👤 C H Day

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor whose disruption causes obesity. We functionally characterized 61 MC4R variants identified in 0.5 million people from UK Biobank and examined their associations with body mass index (BMI) and obesity-related cardiometabolic diseases. We found that the maximal efficacy of β-arrestin recruitment to MC4R, rather than canonical Gα Show less

Truncation mutations in the MYBPC3 gene, encoding for cardiac myosin-binding protein C (MyBP-C), are the leading cause of hypertrophic cardiomyopathy (HCM). Whole heart, fiber and molecular studies demonstrate that MyBP-C is a potent modulator of cardiac contractility, but how these mutations contribute to HCM is unresolved. To readdress whether MYBPC3 truncation mutations result in loss of MyBP-C content and/or the expression of truncated MyBP-C from the mutant allele and determine how these mutations effect myofilament sliding in human myocardium. Septal wall tissue samples were obtained from HCM patients undergoing myectomy (n = 18) and donor controls (n = 8). The HCM samples contained 40% less MyBP-C and reduced levels of MyBP-C phosphorylation, when compared to the donor control samples using quantitative mass spectrometry. These differences occurred in the absence of changes in the stoichiometry of other myofilament proteins or production of truncated MyBP-C from the mutant MYBPC3 allele. The functional impact of MYBPC3 truncation mutations on myofilament sliding was determined using a total internal reflection microscopy (TIRFM) single particle assay. Myosin-thick filaments containing their native complement of MyBP-C, and actin-thin filaments decorated with the troponin/tropomyosin calcium regulatory proteins, were isolated from a subgroup of the HCM (n = 4) and donor (n = 5) heart samples. The maximal sliding velocity of native thin filaments was enhanced within the C-zones of the native thick filaments isolated from the HCM samples, when compared to velocity within the C-zones of thick filaments isolated from the donor samples. Analytical modeling demonstrated that the 40% reduction in MyBP-C content was sufficient to enhance the myofilament sliding velocity, as observed in the TIRFM assay. HCM-causing MYBPC3 truncation mutations result in a loss of MyBP-C content that enhances maximal myofilament sliding velocities, only where MyBP-C is localized within the C-zone. These findings support therapeutic rationale for restoring normal levels of MyBP-C and/or dampening maximal contractile velocities for the treatment of human HCM. Show less

Mutations in cardiac myosin binding protein C (MYBPC3) represent the most frequent cause of familial hypertrophic cardiomyopathy (HCM), making up approximately 50% of identified HCM mutations. MYBPC3 is distinct among other sarcomere genes associated with HCM in that truncating mutations make up the vast majority, whereas nontruncating mutations predominant in other sarcomere genes. Several studies using myocardial tissue from HCM patients have found reduced abundance of wild-type MYBPC3 compared to control hearts, suggesting haploinsufficiency of full-length MYBPC3. Further, decreased mutant versus wild-type mRNA and lack of truncated mutant MYBPC3 protein has been demonstrated, highlighting the presence of allelic imbalance. In this review, we will begin by introducing allelic imbalance and haploinsufficiency, highlighting the broad role each plays within the spectrum of human disease. We will subsequently focus on the roles allelic imbalance and haploinsufficiency play within MYBPC3-linked HCM. Finally, we will explore the implications of these findings on future directions of HCM research. An improved understanding of allelic imbalance and haploinsufficiency may help us better understand genotype-phenotype relationships in HCM and develop novel targeted therapies, providing exciting future research opportunities. Show less

