👤 Kathleen M Harris

Dipeptidyl peptidase 4 inhibitors (DPP4i) may be cardioprotective based on several small animal and clinical studies, though randomized control trials have demonstrated limited benefit. Given these discrepant findings, the role of these agents in chronic myocardial disease, particularly in the absence of diabetes, is still poorly understood. The purpose of this study was to determine the effects of sitagliptin, a DPP4i, on myocardial perfusion and microvessel density in a clinically relevant large animal model of chronic myocardial ischemia. Normoglycemic Yorkshire swine underwent ameroid constrictor placement to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, pigs received either no drug (CON, n = 8) or 100 mg oral sitagliptin (SIT) daily (n = 5). Treatment continued for 5 weeks, followed by hemodynamic measurements, euthanasia, and tissue harvest of ischemic myocardium. There were no significant differences in myocardial function between CON and SIT as measured by stroke work (p > 0.5), cardiac output (p = 0.22), and end-systolic elastance (p = 0.17). SIT was associated with increased absolute blood flow at rest (17% increase, IQR 12-62, p = 0.045) and during pacing (89% increase, IQR 83-105, p = 0.002). SIT was also associated with improved arteriolar density (p = 0.045) compared with CON, without changes in capillary density (p = 0.72). SIT was associated with increased expression of pro-arteriogenic markers MCP-1 (p = 0.003), TGFß (p = 0.03), FGFR1 (p = 0.002), and ICAM-1 (p = 0.03), with a trend toward an increase in the ratio of phosphorylated/active PLCγ1 to total PLCγ1 (p = 0.11) compared with CON. In conclusion, in chronically ischemic myocardium, sitagliptin improves myocardial perfusion and arteriolar collateralization via the activation of pro-arteriogenic signaling pathways. Show less

Endometriosis is a common condition associated with debilitating pelvic pain and infertility. A genome-wide association study meta-analysis, including 60,674 cases and 701,926 controls of European and East Asian descent, identified 42 genome-wide significant loci comprising 49 distinct association signals. Effect sizes were largest for stage 3/4 disease, driven by ovarian endometriosis. Identified signals explained up to 5.01% of disease variance and regulated expression or methylation of genes in endometrium and blood, many of which were associated with pain perception/maintenance (SRP14/BMF, GDAP1, MLLT10, BSN and NGF). We observed significant genetic correlations between endometriosis and 11 pain conditions, including migraine, back and multisite chronic pain (MCP), as well as inflammatory conditions, including asthma and osteoarthritis. Multitrait genetic analyses identified substantial sharing of variants associated with endometriosis and MCP/migraine. Targeted investigations of genetically regulated mechanisms shared between endometriosis and other pain conditions are needed to aid the development of new treatments and facilitate early symptomatic intervention. Show less

We sought to establish a large animal model of inherited hypertrophic cardiomyopathy (HCM) with sufficient disease severity and early penetrance for identification of novel therapeutic strategies. HCM is the most common inherited cardiac disorder affecting 1 in 250-500 people, yet few therapies for its treatment or prevention are available. A research colony of purpose-bred cats carrying the A31P mutation in MYBPC3 was founded using sperm from a single heterozygous male cat. Cardiac function in four generations was assessed by periodic echocardiography and measurement of blood biomarkers. Results showed that HCM penetrance was age-dependent, and that penetrance occurred earlier and was more severe in successive generations, especially in homozygotes. Homozygosity was also associated with progression from preclinical to clinical disease. A31P homozygous cats represent a heritable model of HCM with early disease penetrance and a severe phenotype necessary for interventional studies aimed at altering disease progression. The occurrence of a more severe phenotype in later generations of cats, and the occasional occurrence of HCM in wildtype cats suggests the presence of at least one gene modifier or a second causal variant in this research colony that exacerbates the HCM phenotype when inherited in combination with the A31P mutation. Show less

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiac disease in humans and cats and lacks efficacious pharmacologic interventions in the preclinical phase of disease. LV outflow tract obstruction (LVOTO) is commonly observed in HCM-affected patients and is a primary driver of heart failure symptoms and reduced quality of life. Novel small-molecule cardiac myosin inhibitors target actin-myosin interactions to alleviate overactive protein interactions. A prospective, randomized, controlled cross-over study was performed to evaluate pharmacodynamic effects of two doses (0.3 and 1 mg/kg) of a next-in-class cardiac myosin inhibitor, aficamten (CK-3773274, CK-274), on cardiac function in cats with the A31P MYBPC3 mutation and oHCM. Dose-dependent reductions in LV systolic function, LVOT pressure gradient, and isovolumetric relaxation times compared to baseline were observed. Promising beneficial effects of reduced systolic function warrant further studies of this next-in-class therapeutic to evaluate the benefit of long-term administration in this patient population. Show less

