👤 Ruth McPherson

Constitutive Photomorphogenesis Protein 1 homolog (COP1) is a conserved E3 ligase with key roles in several biological systems. Prior work in hepatocyte-derived tumors categorized COP1 as an oncogene, but its role in untransformed hepatocytes remains largely unexplored. Here, we have investigated the role of COP1 in primary human hepatocytes and two transformed hepatocyte models, HepG2 and HuH-7 cells. The role of COP1 was tested by silencing and transduction experiments in HepG2, HuH-7, and primary human hepatocytes. Transcription array data of COP1-suppressed cells were generated and analyzed using clustering analyses. Cellular impacts were examined by proliferation assays, qRT-PCR, western blotting, reporter assays, and APOB enzyme-linked immunosorbent assays. COP1 suppression had no noticeable impact on HepG2 and HuH-7 proliferation and was associated with contrasting rather than congruent transcriptome changes. Transcriptomic changes were consistent with perturbed metabolism in primary hepatocytes and HepG2 cells and impaired cell cycle regulation in HuH-7 cells. In HepG2 and primary hepatocytes but not in HuH-7 cells, COP1 suppression reduced the expression of important hepatic regulators and markers. COP1 downregulation reduced hepatic nuclear factor-4 alpha (HNF4A) abundance and function, as assessed by a lower abundance of key HNF4A targets, reduced APOB secretion, and reporter assays. HNF4A function could be restored by introducing a siRNA-resistant COP1 transgene, whereas HNF4A restoration partially rescued COP1 silencing in HepG2 cells. Our results identify and detail a pivotal regulatory role of COP1 in hepatocytes, in part through HNF4A. Show less

We recently reported that the long non-coding RNA TRIBAL/TRIB1AL was required to sustain key hepatocyte functions. Here, we identify HepaRG cells as a model for studying TRIBAL and provide additional validation and functional insights. In contrast to HepG2 and HuH-7 cells, differentiated HepaRG cells showed similarities to primary hepatocytes in response to TRIBAL suppression. TRIBAL suppression was associated with reduced HNF4A and MLXIPL abundance in hepatocytes and HepaRG cells. TRIBAL targeting using a panel of cognate antisense oligonucleotides confirmed specificity. A comparison of TRIBAL-suppressed hepatocyte and HepaRG transcriptomics identified extensive functional overlap. Biological ontologies associated with key hepatic metabolic functions were predicted to be inhibited in both models. Comparative analyses with TRIB1-suppressed HepaRG cells, a central metabolic regulator vicinal to TRIBAL, also revealed extensive functional congruence with TRIBAL. Interestingly, TRIBAL transduction failed to restore function in TRIBAL-suppressed cells, which may be linked to structural differences, as supported by contrasting RNAse R sensitivities between the endogenous and transduced forms. In summary, these findings support the use of HepaRG cells as an experimental model to study TRIBAL and underscore its importance in regulating key hepatocyte genes essential for metabolic function. Show less

Genome-wide association studies identified a 20-Kb region of chromosome 8 (8q24.13) associated with plasma lipids, hepatic steatosis, and risk for coronary artery disease. The region is proximal to Using recently available expression quantitative trait loci data and hepatocyte models, we further investigated this locus by Mendelian randomization analysis. Following antisense oligonucleotide targeting of TRIBAL, transcription array, quantitative reverse transcription polymerase chain reaction, and enrichment analyses were performed and effects on apoB and triglyceride secretion were determined. Mendelian randomization analysis supports a causal relationship between genetically determined hepatic This work identifies Show less

Here, we performed a genome-wide search for methylation sites that contribute to the risk of obesity. We integrated methylation quantitative trait locus (mQTL) data with BMI GWAS information through a SNP-based multiomics approach to identify genomic regions where mQTLs for a methylation site co-localize with obesity risk SNPs. We then tested whether the identified site contributed to BMI through Mendelian randomization. We identified multiple methylation sites causally contributing to the risk of obesity. We validated these findings through a replication stage. By integrating expression quantitative trait locus (eQTL) data, we noted that lower methylation at cg21178254 site upstream of Show less