Pharmacological enhancers of lipoprotein lipase (LPL) are in preclinical or early clinical development for cardiovascular prevention. Studying whether these agents will reduce cardiovascular events or diabetes risk when added to existing lipid-lowering drugs would require large outcome trials. Human genetics studies can help prioritize or deprioritize these resource-demanding endeavors. To investigate the independent and combined associations of genetically determined differences in LPL-mediated lipolysis and low-density lipoprotein cholesterol (LDL-C) metabolism with risk of coronary disease and diabetes. In this genetic association study, individual-level genetic data from 392 220 participants from 2 population-based cohort studies and 1 case-cohort study conducted in Europe were included. Data were collected from January 1991 to July 2018, and data were analyzed from July 2014 to July 2018. Six conditionally independent triglyceride-lowering alleles in LPL, the p.Glu40Lys variant in ANGPTL4, rare loss-of-function variants in ANGPTL3, and LDL-C-lowering polymorphisms at 58 independent genomic regions, including HMGCR, NPC1L1, and PCSK9. Odds ratio for coronary artery disease and type 2 diabetes. Of the 392 220 participants included, 211 915 (54.0%) were female, and the mean (SD) age was 57 (8) years. Triglyceride-lowering alleles in LPL were associated with protection from coronary disease (approximately 40% lower odds per SD of genetically lower triglycerides) and type 2 diabetes (approximately 30% lower odds) in people above or below the median of the population distribution of LDL-C-lowering alleles at 58 independent genomic regions, HMGCR, NPC1L1, or PCSK9. Associations with lower risk were consistent in quintiles of the distribution of LDL-C-lowering alleles and 2 × 2 factorial genetic analyses. The 40Lys variant in ANGPTL4 was associated with protection from coronary disease and type 2 diabetes in groups with genetically higher or lower LDL-C. For a genetic difference of 0.23 SDs in LDL-C, ANGPTL3 loss-of-function variants, which also have beneficial associations with LPL lipolysis, were associated with greater protection against coronary disease than other LDL-C-lowering genetic mechanisms (ANGPTL3 loss-of-function variants: odds ratio, 0.66; 95% CI, 0.52-0.83; 58 LDL-C-lowering variants: odds ratio, 0.90; 95% CI, 0.89-0.91; P for heterogeneity = .009). Triglyceride-lowering alleles in the LPL pathway are associated with lower risk of coronary disease and type 2 diabetes independently of LDL-C-lowering genetic mechanisms. These findings provide human genetics evidence to support the development of agents that enhance LPL-mediated lipolysis for further clinical benefit in addition to LDL-C-lowering therapy. Show less

Background Although atrial fibrillation (AF) is common in hypertrophic cardiomyopathy (HCM) patients, the relationship between genetic variation and AF has been poorly defined. Characterizing genetic subtypes of HCM and their associations with AF may help to improve personalized medical care. We aimed to investigate the link between sarcomeric gene variation and incident AF in HCM patients. Methods and Results Patients from the multinational Sarcomeric Human Cardiomyopathy Registry were followed for incident AF. Those with likely pathogenic or pathogenic variants in sarcomeric genes were included. The AF incidence was ascertained by review of medical records and electrocardiograms at each investigative site. One thousand forty adult HCM patients, without baseline AF and with likely pathogenic or pathogenic variation in either MYH7 (n=296), MYBPC3 (n=659), or thin filament genes (n=85), were included. Compared with patients with variation in other sarcomeric genes, those with MYH7 variants were younger on first clinical encounter at the Sarcomeric Human Cardiomyopathy Registry site and more likely to be probands than the MYBPC3 variants. During an average follow-up of 7.2 years, 198 incident AF events occurred. Patients with likely pathogenic or pathogenic mutations in MYH7 had the highest incidence of AF after adjusting for age, sex, proband status, left atrial size, maximal wall thickness, and peak pressure gradient (hazard ratio, 1.7; 95% CI, 1.1-2.6; P=0.009). Conclusions During a mean follow-up of 7.2 years, new-onset AF developed in 19% of HCM patients with sarcomeric mutations. Compared with other sarcomeric genes, patients with likely pathogenic or pathogenic variation in MYH7 had a higher rate of incident AF independent of clinical and echocardiographic factors. Show less

This study demonstrates that significantly shortened telomeres are a hallmark of cardiomyocytes (CMs) from individuals with end-stage hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM) as a result of heritable defects in cardiac proteins critical to contractile function. Positioned at the ends of chromosomes, telomeres are DNA repeats that serve as protective caps that shorten with each cell division, a marker of aging. CMs are a known exception in which telomeres remain relatively stable throughout life in healthy individuals. We found that, relative to healthy controls, telomeres are significantly shorter in CMs of genetic HCM and DCM patient tissues harboring pathogenic mutations: Show less

Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 accelerated degradation. HSC70 was expressed in discrete striations in the sarcomere. Expression of mutant MYBPC3 did not affect HSC70 localization, nor did it induce a protein folding stress response or ubiquitin proteasome dysfunction. Together these data suggest that WT and mutant MYBPC3 proteins are clients for HSC70, and that the HSC70 chaperone system plays a major role in regulating MYBPC3 protein turnover. Show less

