Differential Gene Expression in Macrophages From Human Atherosclerotic Plaques Shows Convergence on Pathways Implicated by Genome-Wide Association Study Risk Variants.

Objective- Plaque macrophages are intricately involved in atherogenesis and plaque destabilization. We sought to identify functional pathways in human plaque macrophages that are differentially regulated in respect of (1) plaque stability and (2) lipid content. We hypothesized that differentially regulated macrophage gene sets would relate to genome-wide association study variants associated with risk of acute complications of atherosclerosis. Approach and Results- Forty patients underwent carotid magnetic resonance imaging for lipid quantification before endarterectomy. Carotid plaque macrophages were procured by laser capture microdissection from (1) lipid core and (2) cap region, in 12 recently symptomatic and 12 asymptomatic carotid plaques. Applying gene set enrichment analysis, a number of gene sets were found to selectively upregulate in symptomatic plaque macrophages, which corresponded to 7 functional pathways: inflammation, lipid metabolism, hypoxic response, cell proliferation, apoptosis, antigen presentation, and cellular energetics. Predicted upstream regulators included IL-1ß, TNF-α, and NF-κB. In vivo lipid quantification by magnetic resonance imaging correlated most strongly with the upregulation of genes of the IFN/ STAT1 pathways. Cross-interrogation of gene set enrichment analysis and meta-analysis gene set enrichment of variant associations showed lipid metabolism pathways, driven by genes coding for APOE and ABCA1/G1 coincided with known risk-associated SNPs (single nucleotide polymorphisms) from genome-wide association studies. Conclusions- Macrophages from recently symptomatic carotid plaques show differential regulation of functional gene pathways. There were additional quantitative relationships between plaque lipid content and key gene sets. The data show a plausible mechanism by which known genome-wide association study risk variants for atherosclerotic complications could be linked to (1) a relevant cellular process, in (2) the key cell type of atherosclerosis, in (3) a human disease-relevant setting.

JCAD, a Gene at the 10p11 Coronary Artery Disease Locus, Regulates Hippo Signaling in Endothelial Cells.

Objective- A large number of genetic loci have been associated with risk of coronary artery disease (CAD) through genome-wide association studies, however, for most loci the underlying biological mechanism is unknown. Determining the molecular pathways and cellular processes affected by these loci will provide new insights into CAD pathophysiology and may lead to new therapies. The CAD-associated variants at 10p11.23 fall in JCAD, which encodes an endothelial junction protein, however, its molecular function in endothelial cells is not known. In this study, we characterize the molecular role of JCAD (junctional cadherin 5 associated) in endothelial cells. Approach and Results- We show that JCAD knockdown in endothelial cells affects key phenotypes related to atherosclerosis including proliferation, migration, apoptosis, tube formation, and monocyte binding. We demonstrate that JCAD interacts with LATS2 (large tumor suppressor kinase 2) and negatively regulates Hippo signaling leading to increased activity of YAP (yes-associated protein), the transcriptional effector of the pathway. We also show by double siRNA knockdown that the phenotypes caused by JCAD knockdown require LATS2 and that JCAD is involved in transmission of RhoA-mediated signals into the Hippo pathway. In human tissues, we find that the CAD-associated lead variant, rs2487928, is associated with expression of JCAD in arteries, including atherosclerotic arteries. Gene co-expression analyses across disease-relevant tissues corroborate our phenotypic findings and support the link between JCAD and Hippo signaling. Conclusions- Our results show that JCAD negatively regulates Hippo signaling in endothelial cells and we suggest that JCAD contributes to atherosclerosis by mediating YAP activity and contributing to endothelial dysfunction.

Mutant Muscle LIM Protein C58G causes cardiomyopathy through protein depletion.