Hypertrophic cardiomyopathy (HCM) is the most prevalent cardiac disease in cats and lacks efficacious preclinical pharmacologic intervention, prompting investigation of novel therapies. Genetic mutations encoding sarcomeric proteins are implicated in the development of HCM and small molecule myosin inhibitors are an emerging class of therapeutics designed to target the interaction of actin and myosin to alleviate the detrimental effects of inappropriate contractile protein interactions. The purpose of this study was to characterize the pharmacodynamic effects of a single oral dose of the novel cardiac myosin inhibitor aficamten (CK-274) on cardiac function in purpose bred cats with naturally occurring A31P MYBPC3 mutation and a clinical diagnosis of HCM with left ventricular outflow tract obstruction (LVOTO). Five purpose bred cats were treated with aficamten (2 mg/kg) or vehicle and echocardiographic evaluations were performed at 0, 6, 24, and 48 h post-dosing. High dose aficamten (2 mg/kg) reduced left ventricular fractional shortening (LVFS%) by increasing the LV systolic internal dimension (LVIDs) and reduced isovolumic relaxation time (IVRT) compared with baseline without significant adverse effects. The marked reduction in systolic function and reduced IVRT coupled with an increased heart rate in treated cats, suggest a lower dose may be optimal. Further studies to determine optimal dosing of aficamten are indicated. Show less

Individuals born very low birthweight (VLBW) are at increased risk of impaired cardiovascular and respiratory function in adulthood. To identify markers to predict future risk for VLBW individuals, we analyzed DNA methylation at birth and at 28 years in the New Zealand (NZ) VLBW cohort (all infants born < 1500 g in NZ in 1986) compared with age-matched, normal birthweight controls. Associations between neonatal methylation and cardiac structure and function (echocardiography), vascular function and respiratory outcomes at age 28 years were documented. Genomic DNA from archived newborn heel-prick blood (n = 109 VLBW, 51 controls) and from peripheral blood at ~ 28 years (n = 215 VLBW, 96 controls) was analyzed on Illumina Infinium MethylationEPIC 850 K arrays. Following quality assurance and normalization, methylation levels were compared between VLBW cases and controls at both ages by linear regression, with genome-wide significance set to p < 0.05 adjusted for false discovery rate (FDR, Benjamini-Hochberg). In neonates, methylation at over 16,400 CpG methylation sites differed between VLBW cases and controls and the canonical pathway most enriched for these CpGs was Cardiac Hypertrophy Signaling (p = 3.44E These findings suggest that methylation patterns in VLBW neonates may be informative about future adult cardiovascular and respiratory outcomes and have value in guiding early preventative care to improve adult health. Show less

Imaging of gene-expression patterns in live animals is difficult to achieve with fluorescent proteins because tissues are opaque to visible light. Imaging of transgene expression with magnetic resonance imaging (MRI), which penetrates to deep tissues, has been limited by single reporter visualization capabilities. Moreover, the low-throughput capacity of MRI limits large-scale mutagenesis strategies to improve existing reporters. Here we develop an MRI system, called GeneREFORM, comprising orthogonal reporters for two-color imaging of transgene expression in deep tissues. Starting from two promiscuous deoxyribonucleoside kinases, we computationally designed highly active, orthogonal enzymes ('reporter genes') that specifically phosphorylate two MRI-detectable synthetic deoxyribonucleosides ('reporter probes'). Systemically administered reporter probes exclusively accumulate in cells expressing the designed reporter genes, and their distribution is displayed as pseudo-colored MRI maps based on dynamic proton exchange for noninvasive visualization of transgene expression. We envision that future extensions of GeneREFORM will pave the way to multiplexed deep-tissue mapping of gene expression in live animals. Show less

