women with and without endometriosis. Cellular localization of these proteins was determined by immunohistochemistry. Results In eutopic endometrial tissue from women without endometriosis, mRNA expression of ABCG1, CYP4B1, and SLC16A6 was significantly up-regulated in the PE to ESE transition. CYP4B1 mRNA in endometrium from women with disease increased significantly compared to samples from women without disease, while there was no change in ABCG1 and SLC16A6 mRNA levels. However, immunodetection of ABCG1, AQP3, and TMED6 was similar across cycle phases, and there was no significant difference in immunostaining intensity or localization between women with and without endometriosis. Conclusions Molecular transporters and CYP4B1 are expressed in human endometrium, and a role for transporters and CYP4B1 in normal endometrial function and in women with endometriosis remains to be determined.

Soy Protein and Fish Oil Independently Decrease Serum Lipid Concentrations but Interactively Reduce Hepatic Enzymatic Activity and Gene Expression Involved in Fatty Acid Synthesis in Rats

The degree of interaction between dietary protein and fat sources to modulate hepatic lipid metabolism was investigated. Male rats were fed diets containing either casein or soy protein isolate as the protein source and either palm or soy oil as the fat source. After 3 wk, the activity and mRNA expression of enzymes involved in hepatic fatty acid synthesis were significantly lower with soy protein than casein when palm oil was the fat source. The same values for the same enzymes were greatly lowered regardless of the protein source when fish oil was the fat source. Both enzymatic activity and mRNA expression for fatty acid oxidation were significantly stimulated by fish oil, but only the former was increased by soy protein. Although both soy protein and fish oil reduced serum lipid concentrations, they worked independently. In soy protein-fed rats, mRNA levels of key enzymes related to cholesterol and bile acid synthesis were decreased and increased, respectively, compared with levels in casein-fed animals. Instead, fish oil strongly induced the mRNA expression of biliary cholesterol transporters, ATP-binding cassette sub-family G, member 5 (ABCG5) and ATP-binding cassette sub-family G, member 8 (ABCG8). Therefore, dietary soy protein and fish oil generally exerted independent hypolipidemic actions in rats. However, the reduction of hepatic fatty acid synthesis caused by the simultaneous ingestion of soy protein and fish oil was smaller than their expected additive reduction, because fish oil strongly decreased the synthesis.

Association of the ABCG1 gene polymorphism with the susceptibility and severity of coronary atherosclerotic disease

To investigate the association of the ATP-binding cassette sub-family G member 1 (ABCG1) gene polymorphisms with coronary atherosclerotic disease (CAD) in Chinese Han population. A population based case-control association study was carried out in 541 patients with CAD and 649 healthy controls from Chinese Han population. Two single nucleotide polymorphisms (SNPs) of the ABCG1 gene were genotyped using polymerase chain reaction-restriction fragment length polymorphism. Logistic regression was used to compare the genotypic and allelic frequency difference. The frequency of allele C of rs225374 was significantly higher in the CAD patients than that in the healthy controls (OR=1.186, 95%CI: 1.009-1.394, P=0.039), while the difference was also significant in the male subgroup (OR=1.236, 95%CI: 1.014-1.506, P=0.036). A statistically higher frequency of rs1044317 allele A was found in the CAD patients in comparison to the healthy controls (OR=1.187, 95%CI: 1.009-1.397, P=0.039). In case-only association study, rs225374 showed significant association in the high Gensini score group compared with the low Gensini score group (OR=1.303, 95%CI: 1.024-1.657, P=0.031). The two SNPs of the ABCG1 gene might be associated with the susceptibility and severity of CAD in Chinese Han population.