Cysteine and glycine rich protein 3 (CSRP3) encodes Muscle LIM Protein (MLP), a well-established disease gene for Hypertrophic Cardiomyopathy (HCM). MLP, in contrast to the proteins encoded by the other recognised HCM disease genes, is non-sarcomeric, and has important signalling functions in cardiomyocytes. To gain insight into the disease mechanisms involved, we generated a knock-in mouse (KI) model, carrying the well documented HCM-causing CSRP3 mutation C58G. In vivo phenotyping of homozygous KI/KI mice revealed a robust cardiomyopathy phenotype with diastolic and systolic left ventricular dysfunction, which was supported by increased heart weight measurements. Transcriptome analysis by RNA-seq identified activation of pro-fibrotic signalling, induction of the fetal gene programme and activation of markers of hypertrophic signalling in these hearts. Further ex vivo analyses validated the activation of these pathways at transcript and protein level. Intriguingly, the abundance of MLP decreased in KI/KI mice by 80% and in KI/+ mice by 50%. Protein depletion was also observed in cellular studies for two further HCM-causing CSRP3 mutations (L44P and S54R/E55G). We show that MLP depletion is caused by proteasome action. Moreover, MLP C58G interacts with Bag3 and results in a proteotoxic response in the homozygous knock-in mice, as shown by induction of Bag3 and associated heat shock proteins. In conclusion, the newly generated mouse model provides insights into the underlying disease mechanisms of cardiomyopathy caused by mutations in the non-sarcomeric protein MLP. Furthermore, our cellular experiments suggest that protein depletion and proteasomal overload also play a role in other HCM-causing CSPR3 mutations that we investigated, indicating that reduced levels of functional MLP may be a common mechanism for HCM-causing CSPR3 mutations.

A common LPA null allele associates with lower lipoprotein(a) levels and coronary artery disease risk.

OBJECTIVE: Increased levels of lipoprotein(a) are a highly heritable risk factor for coronary artery disease (CAD). The genetic determinants of lipoprotein(a) levels are mainly because of genetic variation in the apolipoprotein(a) gene (LPA). We have tested the association of a relatively common null allele of LPA with lipoprotein(a) levels and CAD risk in a large case-control cohort. We have also examined how null allele genotyping complements apolipoprotein(a) isoform typing to refine the relationship between LPA isoform size and circulating lipoprotein(a) levels. APPROACH AND RESULTS: The LPA null allele (rs41272114) was genotyped in the PROCARDIS (Precocious Coronary Artery Disease) case-control cohort (4073 CAD cases and 4225 controls). Lipoprotein(a) levels were measured in 909 CAD cases and 922 controls; apolipoprotein(a) isoform size was estimated using sodium dodecyl sulfate-agarose gel electrophoresis and a high-throughput quantitative polymerase chain reaction-based method. Null carriers are common (null allele frequency, 3%) and have significantly lower circulating lipoprotein(a) levels (P=2.1×10(-10)) and reduced CAD risk (odds ratio, 0.79 [0.66-0.97]; P=0.023) compared with noncarriers. An additive allelic model of apolipoprotein(a) isoform size, refined by null allele genotype and quantitative polymerase chain reaction values, showed a sigmoid relationship with lipoprotein(a) levels, with baseline levels for longer isoform alleles and progressively higher levels of lipoprotein(a) for shorter isoform alleles. CONCLUSIONS: The LPA null allele (rs41272114) is associated with decreased circulating lipoprotein(a) levels and decreased CAD risk. Incorporating rs41272114 refined apolipoprotein(a) isoform size typing obtained by immunoblotting and quantitative polymerase chain reaction. A joint genomic and isoform analysis revealed details of the relationship between apolipoprotein(a) isoform size and circulating lipoprotein(a) level consistent with a threshold effect on lipoprotein secretion.

PHACTR1 modulates vascular compliance but not endothelial function: a translational study.

AIMS: The non-coding locus at 6p24 located in Intron 3 of PHACTR1 has consistently been implicated as a risk allele in myocardial infarction and multiple other vascular diseases. Recent murine studies have identified a role for Phactr1 in the development of atherosclerosis. However, the role of PHACTR1 in vascular tone and in vivo vascular remodelling has yet to be established. The aim of this study was to investigate the role of PHACTR1 in vascular function. METHODS AND RESULTS: Prospectively recruited coronary artery disease (CAD) patients undergoing bypass surgery and retrospectively recruited spontaneous coronary artery dissection (SCAD) patients and matched healthy volunteers were genotyped at the PHACTR1 rs9349379 locus. We observed a significant association between the PHACTR1 loci and changes in distensibility in both the ascending aorta (AA = 0.0053 ± 0.0004, AG = 0.0041 ± 0.003, GG = 0.0034 ± 0.0009, P < 0.05, n = 58, 54, and 7, respectively) and carotid artery (AA = 12.83 ± 0.51, AG = 11.14 ± 0.38, GG = 11.69 ± 0.66, P < 0.05, n = 70, 65, and 18, respectively). This association was not observed in the descending aorta or in SCAD patients. In contrast, the PHACTR1 locus was not associated with changes in endothelial cell function with no association between the rs9349379 locus and in vivo or ex vivo vascular function observed in CAD patients. This finding was confirmed in our murine model where the loss of Phactr1 on the pro-atherosclerosis ApoE-/- background did not alter ex vivo vascular function. CONCLUSION: In conclusion, we have shown a role for PHACTR1 in arterial compliance across multiple vascular beds. Our study suggests that PHACTR1 has a key structural role within the vasculature.