The endothelial glycocalyx regulates vascular permeability, inflammation, and coagulation, and acts as a mechanosensor. The loss of glycocalyx can cause endothelial injury and contribute to several microvascular complications and, therefore, may promote diabetic retinopathy. Studies have shown a partial loss of retinal glycocalyx in diabetes, but with few molecular details of the changes in glycosaminoglycan (GAG) composition. Therefore, the purpose of our study was to investigate the effect of hyperglycemia on GAGs of the retinal endothelial glycocalyx. GAGs were isolated from rat retinal microvascular endothelial cells (RRMECs), media, and retinas, followed by liquid chromatography-mass spectrometry assays. Quantitative real-time polymerase chain reaction was used to study mRNA transcripts of the enzymes involved in GAG biosynthesis. Hyperglycemia significantly increased the shedding of heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). There were no changes to the levels of HS in RRMEC monolayers grown in high-glucose media, but the levels of CS and HA decreased dramatically. Similarly, while HA decreased in the retinas of diabetic rats, the total GAG and CS levels increased. Hyperglycemia in RRMECs caused a significant increase in the mRNA levels of the enzymes involved in GAG biosynthesis (including EXTL-1,2,3, EXT-1,2, ChSY-1,3, and HAS-2,3), with these increases potentially being compensatory responses to overall glycocalyx loss. Both RRMECs and retinas of diabetic rats exhibited glucose-induced alterations in the disaccharide compositions and sulfation of HS and CS, with the changes in sulfation including N,6-O-sulfation on HS and 4-O-sulfation on CS. Show less

Fatty acids exert a range of different biological activities that could be relevant in the development of atopic dermatitis (AD). This study investigated the association of glycerophospholipid fatty acids (GPL-FA) with AD, and their interactions with single nucleotide polymorphisms (SNP) of the Show less

This study summarises the diagnostic validity and clinical utility of genetic testing for patients with hypertrophic cardiomyopathy (HCM) and their at-risk relatives. A systematic search was performed in PubMed (MEDLINE), Embase, CINAHL and Cochrane Central Library databases from inception through 2 March 2020. Subgroup and sensitivity analyses were prespecified for individual sarcomere genes, presence/absence of pathogenic variants, paediatric and adult cohorts, family history, inclusion of probands, and variant classification method. Study quality was assessed using the Newcastle-Ottawa tool. A total of 132 articles met inclusion criteria. The detection rate based on pathogenic and likely pathogenic variants was significantly higher in paediatric cohorts compared with adults (56% vs 42%; p=0.01) and in adults with a family history compared with sporadic cases (59% vs 33%; p=0.005). When studies applied current, improved, variant interpretation standards, the adult detection rate significantly decreased from 42% to 33% (p=0.0001) because less variants met criteria to be considered pathogenic. The mean difference in age-of-onset in adults was significantly earlier for genotype-positive versus genotype-negative cohorts (8.3 years; p<0.0001), This systematic review and meta-analysis is the first, to our knowledge, to collectively quantify historical understandings of detection rate, genotype-phenotype associations and disease penetrance for HCM, while providing the answers to important routine clinical questions and highlighting key areas for future study. Show less

Understanding whether a person has consumed alcohol or not, as well as quantitative assessment of alcohol use, are often based on self-reported measures, which may be subject to recall bias, among other challenges. Although not without limitations, blood biomarkers may complement self-reported assessments to provide a more accurate determination of the presence and quantity of alcohol use. The aim of this review is to provide a critical overview of the current knowledge and research on biomarkers of alcohol use, with a particular focus on blood tests. This scoping review summarizes the published work on blood tests currently used in clinical practice, including phosphatidyl ethanol (PEth), fatty acid ethyl ester (FAEE), carbohydrate-deficient transferrin (CDT), total serum sialic acid (TSA), mean corpuscular volume (MCV), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), and cholesteryl ester transfer protein (CETP). Emerging blood biomarkers with a potential use to assess alcohol drinking are also briefly reviewed, including N-Acetyl-β-Hexosaminidase (Beta-Hex), macrophage migration inhibitory factor (MIF), and D-dopachrome tautomerase (DDT). We discuss the aforementioned biomarkers in the context of their clinical implications, characteristics, strengths, and limitations. The available blood biomarkers considerably vary in the time period in which they detect alcohol use and the amount of alcohol they are sensitive to. While currently available biomarkers provide useful information, especially in combination with self-reported measures, future work is needed to identify more sensitive and specific blood biomarkers for different levels and patterns of alcohol use. Integration of such biomarkers into clinical practice and research will increase the accuracy and richness of the data and may guide more effective and targeted strategies for prevention, diagnosis, and treatment of excessive alcohol use. Show less