Dual specificity mitogen-activated protein kinase (MAPK) phosphatases [dual specificity phosphatase/MAP kinase phosphatase (DUSP-MKP)] have been hypothesized to maintain cancer cell survival by buffering excessive MAPK signaling caused by upstream activating oncogenic products. A large and diverse body of literature suggests that genetic depletion of DUSP-MKPs can reduce tumorigenicity, suggesting that hyperactivating MAPK signaling by DUSP-MKP inhibitors could be a novel strategy to selectively affect the transformed phenotype. Through in vivo structure-activity relationship studies in transgenic zebrafish we recently identified a hyperactivator of fibroblast growth factor signaling [(E)-2-benzylidene-5-bromo-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI-215)] that is devoid of developmental toxicity and restores defective MAPK activity caused by overexpression of DUSP1 and DUSP6 in mammalian cells. Here, we hypothesized that BCI-215 could selectively affect survival of transformed cells. In MDA-MB-231 human breast cancer cells, BCI-215 inhibited cell motility, caused apoptosis but not primary necrosis, and sensitized cells to lymphokine-activated killer cell activity. Mechanistically, BCI-215 induced rapid and sustained phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) in the absence of reactive oxygen species, and its toxicity was partially rescued by inhibition of p38 but not JNK or ERK. BCI-215 also hyperactivated MKK4/SEK1, suggesting activation of stress responses. Kinase phosphorylation profiling documented BCI-215 selectively activated MAPKs and their downstream substrates, but not receptor tyrosine kinases, SRC family kinases, AKT, mTOR, or DNA damage pathways. Our findings support the hypothesis that BCI-215 causes selective cancer cell cytotoxicity in part through non-redox-mediated activation of MAPK signaling, and the findings also identify an intersection with immune cell killing that is worthy of further exploration. Show less

Hand grip strength is a widely used proxy of muscular fitness, a marker of frailty, and predictor of a range of morbidities and all-cause mortality. To investigate the genetic determinants of variation in grip strength, we perform a large-scale genetic discovery analysis in a combined sample of 195,180 individuals and identify 16 loci associated with grip strength (P<5 × 10 Show less

Mutations of cardiac sarcomere genes have been identified to cause HCM, but the molecular mechanisms that lead to cardiomyocyte hypertrophy and risk for sudden death are uncertain. The aim of this study was to examine HCM disease mechanisms at play during cardiac differentiation of human HCM specific pluripotent stem cells. We generated a human embryonic stem cell (hESC) line carrying a naturally occurring mutation of MYPBC3 (c.2905 +1 G >A) to study HCM pathogenesis during cardiac differentiation. HCM-specific hESC-derived cardiomyocytes (hESC-CMs) displayed hallmark aspects of HCM including sarcomere disarray, hypertrophy and impaired calcium impulse propagation. HCM hESC-CMs presented a transient haploinsufficiency of cMyBP-C during cardiomyocyte differentiation, but by day 30 post-differentiation cMyBP-C levels were similar to control hESC-CMs. Gene transfer of full-length MYBPC3 during differentiation prevented hypertrophy, sarcomere disarray and improved calcium impulse propagation in HCM hESC-CMs. These findings point to the critical role of MYBPC3 during sarcomere assembly in cardiac myocyte differentiation and suggest developmental influences of MYBPC3 truncating mutations on the mature hypertrophic phenotype. Show less

The ages of puberty, first sexual intercourse and first birth signify the onset of reproductive ability, behavior and success, respectively. In a genome-wide association study of 125,667 UK Biobank participants, we identify 38 loci associated (P < 5 × 10(-8)) with age at first sexual intercourse. These findings were taken forward in 241,910 men and women from Iceland and 20,187 women from the Women's Genome Health Study. Several of the identified loci also exhibit associations (P < 5 × 10(-8)) with other reproductive and behavioral traits, including age at first birth (variants in or near ESR1 and RBM6-SEMA3F), number of children (CADM2 and ESR1), irritable temperament (MSRA) and risk-taking propensity (CADM2). Mendelian randomization analyses infer causal influences of earlier puberty timing on earlier first sexual intercourse, earlier first birth and lower educational attainment. In turn, likely causal consequences of earlier first sexual intercourse include reproductive, educational, psychiatric and cardiometabolic outcomes. Show less