Genetic variants associated with Lp(a) lipoprotein level and coronary disease.

BACKGROUND: An increased level of Lp(a) lipoprotein has been identified as a risk factor for coronary artery disease that is highly heritable. The genetic determinants of the Lp(a) lipoprotein level and their relevance for the risk of coronary disease are incompletely understood. METHODS: We used a novel gene chip containing 48,742 single-nucleotide polymorphisms (SNPs) in 2100 candidate genes to test for associations in 3145 case subjects with coronary disease and 3352 control subjects. Replication was tested in three independent populations involving 4846 additional case subjects with coronary disease and 4594 control subjects. RESULTS: Three chromosomal regions (6q26-27, 9p21, and 1p13) were strongly associated with the risk of coronary disease. The LPA locus on 6q26-27 encoding Lp(a) lipoprotein had the strongest association. We identified a common variant (rs10455872) at the LPA locus with an odds ratio for coronary disease of 1.70 (95% confidence interval [CI], 1.49 to 1.95) and another independent variant (rs3798220) with an odds ratio of 1.92 (95% CI, 1.48 to 2.49). Both variants were strongly associated with an increased level of Lp(a) lipoprotein, a reduced copy number in LPA (which determines the number of kringle IV-type 2 repeats), and a small Lp(a) lipoprotein size. Replication studies confirmed the effects of both variants on the Lp(a) lipoprotein level and the risk of coronary disease. A meta-analysis showed that with a genotype score involving both LPA SNPs, the odds ratios for coronary disease were 1.51 (95% CI, 1.38 to 1.66) for one variant and 2.57 (95% CI, 1.80 to 3.67) for two or more variants. After adjustment for the Lp(a) lipoprotein level, the association between the LPA genotype score and the risk of coronary disease was abolished. CONCLUSIONS: We identified two LPA variants that were strongly associated with both an increased level of Lp(a) lipoprotein and an increased risk of coronary disease. Our findings provide support for a causal role of Lp(a) lipoprotein in coronary disease.

Pre-eclampsia and offspring cardiovascular health: mechanistic insights from experimental studies.

Pre-eclampsia is increasingly recognized as more than an isolated disease of pregnancy. Women who have had a pregnancy complicated by pre-eclampsia have a 4-fold increased risk of later cardiovascular disease. Intriguingly, the offspring of affected pregnancies also have an increased risk of higher blood pressure and almost double the risk of stroke in later life. Experimental approaches to identify the key features of pre-eclampsia responsible for this programming of offspring cardiovascular health, or the key biological pathways modified in the offspring, have the potential to highlight novel targets for early primary prevention strategies. As pre-eclampsia occurs in 2-5% of all pregnancies, the findings are relevant to the current healthcare of up to 3 million people in the U.K. and 15 million people in the U.S.A. In the present paper, we review the current literature that concerns potential mechanisms for adverse cardiovascular programming in offspring exposed to pre-eclampsia, considering two major areas of investigation: first, experimental models that mimic features of the in utero environment characteristic of pre-eclampsia, and secondly, how, in humans, offspring cardiovascular phenotype is altered after exposure to pre-eclampsia. We compare and contrast the findings from these two bodies of work to develop insights into the likely key pathways of relevance. The present review and analysis highlights the pivotal role of long-term changes in vascular function and identifies areas of growing interest, specifically, response to hypoxia, immune modification, epigenetics and the anti-angiogenic in utero milieu.