Glaucoma is one of the world's leading causes of irreversible blindness. A complex, multifactorial disease, the underlying pathogenesis and reasons for disease progression are not fully understood. The most common form of glaucoma, primary open-angle glaucoma (POAG), was traditionally understood to be the result of elevated intraocular pressure (IOP), leading to optic nerve damage and functional vision loss. Recently, researchers have suggested that POAG may have an underlying genetic component. In fact, studies of genetic association and heritability have yielded encouraging results showing that glaucoma may be influenced by genetic factors, and estimates for the heritability of POAG and disease-related endophenotypes show encouraging results. However, the vast majority of the underlying genetic variants and their molecular mechanisms have not been elucidated. Several genes have been suggested to have molecular mechanisms contributing to alterations in key endophenotypes such as IOP ( Show less

Genome-wide association studies have identified multiple genomic loci associated with coronary artery disease, but most are common variants in non-coding regions that provide limited information on causal genes and etiology of the disease. To overcome the limited scope that common variants provide, we focused our investigation on low-frequency and rare sequence variations primarily residing in coding regions of the genome. Using samples of individuals of European ancestry from ten cohorts within the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, both cross-sectional and prospective analyses were conducted to examine associations between genetic variants and myocardial infarction (MI), coronary heart disease (CHD), and all-cause mortality following these events. For prevalent events, a total of 27,349 participants of European ancestry, including 1831 prevalent MI cases and 2518 prevalent CHD cases were used. For incident cases, a total of 55,736 participants of European ancestry were included (3,031 incident MI cases and 5,425 incident CHD cases). There were 1,860 all-cause deaths among the 3,751 MI and CHD cases from six cohorts that contributed to the analysis of all-cause mortality. Single variant and gene-based analyses were performed separately in each cohort and then meta-analyzed for each outcome. A low-frequency intronic variant (rs988583) in PLCL1 was significantly associated with prevalent MI (OR = 1.80, 95% confidence interval: 1.43, 2.27; P = 7.12 × 10-7). We conducted gene-based burden tests for genes with a cumulative minor allele count (cMAC) ≥ 5 and variants with minor allele frequency (MAF) < 5%. TMPRSS5 and LDLRAD1 were significantly associated with prevalent MI and CHD, respectively, and RC3H2 and ANGPTL4 were significantly associated with incident MI and CHD, respectively. No loci were significantly associated with all-cause mortality following a MI or CHD event. This study identified one known locus (ANGPTL4) and four new loci (PLCL1, RC3H2, TMPRSS5, and LDLRAD1) associated with cardiovascular disease risk that warrant further investigation. Show less

Aortic stenosis (AS) has no approved medical treatment. Identifying etiological pathways for AS could identify pharmacological targets. To identify novel genetic loci and pathways associated with AS. This genome-wide association study used a case-control design to evaluate 44 703 participants (3469 cases of AS) of self-reported European ancestry from the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort (from January 1, 1996, to December 31, 2015). Replication was performed in 7 other cohorts totaling 256 926 participants (5926 cases of AS), with additional analyses performed in 6942 participants from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium. Follow-up biomarker analyses with aortic valve calcium (AVC) were also performed. Data were analyzed from May 1, 2017, to December 5, 2019. Genetic variants (615 643 variants) and polyunsaturated fatty acids (ω-6 and ω-3) measured in blood samples. Aortic stenosis and aortic valve replacement defined by electronic health records, surgical records, or echocardiography and the presence of AVC measured by computed tomography. The mean (SD) age of the 44 703 GERA participants was 69.7 (8.4) years, and 22 019 (49.3%) were men. The rs174547 variant at the FADS1/2 locus was associated with AS (odds ratio [OR] per C allele, 0.88; 95% CI, 0.83-0.93; P = 3.0 × 10-6), with genome-wide significance after meta-analysis with 7 replication cohorts totaling 312 118 individuals (9395 cases of AS) (OR, 0.91; 95% CI, 0.88-0.94; P = 2.5 × 10-8). A consistent association with AVC was also observed (OR, 0.91; 95% CI, 0.83-0.99; P = .03). A higher ratio of arachidonic acid to linoleic acid was associated with AVC (OR per SD of the natural logarithm, 1.19; 95% CI, 1.09-1.30; P = 6.6 × 10-5). In mendelian randomization, increased FADS1 liver expression and arachidonic acid were associated with AS (OR per unit of normalized expression, 1.31 [95% CI, 1.17-1.48; P = 7.4 × 10-6]; OR per 5-percentage point increase in arachidonic acid for AVC, 1.23 [95% CI, 1.01-1.49; P = .04]; OR per 5-percentage point increase in arachidonic acid for AS, 1.08 [95% CI, 1.04-1.13; P = 4.1 × 10-4]). Variation at the FADS1/2 locus was associated with AS and AVC. Findings from biomarker measurements and mendelian randomization appear to link ω-6 fatty acid biosynthesis to AS, which may represent a therapeutic target. Show less