Dual specificity phosphatase 6 (DUSP6) functions as a feedback attenuator of fibroblast growth factor signaling during development. In vitro high throughput chemical screening attempts to discover DUSP6 inhibitors have yielded limited success. However, in vivo whole-organism screens of zebrafish identified compound 1 (BCI) as an allosteric inhibitor of DUSP6. Here we designed and synthesized a panel of analogues to define the structure-activity relationship (SAR) of DUSP6 inhibition. In vivo high-content analysis in transgenic zebrafish, coupled with cell-based chemical complementation assays, identified structural features of the pharmacophore of 1 that were essential for biological activity. In vitro assays of DUSP hyperactivation corroborated the results from in vivo and cellular SAR. The results reinforce the notion that DUSPs are druggable through allosteric mechanisms and illustrate the utility of zebrafish as a model organism for in vivo SAR analyses. Show less

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions. Show less

Heterozygous mutations in sarcomere genes in hypertrophic cardiomyopathy (HCM) are proposed to exert their effect through gain of function for missense mutations or loss of function for truncating mutations. However, allelic expression from individual mutations has not been sufficiently characterized to support this exclusive distinction in human HCM. Sarcomere transcript and protein levels were analyzed in septal myectomy and transplant specimens from 46 genotyped HCM patients with or without sarcomere gene mutations and 10 control hearts. For truncating mutations in MYBPC3, the average ratio of mutant:wild-type transcripts was ≈1:5, in contrast to ≈1:1 for all sarcomere missense mutations, confirming that nonsense transcripts are uniquely unstable. However, total MYBPC3 mRNA was significantly increased by 9-fold in HCM samples with MYBPC3 mutations compared with control hearts and with HCM samples without sarcomere gene mutations. Full-length MYBPC3 protein content was not different between MYBPC3 mutant HCM and control samples, and no truncated proteins were detected. By absolute quantification of abundance with multiple reaction monitoring, stoichiometric ratios of mutant sarcomere proteins relative to wild type were strikingly variable in a mutation-specific manner, with the fraction of mutant protein ranging from 30% to 84%. These results challenge the concept that haploinsufficiency is a unifying mechanism for HCM caused by MYBPC3 truncating mutations. The range of allelic imbalance for several missense sarcomere mutations suggests that certain mutant proteins may be more or less stable or incorporate more or less efficiently into the sarcomere than wild-type proteins. These mutation-specific properties may distinctly influence disease phenotypes. Show less

Coagulation phenotypes show strong intercorrelations, affect cardiovascular disease risk and are influenced by genetic variants. The objective of this study was to search for novel genetic variants influencing the following coagulation phenotypes: factor VII levels, fibrinogen levels, plasma viscosity and platelet count. We genotyped the British Women's Heart and Health Study (n=3,445) and the Whitehall II study (n=5,059) using the Illumina HumanCVD BeadArray to investigate genetic associations and pleiotropy. In addition to previously reported associations (SH2B3, F7/F10, PROCR, GCKR, FGA/FGB/FGG, IL5), we identified novel associations at GRK5 (rs10128498, p=1.30x10(-6)), GCKR (rs1260326, p=1.63x10(-6)), ZNF259-APOA5 (rs651821, p=7.17x10(-6)) with plasma viscosity; and at CSF1 (rs333948, p=8.88x10(-6)) with platelet count. A pleiotropic effect was identified in GCKR which associated with factor VII (p=2.16x10(-7)) and plasma viscosity (p=1.63x10(-6)), and, to a lesser extent, ZNF259-APOA5 which also associated with factor VII and fibrinogen (p<1.00x10-²) and plasma viscosity (p<1.00x10(-5)). Triglyceride associated variants were overrepresented in factor VII and plasma viscosity associations. Adjusting for triglyceride levels resulted in attenuation of associations at the GCKR and ZNF259-APOA5 loci. In addition to confirming previously reported associations, we identified four single nucleotide polymorphisms (SNPs) associated with plasma viscosity and platelet count and found evidence of pleiotropic effects with SNPs in GCKR and ZNF259-APOA5. These triglyceride-associated, pleiotropic SNPs suggest a possible causal role for triglycerides in coagulation. Show less