Discovery and systematic characterization of risk variants and genes for coronary artery disease in over a million participants.

The discovery of genetic loci associated with complex diseases has outpaced the elucidation of mechanisms of disease pathogenesis. Here we conducted a genome-wide association study (GWAS) for coronary artery disease (CAD) comprising 181,522 cases among 1,165,690 participants of predominantly European ancestry. We detected 241 associations, including 30 new loci. Cross-ancestry meta-analysis with a Japanese GWAS yielded 38 additional new loci. We prioritized likely causal variants using functionally informed fine-mapping, yielding 42 associations with less than five variants in the 95% credible set. Similarity-based clustering suggested roles for early developmental processes, cell cycle signaling and vascular cell migration and proliferation in the pathogenesis of CAD. We prioritized 220 candidate causal genes, combining eight complementary approaches, including 123 supported by three or more approaches. Using CRISPR-Cas9, we experimentally validated the effect of an enhancer in MYO9B, which appears to mediate CAD risk by regulating vascular cell motility. Our analysis identifies and systematically characterizes >250 risk loci for CAD to inform experimental interrogation of putative causal mechanisms for CAD.

A key role for the novel coronary artery disease gene JCAD in atherosclerosis via shear stress mechanotransduction.

AIMS: Genome-wide association studies (GWAS) have consistently identified an association between coronary artery disease (CAD) and a locus on chromosome 10 containing a single gene, JCAD (formerly KIAA1462). However, little is known about the mechanism by which JCAD could influence the development of atherosclerosis. METHODS AND RESULTS: Vascular function was quantified in subjects with CAD by flow-mediated dilatation (FMD) and vasorelaxation responses in isolated blood vessel segments. The JCAD risk allele identified by GWAS was associated with reduced FMD and reduced endothelial-dependent relaxations. To study the impact of loss of Jcad on atherosclerosis, Jcad-/- mice were crossed to an ApoE-/- background and fed a high-fat diet from 6 to16 weeks of age. Loss of Jcad did not affect blood pressure or heart rate. However, Jcad-/-ApoE-/- mice developed significantly less atherosclerosis in the aortic root and the inner curvature of the aortic arch. En face analysis revealed a striking reduction in pro-inflammatory adhesion molecules at sites of disturbed flow on the endothelial cell layer of Jcad-/- mice. Loss of Jcad lead to a reduced recovery perfusion in response to hind limb ischaemia, a model of altered in vivo flow. Knock down of JCAD using siRNA in primary human aortic endothelial cells significantly reduced the response to acute onset of flow, as evidenced by reduced phosphorylation of NF-КB, eNOS, and Akt. CONCLUSION: The novel CAD gene JCAD promotes atherosclerotic plaque formation via a role in the endothelial cell shear stress mechanotransduction pathway.

Neonatal Micro-RNA Profile Determines Endothelial Function in Offspring of Hypertensive Pregnancies.

Offspring of hypertensive pregnancies are at increased risk of developing hypertension in adulthood. In the neonatal period they display endothelial cell dysfunction and altered microvascular development. MicroRNAs, as important endothelial cellular regulators, may play a role in this early endothelial dysfunction. Therefore we identified differential microRNA patterns in endothelial cells from offspring of hypertensive pregnancies and determined their role in postnatal vascular cell function. Studies were performed on human umbilical vein endothelial cell (HUVECs) samples from 57 pregnancies. Unbiased RNA-sequencing identified 30 endothelial-related microRNAs differentially expressed in HUVECs from hypertensive compared to normotensive pregnancies. Quantitative reverse transcription PCR (RT-qPCR) confirmed a significant higher expression level of the top candidate, miR-146a. Combined miR-146a targeted gene expression and pathway analysis revealed significant alterations in genes involved in inflammation, angiogenesis and immune response in the same HUVECs. Elevated miR-146a expression level at birth identified cells with reduced ability for in vitro vascular tube formation, which was rescued by miR-146a inhibition. In contrast, miR-146a overexpression significantly reduced vascular tube formation in HUVECs from normotensive pregnancies. Finally, we confirmed that mir146a levels at birth predicted in vivo microvascular development during the first three postnatal months. Offspring of hypertensive pregnancy have a distinct endothelial regulatory microRNA profile at birth, which is related to altered endothelial cell behaviour, and predicts patterns of microvascular development during the first three months of life. Modification of this microRNA profile in vitro can restore impaired vascular cell function.