Lean body mass (LM) plays an important role in mobility and metabolic function. We previously identified five loci associated with LM adjusted for fat mass in kilograms. Such an adjustment may reduce the power to identify genetic signals having an association with both lean mass and fat mass. To determine the impact of different fat mass adjustments on genetic architecture of LM and identify additional LM loci. We performed genome-wide association analyses for whole-body LM (20 cohorts of European ancestry with n = 38,292) measured using dual-energy X-ray absorptiometry) or bioelectrical impedance analysis, adjusted for sex, age, age2, and height with or without fat mass adjustments (Model 1 no fat adjustment; Model 2 adjustment for fat mass as a percentage of body mass; Model 3 adjustment for fat mass in kilograms). Seven single-nucleotide polymorphisms (SNPs) in separate loci, including one novel LM locus (TNRC6B), were successfully replicated in an additional 47,227 individuals from 29 cohorts. Based on the strengths of the associations in Model 1 vs Model 3, we divided the LM loci into those with an effect on both lean mass and fat mass in the same direction and refer to those as "sumo wrestler" loci (FTO and MC4R). In contrast, loci with an impact specifically on LM were termed "body builder" loci (VCAN and ADAMTSL3). Using existing available genome-wide association study databases, LM increasing alleles of SNPs in sumo wrestler loci were associated with an adverse metabolic profile, whereas LM increasing alleles of SNPs in "body builder" loci were associated with metabolic protection. In conclusion, we identified one novel LM locus (TNRC6B). Our results suggest that a genetically determined increase in lean mass might exert either harmful or protective effects on metabolic traits, depending on its relation to fat mass. Show less

Studies have shown that the reduction in serum TAG concentrations with long-chain n-3 fatty acid supplementation is highly variable among individuals. The objectives of the present study were to compare the proportions of individuals whose TAG concentrations lowered after high-dose DHA and EPA, and to identify the predictors of response to both modalities. In a double-blind, controlled, crossover study, 154 men and women were randomised to three supplemented phases of 10 weeks each: (1) 2·7 g/d of DHA, (2) 2·7 g/d of EPA and (3) 3 g/d of maize oil, separated by 9-week washouts. As secondary analyses, the mean intra-individual variation in TAG was calculated using the standard deviation from the mean of four off-treatment samples. The response remained within the intra-individual variation (±0·25 mmol/l) in 47 and 57 % of participants after DHA and EPA, respectively. Although there was a greater proportion of participants with a reduction >0·25 mmol/l after DHA than after EPA (45 υ. 32 %; P 0·25 mmol/l after both DHA and EPA had higher non-HDL-cholesterol, TAG and insulin concentrations compared with other responders at baseline (all P < 0·05). In conclusion, supplementation with 2·7 g/d DHA or EPA had no meaningful effect on TAG concentrations in a large proportion of individuals with normal mean TAG concentrations at baseline. Although DHA lowered TAG in a greater proportion of individuals compared with EPA, the magnitude of TAG lowering among them was similar. Show less

The objective of this study was to perform a proof-of-concept experiment that validates a precision medicine approach to identify variants associated with hypertrophic cardiomyopathy (HCM). We hypothesized that whole-genome sequencing would identify variant(s) associated with HCM in two affected Maine Coon/Maine Coon cross cats when compared with 79 controls of various breeds. Two affected and two control Maine Coon/Maine Coon cross cats had whole-genome sequencing performed at approximately × 30 coverage. Variants were called in these four cats and 77 cats of various breeds as part of the 99 Lives Cat Genome Sequencing Initiative ( http://felinegenetics.missouri.edu/99lives ) using Platypus v0.7.9.1, annotated with dbSNP ID, and variants' effect predicted by SnpEff. Strict filtering criteria (alternate allele frequency >49%) were applied to identify homozygous-alternate or heterozygous variants in the two HCM-affected samples when compared with 79 controls of various breeds. A total of four variants were identified in the two Maine Coon/Maine Coon cross cats with HCM when compared with 79 controls after strict filtering. Three of the variants identified in genes This proof-of-concept experiment identified the previously reported Show less