17β-Hydroxysteroid dehydrogenases (17β-HSDs) catalyse the 17-position reduction/oxidation of steroids. 17β-HSD type 3 (17β-HSD3) catalyses the reduction of the weakly androgenic androstenedione (adione) to testosterone, suggesting that specific inhibitors of 17β-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia. STX2171 is a novel selective non-steroidal 17β-HSD3 inhibitor with an IC(50) of ∼200 nM in a whole-cell assay. It inhibits adione-stimulated proliferation of 17β-HSD3-expressing androgen receptor-positive LNCaP(HSD3) prostate cancer cells in vitro. An androgen-stimulated LNCaP(HSD3) xenograft proof-of-concept model was developed to study the efficacies of STX2171 and a more established 17β-HSD3 inhibitor, STX1383 (SCH-451659, Schering-Plough), in vivo. Castrated male MF-1 mice were inoculated s.c. with 1×10(7) cells 24 h after an initial daily dose of testosterone propionate (TP) or vehicle. After 4 weeks, tumours had not developed in vehicle-dosed mice, but were present in 50% of those mice given TP. One week after switching the stimulus to adione, mice were dosed additionally with the vehicle or inhibitor for a further 4 weeks. Both TP and adione efficiently stimulated tumour growth and increased plasma testosterone levels; however, in the presence of either 17β-HSD3 inhibitor, adione-dependent tumour growth was significantly inhibited and plasma testosterone levels reduced. Mouse body weights were unaffected. Both inhibitors also significantly lowered plasma testosterone levels in intact mice. In conclusion, STX2171 and STX1383 significantly lower plasma testosterone levels and inhibit androgen-dependent tumour growth in vivo, indicating that 17β-HSD3 inhibitors may have application in the treatment of hormone-dependent prostate cancer. Show less

Glucagon and glucagon-like peptide-1 (GLP-1)are two structurally related hormones that acutely regulate glucose control in opposite directions through homologous receptors. The molecular basis for selectivity between these two hormones and their receptors is of physiological and medicinal importance. The application of co-agonists to enhance body weight reduction and correct multiple abnormalities associated with the metabolic syndrome has recently been reported. Substitution of amino acids 16, 18, and 20 in glucagon with those found in GLP-1 and exendin-4 were identified as partial contributors to balanced, high potency receptor action. The amidation of the C-terminus was an additional glucagon-based structural change observed to be of seminal importance to discriminate recognition by both receptors. In this work, the molecular basis for receptor selectivity associated with differences in C-terminal peptide sequence has been determined. A single charge inversion in glucagon and GLP-1 receptor sequence at position 68* was determined to significantly alter hormone action. Changing E68* in GLP-1R to the corresponding Lys of GCGR reduced receptor activity for natural GLP-1 hormones by eightfold. The enhanced C-terminal positive charges in GLP-1 peptides favor the native receptor's negative charge at position 68*, while the unfavorable interaction with the C-terminal acid of native glucagon is minimized by amidation. The extension of these observations to other glucagon-related hormones such as oxyntomodulin and exendin, as well as other related receptors such as GIPR, should assist in the assembly of additional hormones with broadened pharmacology. Show less

The T-455C and C-482T APOC3 promoter region polymorphisms (SNPs) have recently been reported to predispose to dyslipidemia, insulin resistance, and nonalcoholic fatty liver disease (NAFLD) in Indian subjects, but the association with liver damage has not been evaluated so far. The aim was to assess the association between APOC3 SNPs and liver damage in Caucasian patients. We considered 437 Italian patients with histological diagnosis of NAFLD (including 137 children, 120 morbid obese) and 316 healthy controls, 71 Italian family trios, and 321 patients from the UK. APOC3 SNPs were determined by sequencing, allele-specific oligonucleotide probes and PCR-restriction fragment length polymorphism analysis, hepatic APOC3 mRNA levels by real-time PCR. APOC3 SNPs were not associated with NAFLD in Italian subjects, although a borderline significance for the transmission of the -455T allele was observed in the family study. Homozygosity for the APOC3 wild-type genotype (APOC3 WT) was associated with a more favorable lipid profile in control subjects, and consistently with lower hepatic APOC3 mRNA levels in obese patients without diabetes. However, APOC3 SNPs, alone or in combination, were not associated with insulin resistance, altered lipid levels, liver enzymes, and with liver damage (severity of steatosis, nonalcoholic steatohepatitis, and moderate/severe fibrosis) in Italian as well as in UK patients, and in the whole cohort. Stratification for the I148M PNPLA3 mutation, associated with the susceptibility to NASH, did not alter the results. APOC3 genotype is not associated with progressive liver damage in Caucasian patients with NAFLD. Show less

Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ∼ 2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10⁻⁸), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation. 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