Network analysis of coronary artery disease risk genes elucidates disease mechanisms and druggable targets.

Genome-wide association studies (GWAS) have identified over two hundred chromosomal loci that modulate risk of coronary artery disease (CAD). The genes affected by variants at these loci are largely unknown and an untapped resource to improve our understanding of CAD pathophysiology and identify potential therapeutic targets. Here, we prioritized 68 genes as the most likely causal genes at genome-wide significant loci identified by GWAS of CAD and examined their regulatory roles in 286 metabolic and vascular tissue gene-protein sub-networks ("modules"). The modules and genes within were scored for CAD druggability potential. The scoring enriched for targets of cardiometabolic drugs currently in clinical use and in-depth analysis of the top-scoring modules validated established and revealed novel target tissues, biological processes, and druggable targets. This study provides an unprecedented resource of tissue-defined gene-protein interactions directly affected by genetic variance in CAD risk loci.

Association analyses based on false discovery rate implicate new loci for coronary artery disease.

Genome-wide association studies (GWAS) in coronary artery disease (CAD) had identified 66 loci at 'genome-wide significance' (P < 5 × 10-8) at the time of this analysis, but a much larger number of putative loci at a false discovery rate (FDR) of 5% (refs. 1,2,3,4). Here we leverage an interim release of UK Biobank (UKBB) data to evaluate the validity of the FDR approach. We tested a CAD phenotype inclusive of angina (SOFT; ncases = 10,801) as well as a stricter definition without angina (HARD; ncases = 6,482) and selected cases with the former phenotype to conduct a meta-analysis using the two most recent CAD GWAS. This approach identified 13 new loci at genome-wide significance, 12 of which were on our previous list of loci meeting the 5% FDR threshold, thus providing strong support that the remaining loci identified by FDR represent genuine signals. The 304 independent variants associated at 5% FDR in this study explain 21.2% of CAD heritability and identify 243 loci that implicate pathways in blood vessel morphogenesis as well as lipid metabolism, nitric oxide signaling and inflammation.

Genotypic effect of the -565C>T polymorphism in the ABCA1 gene promoter on ABCA1 expression and severity of atherosclerosis.

OBJECTIVE: Loss-of-function mutations of the ATP-binding cassette transporter A1 (ABCA1) gene cause Tangier disease, a rare genetic disorder with accumulation of lipid-laden macrophages and increased risk of atherosclerosis. Common variants of this gene may be a genetic factor for atherosclerosis in the general population. This study was performed to test the reported association between the -565C>T polymorphism and atherosclerosis severity and to investigate whether this variant per se had an effect on promoter activity of the ABCA1 gene. METHODS AND RESULTS: A cohort of patients with coronary atherosclerosis were genotyped for the -565C>T polymorphism. Logistic regression analyses showed that homozygotes of the -565T allele had greatest mean number of diseased coronary arteries, particular in nonsmokers. Real-time reverse-transcriptase polymerase chain reaction showed that in atherosclerotic plaques removed from patients undergoing endarteretomy, ABCA1 expression levels were lowest in those who had the T/T genotype and highest in those of the C/C genotype. Transfection and reporter assays demonstrated that in cultured macrophages, the -565T allelic promoter had a lower activity in driving gene expression than the -565C allelic promoter. Electrophoretic mobility shift assays displayed differential binding of nuclear proteins to the 2 alleles. CONCLUSIONS: These results indicate that the -565C>T polymorphism has an allele-specific effect on ABCA1 gene expression and provide further evidence of a genotypic effect on coronary atherosclerosis severity. The study showed that the ABCA1 gene -565C>T polymorphism was associated with severity of coronary atherosclerosis in a cohort of patients from Southern England and that this sequence variant per se had an effect on promoter activity of the ABCA1 gene. The data support the notion that common ABCA1 gene variants can contribute to interindividual variability in atherosclerosis susceptibility and severity.

Association of Maternal Antiangiogenic Profile at Birth With Early Postnatal Loss of Microvascular Density in Offspring of Hypertensive Pregnancies.