Genome-wide association studies (GWAS) have identified >250 loci for body mass index (BMI), implicating pathways related to neuronal biology. Most GWAS loci represent clusters of common, noncoding variants from which pinpointing causal genes remains challenging. Here we combined data from 718,734 individuals to discover rare and low-frequency (minor allele frequency (MAF) < 5%) coding variants associated with BMI. We identified 14 coding variants in 13 genes, of which 8 variants were in genes (ZBTB7B, ACHE, RAPGEF3, RAB21, ZFHX3, ENTPD6, ZFR2 and ZNF169) newly implicated in human obesity, 2 variants were in genes (MC4R and KSR2) previously observed to be mutated in extreme obesity and 2 variants were in GIPR. The effect sizes of rare variants are ~10 times larger than those of common variants, with the largest effect observed in carriers of an MC4R mutation introducing a stop codon (p.Tyr35Ter, MAF = 0.01%), who weighed ~7 kg more than non-carriers. Pathway analyses based on the variants associated with BMI confirm enrichment of neuronal genes and provide new evidence for adipocyte and energy expenditure biology, widening the potential of genetically supported therapeutic targets in obesity. Show less

Alzheimer's disease (AD) is a public health priority for the 21st century. Risk reduction currently revolves around lifestyle changes with much research trying to elucidate the biological underpinnings. We show that self-report of parental history of Alzheimer's dementia for case ascertainment in a genome-wide association study of 314,278 participants from UK Biobank (27,696 maternal cases, 14,338 paternal cases) is a valid proxy for an AD genetic study. After meta-analysing with published consortium data (n = 74,046 with 25,580 cases across the discovery and replication analyses), three new AD-associated loci (P < 5 × 10 Show less

In clinical trials, inhibition of cholesteryl ester transfer protein (CETP) raises HDL cholesterol levels but does not robustly improve cardiovascular outcomes. Approximately two-thirds of trial participants are obese. Lower plasma CETP activity is associated with increased cardiovascular risk in human studies, and protective aspects of CETP have been observed in mice fed a high-fat diet (HFD) with regard to metabolic outcomes. To define whether CETP inhibition has different effects depending on the presence of obesity, we performed short-term anacetrapib treatment in chow- and HFD-fed CETP transgenic mice. Anacetrapib raised HDL cholesterol and improved aspects of HDL functionality, including reverse cholesterol transport, and HDL's antioxidative capacity in HFD-fed mice was better than in chow-fed mice. Anacetrapib worsened the anti-inflammatory capacity of HDL in HFD-fed mice. The HDL proteome was markedly different with anacetrapib treatment in HFD- versus chow-fed mice. Despite benefits on HDL, anacetrapib led to liver triglyceride accumulation and insulin resistance in HFD-fed mice. Overall, our results support a physiologic importance of CETP in protecting from fatty liver and demonstrate context selectivity of CETP inhibition that might be important in obese subjects. Show less

We screened variants on an exome-focused genotyping array in >300,000 participants (replication in >280,000 participants) and identified 444 independent variants in 250 loci significantly associated with total cholesterol (TC), high-density-lipoprotein cholesterol (HDL-C), low-density-lipoprotein cholesterol (LDL-C), and/or triglycerides (TG). At two loci (JAK2 and A1CF), experimental analysis in mice showed lipid changes consistent with the human data. We also found that: (i) beta-thalassemia trait carriers displayed lower TC and were protected from coronary artery disease (CAD); (ii) excluding the CETP locus, there was not a predictable relationship between plasma HDL-C and risk for age-related macular degeneration; (iii) only some mechanisms of lowering LDL-C appeared to increase risk for type 2 diabetes (T2D); and (iv) TG-lowering alleles involved in hepatic production of TG-rich lipoproteins (TM6SF2 and PNPLA3) tracked with higher liver fat, higher risk for T2D, and lower risk for CAD, whereas TG-lowering alleles involved in peripheral lipolysis (LPL and ANGPTL4) had no effect on liver fat but decreased risks for both T2D and CAD. Show less