Adipocyte Modulation of High-Density Lipoprotein Cholesterol

Adipose harbors a large depot of free cholesterol. However, a role for adipose in cholesterol lipidation of high-density lipoprotein (HDL) in vivo is not established. We present the first evidence that adipocytes support transfer of cholesterol to HDL in vivo as well as in vitro and implicate ATP-binding cassette subfamily A member 1 (ABCA1) and scavenger receptor class B type I (SR-BI), but not ATP-binding cassette subfamily G member 1 (ABCG1), cholesterol transporters in this process. Cholesterol efflux from wild-type, ABCA1(-/-), SR-BI(-/-), and ABCG1(-/-) adipocytes to apolipoprotein A-I (apoA-I) and HDL3 were measured in vitro. 3T3L1 adipocytes, labeled with (3)H-cholesterol, were injected intraperitoneally into wild-type, apoA-I transgenic, and apoA-I(-/-) mice, and tracer movement onto plasma HDL was monitored. Identical studies were performed with labeled wild-type, ABCA1(-/-), or SR-BI(-/-) mouse embryonic fibroblast adipocytes. The effect of tumor necrosis factor-alpha on transporter expression and cholesterol efflux was monitored during adipocyte differentiation. Cholesterol efflux to apoA-I and HDL3 was impaired in ABCA1(-/-) and SR-BI(-/-) adipocytes, respectively, with no effect observed in ABCG1(-/-) adipocytes. Intraperitoneal injection of labeled 3T3L1 adipocytes resulted in increased HDL-associated (3)H-cholesterol in apoA-I transgenic mice but reduced levels in apoA-I(-/-) animals. Intraperitoneal injection of labeled ABCA1(-/-) or SR-BI(-/-) adipocytes reduced plasma counts relative to their respective controls. Tumor necrosis factor-alpha reduced both ABCA1 and SR-BI expression and impaired cholesterol efflux from partially differentiated adipocytes. These data suggest a novel metabolic function of adipocytes in promoting cholesterol transfer to HDL in vivo and implicate adipocyte SR-BI and ABCA1, but not ABCG1, in this process. Furthermore, adipocyte modulation of HDL may be impaired in adipose inflammatory disease states such as type 2 diabetes mellitus.

Enhanced free cholesterol, SREBP-2 and StAR expression in human NASH

Non-alcoholic fatty liver disease (NAFLD) pathogenesis remains unknown. Due to the emerging role of free cholesterol (FC) in NAFLD, our aim was to examine the correlation between FC accumulation in patients with NAFLD and the expression of enzymes that regulate cholesterol homeostasis. Filipin staining, indicative of FC accumulation, and real-time PCR analyses were performed in 31 NAFLD patients and in seven controls. All NASH patients (n=14) and 4 out of 17 patients with steatosis exhibited filipin staining compared to controls (0 out of 7 subjects with normal liver histology and BMI). Sterol regulatory element-binding protein-2 (SREBP-2) mRNA levels were 7- and 3-fold higher in NASH and steatosis patients, respectively, compared to controls. Since hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the key enzyme in cholesterol synthesis and transcriptionally controlled by SREBP-2 we measured its mRNA levels, being 3- to 4-fold higher in NAFLD compared to controls, without any difference between NASH and steatosis patients. Fatty acid synthase (FAS) and SREBP-1c expression were not significantly induced in NAFLD, while ATP-binding cassette sub-family G member 1 (ABCG1), a transporter involved in cholesterol egress, and acyl-CoA-cholesterol acyltransferase mRNA levels were modestly increased (1.5- to 2.5-fold, p<0.05), regardless of fibrosis. Interestingly, mRNA levels of steroidogenic acute regulatory protein (StAR), a mitochondrial-cholesterol transporting polypeptide, increased 7- and 15-fold in steatosis and NASH patients, respectively, compared to controls. FC increases in NASH and correlates with SREBP-2 induction. Moreover, StAR overexpression in NASH suggests that mitochondrial FC may be a player in disease progression and a novel target for intervention.

Phytosterolemia on the island of Kosrae: founder effect for a novel ABCG8 mutation results in high carrier rate and increased plasma plant sterol levels

Screening of 932 adults on the Pacific island of Kosrae for plasma plant sterol levels disclosed three subjects, two of them asymptomatic, with phytosterolemia. Sequencing the ATP binding cassette subfamily G member 8 (ABCG8) gene revealed a novel exon 2 mutation that causes a change in codon 24 from glutamine to histidine and a frame shift followed by a premature stop codon, precluding the formation of a functional ABCG8 protein. Genotyping of 1,090 Kosraens revealed 150 as carriers, a 13.8% carrier rate. DNA sequencing of 67 carriers revealed the same mutation as in the probands. In carriers, plasma campesterol and sitosterol levels were 55% and 30% higher, respectively, than in noncarriers. Moreover, compared with noncarriers, carriers showed 21% lower plasma levels of lathosterol, a surrogate marker for cholesterol biosynthesis. There was no difference between the groups in plasma total cholesterol, triglycerides, apolipoprotein B, or apolipoprotein A-I levels. In summary, on the island of Kosrae, a strong founder effect of a mutant ABCG8 allele results in a large number of carriers with increased plasma plant sterol levels and decreased lathosterol levels. The latter finding suggests that heterozygosity for a mutated ABCG8 allele results in a modest increase in dietary cholesterol absorption and a decrease in cholesterol biosynthesis.