OBJECTIVE: Leveraging microRNA-Seq data and the 1000 Genomes imputed genotypes, we identified rs174561 as a strong microRNA quantitative trait loci for circulating microRNA-1908-5p with higher miR-1908-5p and reduced LDL (lowdensity lipoprotein)-cholesterol, fasting glucose and A1c concentrations in carriers of the rs-174561-C allele. Here, we have investigated the molecular mechanism(s) linking miR-1908-5p to LDL-C concentrations. APPROACH AND RESULTS: Transfection experiments demonstrate that the presence of the C allele significantly increases miR- 1908-5p abundance relative to the T allele. LDLR mRNA and low-density lipoprotein receptor (LDLR) total protein were unchanged in response to differential miR-1908-5p expression. However, the ratio of the cleaved to full-length form of LDLR decreased with miR-1908-5p mimic and increased with miR-1908-5p inhibitor treatment. BMP1 (bone morphogenetic protein 1) is a protease responsible for LDLR cleavage, and we show that miR-1908-5p mimic reduces BMP1 mRNA. Using a reporter array, we identified the TGF-β (transforming growth factor-beta) signaling pathway activity to be reduced by miR- 1908-5p mimic treatment, and this was associated with reduced TGFB1 expression. TGF-β signaling increases BMP1, and we further demonstrate that the effect of miR-1908-5p on LDLR cleavage is abolished by exogenous TGF-β treatment. CONCLUSIONS: These findings uncover a mechanism whereby miR-1908-5p reduces TGFB1 abundance resulting in lower expression of BMP1, ultimately leading to reduced LDLR cleavage. Cleavage of the mature LDLR is known to reduce cell surface affinity for LDL, thereby linking miR-1908-5p to lower circulating LDL-cholesterol levels. Show less

In this study, we aimed to investigate functional mechanisms underlying coronary artery disease (CAD) loci and find molecular biomarkers for CAD. We devised a multiomics data analysis approach based on Mendelian randomization and utilized it to search for molecular biomarkers causally associated with the risk of CAD within genomic regions known to be associated with CAD. Through our CAD-centered multiomics data analysis approach, we identified 33 molecular biomarkers (probes) that were causally associated with the risk of CAD. The majority of these (N=19) were methylation probes; moreover, methylation was often behind the causal effect of expression/protein probes. We identified a number of novel loci that have a causal impact on CAD including We demonstrate that multiomics data analysis is a powerful approach to unravel the functional mechanisms underlying CAD loci and to identify novel molecular biomarkers. Our results indicate epigenetic modifications are important in the pathogenesis of CAD and identifying and targeting these sites is of potential therapeutic interest to address the detrimental effects of both environmental and genetic factors. Show less

Therapies that inhibit CETP (cholesteryl ester transfer protein) have failed to demonstrate a reduction in risk for coronary heart disease (CHD). Human DNA sequence variants that truncate the To test whether protein-truncating variants (PTVs) at the We sequenced the exons of the Compared with noncarriers, carriers of PTV at Show less

Genome-wide association studies have so far identified 56 loci associated with risk of coronary artery disease (CAD). Many CAD loci show pleiotropy; that is, they are also associated with other diseases or traits. This study sought to systematically test if genetic variants identified for non-CAD diseases/traits also associate with CAD and to undertake a comprehensive analysis of the extent of pleiotropy of all CAD loci. In discovery analyses involving 42,335 CAD cases and 78,240 control subjects we tested the association of 29,383 common (minor allele frequency >5%) single nucleotide polymorphisms available on the exome array, which included a substantial proportion of known or suspected single nucleotide polymorphisms associated with common diseases or traits as of 2011. Suggestive association signals were replicated in an additional 30,533 cases and 42,530 control subjects. To evaluate pleiotropy, we tested CAD loci for association with cardiovascular risk factors (lipid traits, blood pressure phenotypes, body mass index, diabetes, and smoking behavior), as well as with other diseases/traits through interrogation of currently available genome-wide association study catalogs. We identified 6 new loci associated with CAD at genome-wide significance: on 2q37 (KCNJ13-GIGYF2), 6p21 (C2), 11p15 (MRVI1-CTR9), 12q13 (LRP1), 12q24 (SCARB1), and 16q13 (CETP). Risk allele frequencies ranged from 0.15 to 0.86, and odds ratio per copy of the risk allele ranged from 1.04 to 1.09. Of 62 new and known CAD loci, 24 (38.7%) showed statistical association with a traditional cardiovascular risk factor, with some showing multiple associations, and 29 (47%) showed associations at p < 1 × 10 We identified 6 loci associated with CAD at genome-wide significance. Several CAD loci show substantial pleiotropy, which may help us understand the mechanisms by which these loci affect CAD risk. Show less