There are no validated molecular methods that prospectively identify patients with surgically resected lung squamous cell carcinoma (SCC) at high risk for recurrence. By focusing on the expression of genes with known functions in development of lung SCC and prognosis, we sought to develop a robust prognostic classifier of early-stage lung SCC. The expression of 253 genes selected by literature search was evaluated in microarrays from 107 stage I/II tumors. Associations with survival were evaluated by Cox regression and Kaplan-Meier survival analyses in two independent cohorts of 121 and 91 patients with SCC, respectively. A classifier score based on multivariable Cox regression was derived and examined in six additional publicly available data sets of stage I/II lung SCC expression profiles (n = 358). The prognostic value of this classifier was evaluated in meta-analysis of patients with stage I/II (n = 479) and stage I (n = 326) lung SCC. Dual specificity phosphatase 6 gene (DUSP6) and actinin alpha 4 gene (ACTN4) were associated with prognostic outcome in two independent patient cohorts. Their expression values were utilized to develop a classifier that identified patients with stage I/II lung SCC at high risk for recurrence (hazard ratio [HR] = 4.7, p = 0.018) or cancer-specific mortality (HR = 3.5, p = 0.016). This classifier also identified patients at high risk for recurrence (HR = 2.7, p = 0.008) or death (HR = 2.2, p = 0.001) in publicly available data sets of stage I/II and in meta-analysis of stage I patients. We have established and validated a prognostic classifier to inform clinical management of patients with lung SCC after surgical resection. Show less

The "super-relaxed state" (SRX) of myosin represents a 'reserve' of motors in the heart. Myosin heads in the SRX are bound to the thick filament and have a very low ATPase rate. Changes in the SRX are likely to modulate cardiac contractility. We previously demonstrated that the SRX is significantly reduced in mouse cardiomyocytes lacking cardiac myosin binding protein-C (cMyBP-C). Here, we report the effect of mutations in the cMyBP-C gene (MYBPC3) using samples from human patients with hypertrophic cardiomyopathy (HCM). Left ventricular (LV) samples from 11 HCM patients were obtained following myectomy surgery to relieve LV outflow tract obstruction. HCM samples were genotyped as either MYBPC3 mutation positive (MYBPC3mut) or negative (HCMsmn) and were compared to eight non-failing donor hearts. Compared to donors, only MYBPC3mut samples display a significantly diminished SRX, characterised by a decrease in both the number of myosin heads in the SRX and the lifetime of ATP turnover. These changes were not observed in HCMsmn samples. There was a positive correlation (p < 0.01) between the expression of cMyBP-C and the proportion of myosin heads in the SRX state, suggesting cMyBP-C modulates and maintains the SRX. Phosphorylation of the myosin regulatory light chain in MYBPC3mut samples was significantly decreased compared to the other groups, suggesting a potential mechanism to compensate for the diminished SRX. We conclude that by altering both contractility and sarcomeric energy requirements, a reduced SRX may be an important disease mechanism in patients with MYBPC3 mutations. Show less

The association of obesity susceptibility variants with change in body mass index (BMI) across the life course is not well understood. In ancestry-stratified models of 5962 European American (EA), 2080 African American (AA) and 1582 Hispanic American (HA) individuals from the National Longitudinal Study of Adolescent to Adult Health (Add Health), we examined associations between 34 obesity single-nucleotide polymorphisms (SNPs) with per year change in BMI, measured by the slope from a growth-curve analysis of two or more BMI measurements between adolescence and young adulthood. For SNPs nominally associated with BMI change (P<0.05), we interrogated age differences within data collection Wave and time differences between age categories that overlapped between Waves. We found SNPs in/near FTO, MC4R, MTCH2, TFAP2B, SEC16B and TMEM18 were significantly associated (P<0.0015≈0.05/34) with BMI change in EA and the ancestry-combined meta-analysis. rs9939609 in FTO met genome-wide significance at P<5e-08 in the EA and ancestry-combined analysis, respectively [Beta(se)=0.025(0.004);Beta(se)=0.021(0.003)]. No SNPs were significant after Bonferroni correction in AA or HA, although five SNPs in AA and four SNPs in HA were nominally significant (P<0.05). In EA and the ancestry-combined meta-analysis, rs3817334 near MTCH2 showed larger effects in younger respondents, whereas rs987237 near TFAP2B, showed larger effects in older respondents across all Waves. Differences in effect estimates across time for MTCH2 and TFAP2B are suggestive of either era or cohort effects. The observed association between variants in/near FTO, MC4R, MTCH2, TFAP2B, SEC16B and TMEM18 with change in BMI from adolescence to young adulthood suggest that the genetic effect of BMI loci varies over time in a complex manner, highlighting the importance of investigating loci influencing obesity risk across the life course. Show less