Blood lipids are important cardiovascular disease (CVD) risk factors with both genetic and environmental determinants. The Whitehall II study (n=5592) was genotyped with the gene-centric HumanCVD BeadChip (Illumina). We identified 195 SNPs in 16 genes/regions associated with 3 major lipid fractions and 2 apolipoprotein components at p<10(-5), with the associations being broadly concordant with prior genome-wide analysis. SNPs associated with LDL cholesterol and apolipoprotein B were located in LDLR, PCSK9, APOB, CELSR2, HMGCR, CETP, the TOMM40-APOE-C1-C2-C4 cluster, and the APOA5-A4-C3-A1 cluster; SNPs associated with HDL cholesterol and apolipoprotein AI were in CETP, LPL, LIPC, APOA5-A4-C3-A1, and ABCA1; and SNPs associated with triglycerides in GCKR, BAZ1B, MLXIPL, LPL, and APOA5-A4-C3-A1. For 48 SNPs in previously unreported loci that were significant at p<10(-4) in Whitehall II, in silico analysis including the British Women's Heart and Health Study, BRIGHT, ASCOT, and NORDIL studies (total n>12,500) revealed previously unreported associations of SH2B3 (p<2.2x10(-6)), BMPR2 (p<2.3x10(-7)), BCL3/PVRL2 (flanking APOE; p<4.4x10(-8)), and SMARCA4 (flanking LDLR; p<2.5x10(-7)) with LDL cholesterol. Common alleles in these genes explained 6.1%-14.7% of the variance in the five lipid-related traits, and individuals at opposite tails of the additive allele score exhibited substantial differences in trait levels (e.g., >1 mmol/L in LDL cholesterol [approximately 1 SD of the trait distribution]). These data suggest that multiple common alleles of small effect can make important contributions to individual differences in blood lipids potentially relevant to the assessment of CVD risk. These genes provide further insights into lipid metabolism and the likely effects of modifying the encoded targets therapeutically. Show less

The dual-specificity phosphatase 6 (Dusp6) functions as a feedback regulator of fibroblast growth factor (FGF) signaling to limit the activity of extracellular signal-regulated kinases (ERKs) 1 and 2. We have identified a small-molecule inhibitor of Dusp6-(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI)-using a transgenic zebrafish chemical screen. BCI treatment blocked Dusp6 activity and enhanced FGF target gene expression in zebrafish embryos. Docking simulations predicted an allosteric binding site for BCI within the phosphatase domain. In vitro studies supported a model in which BCI inhibits Dusp6 catalytic activation by ERK2 substrate binding. We used BCI treatment at varying developmental stages to uncover a temporal role for Dusp6 in restricting cardiac progenitors and controlling heart organ size. This study highlights the power of in vivo zebrafish chemical screens to identify new compounds targeting Dusp6, a component of the FGF signaling pathway that has eluded traditional high-throughput in vitro screens. Show less

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are responsible for the pre-receptor reduction/oxidation of steroids at the 17-position into active/inactive hormones, and the 15 known enzymes vary in their substrate specificity, localisation, and directional activity. 17beta-HSD Type 3 (17beta-HSD3) has been seen to be over-expressed in prostate cancer, and catalyses the reduction of androstenedione (Adione) to testosterone (T), which stimulates prostate tumour growth. Specific inhibitors of 17beta-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia, and also have potential as male anti-fertility agents. A 293-EBNA-based cell line with stable expression of transfected human 17beta-HSD3 was created and used to develop a whole cell radiometric TLC-based assay to assess the 17beta-HSD3 inhibitory potency of a series of compounds. STX2171 and STX2624 (IC(50) values in the 200-450nM range) were two of several active inhibitors identified. In similar TLC-based assays these compounds were found to be inactive against 17beta-HSD1 and 17beta-HSD2, indicating selectivity. A novel proof of concept model was developed to study the efficacy of the compounds in vitro using the androgen receptor positive hormone-dependent prostate cancer cell line, LNCaPwt, and its derivative, LNCaP[17beta-HSD3], transfected and selected for stable expression of 17beta-HSD3. The proliferation of the parental cell line was most efficiently stimulated by 5alpha-dihydrotestosterone (DHT), but the LNCaP[17beta-HSD3] cells were equally stimulated by Adione, indicating that 17beta-HSD3 efficiently converts Adione to T in this model. Adione-stimulated proliferation of LNCaP[17beta-HSD3] cells was inhibited in the presence of either STX2171 or STX2624. The compounds alone neither stimulated proliferation of the cells nor caused significant cell death, indicating that they are non-androgenic with low cytotoxicity. STX2171 inhibited Adione-stimulated growth of xenografts established from LNCaPwt cells in castrated mice in vivo. In conclusion, a primary screening assay and proof of concept model have been developed to study the efficacy of 17beta-HSD3 inhibitory compounds, which may have a role in the treatment of hormone-dependent cancer. Active compounds are selective for 17beta-HSD3 over 17beta-HSD1 and 17beta-HSD2, non-androgenic with low toxicity, and efficacious in both an in vitro proof of concept model and in an in vivo tumour model. Show less