Metabolites derived from dietary choline and L-carnitine, such as trimethylamine N-oxide and betaine, have recently been identified as novel risk factors for atherosclerosis in mice and humans. We sought to identify genetic factors associated with plasma betaine levels and determine their effect on risk of coronary artery disease (CAD). A two-stage genome-wide association study (GWAS) identified two significantly associated loci on chromosomes 2q34 and 5q14.1. The lead variant on 2q24 (rs715) localizes to carbamoyl-phosphate synthase 1 (CPS1), which encodes a mitochondrial enzyme that catalyses the first committed reaction and rate-limiting step in the urea cycle. Rs715 is also significantly associated with decreased levels of urea cycle metabolites and increased plasma glycine levels. Notably, rs715 yield a strikingly significant and protective association with decreased risk of CAD in only women. These results suggest that glycine metabolism and/or the urea cycle represent potentially novel sex-specific mechanisms for the development of atherosclerosis. Show less

Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance. When MI occurs early in life, genetic inheritance is a major component to risk. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families, whereas common variants at more than 45 loci have been associated with MI risk in the population. Here we evaluate how rare mutations contribute to early-onset MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes in which rare coding-sequence mutations were more frequent in MI cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare non-synonymous mutations were at 4.2-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). Approximately 2% of early MI cases harbour a rare, damaging mutation in LDLR; this estimate is similar to one made more than 40 years ago using an analysis of total cholesterol. Among controls, about 1 in 217 carried an LDLR coding-sequence mutation and had plasma LDL cholesterol > 190 mg dl(-1). At apolipoprotein A-V (APOA5), carriers of rare non-synonymous mutations were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol, whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding-sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase and apolipoprotein C-III (refs 18, 19). Combined, these observations suggest that, as well as LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk. Show less

Recent genome-wide association studies have identified multiple loci robustly associated with plasma lipids, which also contribute to extreme lipid phenotypes. However, these common genetic variants explain <12% of variation in lipid traits. Adiposity is also an important determinant of plasma lipoproteins, particularly plasma TGs and HDL cholesterol (HDLc) concentrations. Thus, interactions between genes and clinical phenotypes may contribute to this unexplained heritability. We have applied a weighted genetic risk score (GRS) for both plasma TGs and HDLc in two large cohorts at the extremes of BMI. Both BMI and GRS were strongly associated with these lipid traits. A significant interaction between obese/lean status and GRS was noted for each of TG (P(Interaction) = 2.87 × 10(-4)) and HDLc (P(Interaction) = 1.05 × 10(-3)). These interactions were largely driven by SNPs tagging APOA5, glucokinase receptor (GCKR), and LPL for TG, and cholesteryl ester transfer protein (CETP), GalNAc-transferase (GALNT2), endothelial lipase (LIPG), and phospholipid transfer protein (PLTP) for HDLc. In contrast, the GRSLDL cholesterol × adiposity interaction was not significant. Sexual dimorphism was evident for the GRSHDL on HDLc in obese (P(Interaction) = 0.016) but not lean subjects. SNP by BMI interactions may provide biological insight into specific genetic associations and missing heritability. Show less

Plasma triglyceride levels are heritable and are correlated with the risk of coronary heart disease. Sequencing of the protein-coding regions of the human genome (the exome) has the potential to identify rare mutations that have a large effect on phenotype. We sequenced the protein-coding regions of 18,666 genes in each of 3734 participants of European or African ancestry in the Exome Sequencing Project. We conducted tests to determine whether rare mutations in coding sequence, individually or in aggregate within a gene, were associated with plasma triglyceride levels. For mutations associated with triglyceride levels, we subsequently evaluated their association with the risk of coronary heart disease in 110,970 persons. An aggregate of rare mutations in the gene encoding apolipoprotein C3 (APOC3) was associated with lower plasma triglyceride levels. Among the four mutations that drove this result, three were loss-of-function mutations: a nonsense mutation (R19X) and two splice-site mutations (IVS2+1G→A and IVS3+1G→T). The fourth was a missense mutation (A43T). Approximately 1 in 150 persons in the study was a heterozygous carrier of at least one of these four mutations. Triglyceride levels in the carriers were 39% lower than levels in noncarriers (P<1×10(-20)), and circulating levels of APOC3 in carriers were 46% lower than levels in noncarriers (P=8×10(-10)). The risk of coronary heart disease among 498 carriers of any rare APOC3 mutation was 40% lower than the risk among 110,472 noncarriers (odds ratio, 0.60; 95% confidence interval, 0.47 to 0.75; P=4×10(-6)). Rare mutations that disrupt APOC3 function were associated with lower levels of plasma triglycerides and APOC3. Carriers of these mutations were found to have a reduced risk of coronary heart disease. (Funded by the National Heart, Lung, and Blood Institute and others.). Show less