Delta-5 desaturase (D5D) and delta-6 desaturase (D6D), encoded by fatty acid desaturase 1 (FADS1) and FADS2 genes, respectively, are enzymes in the synthetic pathways for ω3, ω6, and ω9 polyunsaturated fatty acids (PUFAs). Although PUFAs appear to be involved in mammalian metabolic pathways, the physiologic effect of isolated D5D deficiency on these pathways is unclear. After generating >4,650 knockouts (KOs) of independent mouse genes and analyzing them in our high-throughput phenotypic screen, we found that Fads1 KO mice were among the leanest of 3,651 chow-fed KO lines analyzed for body composition and were among the most glucose tolerant of 2,489 high-fat-diet-fed KO lines analyzed by oral glucose tolerance test. In confirmatory studies, chow- or high-fat-diet-fed Fads1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 38% and 31% lower in Fads1 male and female KO mice, respectively. Fads1 KO mice also had lower glucose and insulin excursions during oral glucose tolerance tests along with lower fasting glucose, insulin, triglyceride, and total cholesterol levels. In additional studies using a vascular injury model, Fads1 KO mice had significantly decreased femoral artery intima/media ratios consistent with a decreased inflammatory response in their arterial wall. Based on this result, we bred Fads1 KO and WT mice onto an ApoE KO background and fed them a Western diet for 14 weeks; in this atherogenic environment, aortic trees of Fads1 KO mice had 40% less atheromatous plaque compared to WT littermates. Importantly, PUFA levels measured in brain and liver phospholipid fractions of Fads1 KO mice were consistent with decreased D5D activity and normal D6D activity. The beneficial metabolic phenotype demonstrated in Fads1 KO mice suggests that selective D5D inhibitors may be useful in the treatment of human obesity, diabetes, and atherosclerotic cardiovascular disease. Show less

White blood cells play diverse roles in innate and adaptive immunity. Genetic association analyses of phenotypic variation in circulating white blood cell (WBC) counts from large samples of otherwise healthy individuals can provide insights into genes and biologic pathways involved in production, differentiation, or clearance of particular WBC lineages (myeloid, lymphoid) and also potentially inform the genetic basis of autoimmune, allergic, and blood diseases. We performed an exome array-based meta-analysis of total WBC and subtype counts (neutrophils, monocytes, lymphocytes, basophils, and eosinophils) in a multi-ancestry discovery and replication sample of ∼157,622 individuals from 25 studies. We identified 16 common variants (8 of which were coding variants) associated with one or more WBC traits, the majority of which are pleiotropically associated with autoimmune diseases. Based on functional annotation, these loci included genes encoding surface markers of myeloid, lymphoid, or hematopoietic stem cell differentiation (CD69, CD33, CD87), transcription factors regulating lineage specification during hematopoiesis (ASXL1, IRF8, IKZF1, JMJD1C, ETS2-PSMG1), and molecules involved in neutrophil clearance/apoptosis (C10orf54, LTA), adhesion (TNXB), or centrosome and microtubule structure/function (KIF9, TUBD1). Together with recent reports of somatic ASXL1 mutations among individuals with idiopathic cytopenias or clonal hematopoiesis of undetermined significance, the identification of a common regulatory 3' UTR variant of ASXL1 suggests that both germline and somatic ASXL1 mutations contribute to lower blood counts in otherwise asymptomatic individuals. These association results shed light on genetic mechanisms that regulate circulating WBC counts and suggest a prominent shared genetic architecture with inflammatory and autoimmune diseases. Show less

Clopidogrel is commonly prescribed to cats with perceived increased risk of thromboembolic events, but little information exists regarding its antiplatelet effects. To determine effects of clopidogrel on platelet responsiveness in cats with or without the A31P mutation in the MYBPC3 gene. A secondary aim was to characterize variability in feline platelet responses to clopidogrel. Fourteen healthy cats from a Maine Coon/outbred mixed Domestic cat colony: 8 cats homozygous for A31P mutation in the MYPBC3 gene and 6 wild-type cats without the A31P mutation. Ex vivo study. All cats received clopidogrel (18.75 mg PO q24h) for 14 days. Before and after clopidogrel treatment, adenosine diphosphate (ADP)-induced P-selectin expression was evaluated. ADP- and thrombin-induced platelet aggregation was measured by optical aggregometry (OA). Platelet pVASP and ADP receptor response index (ARRI) were measured by Western blot analysis. Platelet activation from cats with the A31P mutation was significantly (P = .0095) increased [35.55% (18.58-48.55) to 58.90% (24.85-69.90)], in response to ADP. Clopidogrel treatment attenuated ADP-induced P-selectin expression and platelet aggregation. ADP- and PGE Clopidogrel is effective at attenuating platelet activation and aggregation in some cats. Cats with A31P mutation had increased platelet activation relative to the variable response seen in wild-type cats. Show less

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase. Show less

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function. Show less