17beta-Hydroxysteroid dehydrogenase type 3 (17beta-HSD3) is expressed at high levels in the testes and seminal vesicles but has also been shown to be present in prostate tissue, suggesting its potential involvement in both gonadal and non-gonadal testosterone biosynthesis. The role of 17beta-HSD3 in testosterone biosynthesis makes this enzyme an attractive molecular target for small molecule inhibitors for the treatment of prostate cancer. Here we report the design of selective inhibitors of 17beta-HSD3 as potential anti-cancer agents. Due to 17beta-HSD3 being a membrane-bound protein a crystal structure is not yet available. A homology model of 17beta-HSD3 has been built to aid structure-based drug design. This model has been used with docking studies to identify a series of lead compounds that may give an insight as to how inhibitors interact with the active site. Compound 1 was identified as a potent selective inhibitor of 17beta-HSD3 with an IC(50)=700nM resulting in the discovery of a novel lead series for further optimisation. Using our homology model as a tool for inhibitor design compound 5 was discovered as a novel potent and selective inhibitor of 17beta-HSD3 with an IC(50) approximately 200nM. Show less

Oestradiol (E2) stimulates the growth of hormone-dependent breast cancer. 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the pre-receptor activation/inactivation of hormones and other substrates. 17beta-HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17beta-HSD, 17beta-HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17beta-HSD1 which is important for E2 production and report the inhibition of E1-stimulated breast tumor growth by STX1040, a non-oestrogenic selective inhibitor of 17beta-HSD1, using a novel murine model. 17beta-HSD1 and 17beta-HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17beta-HSD12 was highly expressed in breast cancer cell lines, only 17beta-HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1-stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 microg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1-stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17beta-HSD, 17beta-HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17beta-HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone-dependent breast cancer. Show less

Cohort and case-control genetic association studies offer the greatest power to detect small genotypic influences on disease phenotypes, relative to family-based designs. However, genetic subdivisions could confound studies involving unrelated individuals, but the topic has been little investigated. We examined geographical and interallelic association of SNP and microsatellite haplotypes of the Y chromosome, of regions of chromosome 11, and of autosomal SNP genotypes relevant to cardiovascular risk traits in a UK-wide epidemiological survey. We show evidence (p = 0.00001) of the Danelaw history of the UK, marked by a two-fold excess of a Viking Y haplotype in central England. We also found evidence for a (different) single-centre geographical over-representation of one haplotype, both for APOC3-A4-A5 and for IGF2. The basis of this remains obscure but neither reflect genotyping error nor correlate with the phenotypic associations by centre of these markers. A panel of SNPs relevant to cardiovascular risks traits showed neither association with geographical location nor with Y haplotypes. Combinations of Y haplotyping, autosomal haplotyping, and genome-wide SNP typing, taken together with phenotypic2 associations, should improve epidemiological recognition and interpretation of possible confounding by genetic subdivision. Show less

In the yeast Saccharomyces cerevisiae, mitotic cell cycle progression depends upon the G(1)-phase cyclin-dependent kinase Cln-Cdc28 and cell growth to a minimum cell size. In contrast, Cln-Cdc28 inhibits entry into meiosis, and a cell growth requirement for sporulation has not been established. Here, we report that entry into meiosis also depends upon cell growth. Moreover, sporulation and cell growth rates were proportional to cell size; large cells grew rapidly and sporulated sooner while smaller cells grew slowly and sporulated later. In addition, Cln2 protein levels were higher in smaller cells suggesting that Cln-Cdc28 activity represses meiosis in smaller cells by preventing cell growth. In support of this hypothesis, loss of Clns, or the presence of a cdc28 mutation increased cell growth specifically in smaller cells and accelerated meiosis in these cells. Finally, overexpression of CLNs repressed meiosis in smaller cells, but not in large cells. Taken together, these results demonstrate that Cln-Cdc28 represses entry into meiosis in part by inhibiting cell growth. Show less