Offspring of hypertensive pregnancies are more likely to have microvascular rarefaction and increased blood pressure in later life. We tested the hypothesis that maternal angiogenic profile during a hypertensive pregnancy is associated with fetal vasculogenic capacity and abnormal postnatal microvascular remodeling. Infants (n=255) born after either hypertensive or normotensive pregnancies were recruited for quantification of postnatal dermal microvascular structure at birth and 3 months of age. Vasculogenic cell potential was assessed in umbilical vein endothelial cells from 55 offspring based on in vitro microvessel tube formation and proliferation assays. Maternal angiogenic profile (soluble fms-like tyrosine kinase-1, soluble endoglin, vascular endothelial growth factor, and placental growth factor) was measured from postpartum plasma samples to characterize severity of pregnancy disorder. At birth, offspring born after hypertensive pregnancy had similar microvessel density to those born after a normotensive pregnancy, but during the first 3 postnatal months, they had an almost 2-fold greater reduction in total vessel density (-17.7±16.4% versus -9.9±18.7%; P=0.002). This postnatal loss varied according to the vasculogenic capacity of the endothelial cells of the infant at birth (r=0.49; P=0.02). The degree of reduction in both in vitro and postnatal in vivo vascular development was proportional to levels of antiangiogenic factors in the maternal circulation. In conclusion, our data indicate that offspring born to hypertensive pregnancies have reduced vasculogenic capacity at birth that predicts microvessel density loss over the first 3 postnatal months. Degree of postnatal microvessel reduction is proportional to levels of antiangiogenic factors in the maternal circulation at birth.

Chronic Activation of γ2 AMPK Induces Obesity and Reduces ß Cell Function.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

No Association of Coronary Artery Disease with X-Chromosomal Variants in Comprehensive International Meta-Analysis.

In recent years, genome-wide association studies have identified 58 independent risk loci for coronary artery disease (CAD) on the autosome. However, due to the sex-specific data structure of the X chromosome, it has been excluded from most of these analyses. While females have 2 copies of chromosome X, males have only one. Also, one of the female X chromosomes may be inactivated. Therefore, special test statistics and quality control procedures are required. Thus, little is known about the role of X-chromosomal variants in CAD. To fill this gap, we conducted a comprehensive X-chromosome-wide meta-analysis including more than 43,000 CAD cases and 58,000 controls from 35 international study cohorts. For quality control, sex-specific filters were used to adequately take the special structure of X-chromosomal data into account. For single study analyses, several logistic regression models were calculated allowing for inactivation of one female X-chromosome, adjusting for sex and investigating interactions between sex and genetic variants. Then, meta-analyses including all 35 studies were conducted using random effects models. None of the investigated models revealed genome-wide significant associations for any variant. Although we analyzed the largest-to-date sample, currently available methods were not able to detect any associations of X-chromosomal variants with CAD.

Regulation of corticotropin-releasing hormone type 2 receptors by multiple promoters and alternative splicing: identification of multiple splice variants.

We demonstrate that multiple promoters and alternate splicing regulate expression of the human CRH receptor type 2 (CRHR2) gene. We show that flanking regions to the first exons drive promoter activity in both endogenously and nonendogenously expressing cell lines. Putative promoter elements have been identified that are conserved between species, including the comparison of CRHR2gamma in nonhuman primates that was previously known only in humans, which may be responsible for subtype tissue specific regulation. We have identified novel transcripts produced by alternate splicing of the first exon of CRHR2beta (beta1a) with various combinations of the 5' exons including a novel exon (beta1c) spliced to the common exons. The 5' structure of the gene permits many other combinations of alternate splicing that may arise as part of a regulatory mechanism controlling functional receptor expression. The 5'-untranslated region of the first exons has been extended; and 3' acceptor sites identified within the 5' untranslated region of CRHR2gamma and CRHR2alpha are used during alternate splicing of CRHR2beta upstream exons. This has important implications because various reports on the expression of CRHR2gamma and CRHR2alpha have been unable to discriminate between the functional receptor and CRHR2beta alternate splice variants. Only the described sequences upstream of the 3' splice site are unique to CRHR2gamma and CRHR2alpha.

A comprehensive 1,000 Genomes-based genome-wide association meta-analysis of coronary artery disease.