Genetic studies might provide new insights into the biological mechanisms underlying lipid metabolism and risk of CAD. We therefore conducted a genome-wide association study to identify novel genetic determinants of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides. We combined genome-wide association data from 8 studies, comprising up to 17 723 participants with information on circulating lipid concentrations. We did independent replication studies in up to 37 774 participants from 8 populations and also in a population of Indian Asian descent. We also assessed the association between single-nucleotide polymorphisms (SNPs) at lipid loci and risk of CAD in up to 9 633 cases and 38 684 controls. We identified 4 novel genetic loci that showed reproducible associations with lipids (probability values, 1.6×10(-8) to 3.1×10(-10)). These include a potentially functional SNP in the SLC39A8 gene for HDL-C, an SNP near the MYLIP/GMPR and PPP1R3B genes for LDL-C, and at the AFF1 gene for triglycerides. SNPs showing strong statistical association with 1 or more lipid traits at the CELSR2, APOB, APOE-C1-C4-C2 cluster, LPL, ZNF259-APOA5-A4-C3-A1 cluster and TRIB1 loci were also associated with CAD risk (probability values, 1.1×10(-3) to 1.2×10(-9)). We have identified 4 novel loci associated with circulating lipids. We also show that in addition to those that are largely associated with LDL-C, genetic loci mainly associated with circulating triglycerides and HDL-C are also associated with risk of CAD. These findings potentially provide new insights into the biological mechanisms underlying lipid metabolism and CAD risk. Show less

Levels of circulating glucose are tightly regulated. To identify new loci influencing glycemic traits, we performed meta-analyses of 21 genome-wide association studies informative for fasting glucose, fasting insulin and indices of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 nondiabetic participants. Follow-up of 25 loci in up to 76,558 additional subjects identified 16 loci associated with fasting glucose and HOMA-B and two loci associated with fasting insulin and HOMA-IR. These include nine loci newly associated with fasting glucose (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and C2CD4B) and one influencing fasting insulin and HOMA-IR (near IGF1). We also demonstrated association of ADCY5, PROX1, GCK, GCKR and DGKB-TMEM195 with type 2 diabetes. Within these loci, likely biological candidate genes influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Our results demonstrate that genetic studies of glycemic traits can identify type 2 diabetes risk loci, as well as loci containing gene variants that are associated with a modest elevation in glucose levels but are not associated with overt diabetes. Show less

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone, which is secreted from endocrine cells in the small intestine after meal ingestion. GIP has been shown to affect osteoblastic function in vitro; however, the in vivo effects of GIP on bone remodeling remain unclear. In the present study, we investigated the role of GIP in modulating bone turnover, by evaluating serum markers of bone turnover, bone density, bone morphology, and changes in biomechanical bone strength over time (one to five months) in GIP receptor knockout mice (GIPR-/- mice). The GIPR-/- mice showed a decreased bone size, lower bone mass, altered bone microarchitecture and biomechanical properties, and altered parameters for bone turnover, especially in bone formation. Moreover, the effects of GIP on bone mass were site-specific and compensatory mechanism developed over time and ameliorated the impact of the loss of GIP signaling on bone mass. Further, GIPR-/- mice had earlier age-related changes than wild-type mice in body composition, including bone mass, lean body mass, and fat percentage. In summary, our results indicate that GIP has an anabolic effect on bone mass and bone quality and suggests that GIP may be a hormonal link between nutrient ingestion and utilization. Show less