Existing knowledge of genetic variants affecting risk of coronary artery disease (CAD) is largely based on genome-wide association study (GWAS) analysis of common SNPs. Leveraging phased haplotypes from the 1000 Genomes Project, we report a GWAS meta-analysis of ∼185,000 CAD cases and controls, interrogating 6.7 million common (minor allele frequency (MAF) > 0.05) and 2.7 million low-frequency (0.005 < MAF < 0.05) variants. In addition to confirming most known CAD-associated loci, we identified ten new loci (eight additive and two recessive) that contain candidate causal genes newly implicating biological processes in vessel walls. We observed intralocus allelic heterogeneity but little evidence of low-frequency variants with larger effects and no evidence of synthetic association. Our analysis provides a comprehensive survey of the fine genetic architecture of CAD, showing that genetic susceptibility to this common disease is largely determined by common SNPs of small effect size.

Functional single-nucleotide polymorphism in acetyl-CoA carboxylase ACACB gene promoter.

The acetyl-CoA carboxylase isoform ACC2 expressed in the liver generates malonyl-CoA, which primarily regulates fatty acid oxidation through inhibition of the mitochondrial carrier carnitine palmitoyl-CoA transferase-I. Activity is initiated by sterol regulatory element-binding protein-1 (SREBP-1) binding to steroid response elements SRE in ACACB gene promoter P-II. We proposed that sequence variation in the promoter might affect expression. We investigated the effect of a single-nucleotide polymorphism -368 C/T (rs16939972) in ACACB P-II on activity in transfected HepG2 hepatoma cells. The T-allele construct showed significantly lower activity than the C-allele (p = 0.016) but only in the presence of SREBP-1a overexpression. Electrophoretic mobility shift assays showed that HepG2 nuclear proteins bound specifically to both allele probes, but with higher affinity to the T-allele. We tested competition for nuclear protein binding between the T-allele probe and unlabeled probes containing consensus sequences for six candidate transcription factors plus SREBP-1a. The SREBP-1a competitor probe had no effect on the shifted complex. GATA, c-Myb, and GR competitor probes abolished the complex; however, these proteins were undetectable in mass spectrometry of gel extracts from shifted bands. In conclusion, the -368 C/T single-nucleotide polymorphism in ACACB P-II binds HepG2 nuclear proteins that affect promoter activity in an allele-specific fashion.

Effect of high-fat feeding on expression of genes controlling availability of dopamine in mouse hypothalamus.

OBJECTIVE: Hypothalamic centers integrate external signals of nutrient availability and energy status and initiate responses to maintain homeostasis. Quantifying changes in hypothalamic gene expression in the presence of nutrient excess may identify novel responsive elements. METHODS: Affymetrix Mouse Genome 430 2.0 oligonucleotide microarrays containing 45 102 probe sets were used to interrogate differential expression of genes in dietary-induced obesity model C57BL6 inbred mice fed a high-fat (35% fat; n=8) or standard (4% fat; n=6) diet from 3 to 15 wk of age. Ontologies of regulated genes were examined and expression of selected genes was validated by quantitative real-time polymerase chain reaction. RESULTS: One thousand two hundred twelve unique gene transcripts showed altered expression on the microarrays. Gene ontology analysis revealed changes in neuropeptide genes responding to leptin, Pomc, Cart, Npy, and Agrp, compatible with a homeostatic response to high-fat intake, although mean weight increased 2.3-fold compared with standard fed mice (P<0.001). Neurotransmitter system ontologies revealed upregulation of five genes controlling availability of dopamine. Changes in Th tyrosine hydroxylase (2.1-fold) and Slc18a2 solute carrier family 18 (vesicular monoamine), member 2 (4.4-fold) controlling synthesis and release, and Slc6a3 solute carrier family 6 (neurotransmitter transporter, dopamine), member 3 (4.8-fold), Snca alpha-synuclein (1.3-fold), and Maoa monoamine oxidase (1.9-fold) limiting availability were confirmed by quantitative real-time polymerase chain reaction. CONCLUSION: Expression of five genes involved in availability of dopamine was increased after a high-fat diet. Failure to reduce dopamine availability sufficiently, to counter the feeding reward effect, could contribute to diet-induced obesity in these mice.