👤 Tina K Jensen

Obesity and insulin resistance may be associated with elevated plasma concentration of branched-chain amino acids (BCAAs) and impaired BCAA metabolism. However, it is unknown whether the insulin-sensitising effect of long-term exercise can be explained by concomitant change in BCAAs and their metabolism. We included 26 sedentary overweight and normal-weight middle-aged men from the MyoGlu clinical trial, with or without dysglycaemia, for 12 weeks of supervised intensive exercise intervention, including two endurance and two resistance sessions weekly. Insulin sensitivity was measured as the glucose infusion rate (GIR) from a hyperinsulinaemic-euglycaemic clamp. In addition, maximum oxygen uptake, upper and lower body strength and adipose tissue depots (using MRI and spectroscopy) were measured, and subcutaneous white adipose tissue (ScWAT) and skeletal muscle (SkM) biopsies were harvested both before and after the 12 week intervention. In the present study we have measured plasma BCAAs and related metabolites using CG-MS/MS and HPLC-MS/MS, and performed global mRNA-sequencing pathway analysis on ScWAT and SkM. In MyoGlu, men with dysglycaemia displayed lower GIR, more fat mass and higher liver fat content than normoglycaemic men at baseline, and 12 weeks of exercise increased GIR, improved body composition and reduced liver fat content similarly for both groups. In our current study we observed higher plasma concentrations of BCAAs (14.4%, p = 0.01) and related metabolites, such as 3-hydroxyisobutyrate (19.4%, p = 0.034) in dysglycaemic vs normoglycaemic men at baseline. Baseline plasma BCAA levels correlated negatively to the change in GIR (ρ = -0.41, p = 0.037) and [Formula: see text] (ρ = -0.47, p = 0.015) after 12 weeks of exercise and positively to amounts of intraperitoneal fat (ρ = 0.40, p = 0.044) and liver fat (ρ = 0.58, p = 0.01). However, circulating BCAAs and related metabolites did not respond to 12 weeks of exercise, with the exception of isoleucine, which increased in normoglycaemic men (10 μmol/l, p = 0.01). Pathway analyses of mRNA-sequencing data implied reduced BCAA catabolism in both SkM and ScWAT in men with dysglycaemia compared with men with normoglycaemia at baseline. Gene expression levels related to BCAA metabolism correlated positively with GIR and markers of mitochondrial content in both SkM and ScWAT, and negatively with fat mass generally, and particularly with intraperitoneal fat mass. mRNA-sequencing pathway analysis also implied increased BCAA metabolism after 12 weeks of exercise in both groups and in both tissues, including enhanced expression of the gene encoding branched-chain α-ketoacid dehydrogenase (BCKDH) and reduced expression of the BCKDH phosphatase in both groups and tissues. Gene expression of SLC25A44, which encodes a mitochondrial BCAA transporter, was increased in SkM in both groups, and gene expression of BCKDK, which encodes BCKDH kinase, was reduced in ScWAT in dysglycaemic men. Mediation analyses indicated a pronounced effect of enhanced SkM (~53%, p = 0.022), and a moderate effect of enhanced ScWAT (~18%, p = 0.018) BCAA metabolism on improved insulin sensitivity after 12 weeks of exercise, based on mRNA sequencing. In comparison, plasma concentration of BCAAs did not mediate any effect in this regard. Plasma BCAA concentration was largely unresponsive to long-term exercise and unrelated to exercise-induced insulin sensitivity. On the other hand, the insulin-sensitising effect of long-term exercise in men may be explained by enhanced SkM and, to a lesser degree, also by enhanced ScWAT BCAA catabolism. Graphical abstract. Show less

Increasing adaptive thermogenesis by stimulating browning in white adipose tissue is a promising method of improving metabolic health. However, the molecular mechanisms underlying this transition remain elusive. Our study examined the molecular determinants driving the differentiation of precursor cells into thermogenic adipocytes. In this study, we conducted temporal high-resolution proteomic analysis of subcutaneous white adipose tissue (scWAT) after cold exposure in mice. This was followed by loss- and gain-of-function experiments using siRNA-mediated knockdown and CRISPRa-mediated induction of gene expression, respectively, to evaluate the function of the transcriptional regulator Y box-binding protein 1 (YBX1) during adipogenesis of brown pre-adipocytes and mesenchymal stem cells. Transcriptomic analysis of mesenchymal stem cells following induction of endogenous Ybx1 expression was conducted to elucidate transcriptomic events controlled by YBX1 during adipogenesis. Our proteomics analysis uncovered 509 proteins differentially regulated by cold in a time-dependent manner. Overall, 44 transcriptional regulators were acutely upregulated following cold exposure, among which included the cold-shock domain containing protein YBX1, peaking after 24 h. Cold-induced upregulation of YBX1 also occurred in brown adipose tissue, but not in visceral white adipose tissue, suggesting a role of YBX1 in thermogenesis. This role was confirmed by Ybx1 knockdown in brown and brite preadipocytes, which significantly impaired their thermogenic potential. Conversely, inducing Ybx1 expression in mesenchymal stem cells during adipogenesis promoted browning concurrent with an increased expression of thermogenic markers and enhanced mitochondrial respiration. At a molecular level, our transcriptomic analysis showed that YBX1 regulates a subset of genes, including the histone H3K9 demethylase Jmjd1c, to promote thermogenic adipocyte differentiation. Our study mapped the dynamic proteomic changes of murine scWAT during browning and identified YBX1 as a novel factor coordinating the genomic mechanisms by which preadipocytes commit to brite/beige lineage. Show less

Previously, we reported that inverse associations of high-density lipoprotein (HDL) with cardiovascular disease and diabetes were only observed for HDL that lacked the pro-inflammatory protein apolipoprotein C3 (apoC3). To provide further insight into the cardiometabolic properties of HDL subspecies defined by the presence or absence of apoC3, we aimed to examine these subspecies with liver fat content and non-alcoholic fatty liver disease (NAFLD). We investigated cross-sectional associations between ELISA-measured plasma levels of apoA1 in HDL that contained or lacked apoC3 and computed tomography-determined liver fat content and NAFLD (<51 HU) at baseline (2000-2002) among 5007 participants in the Multi-Ethnic Study of Atherosclerosis (MESA) without heavy alcohol consumption (>14 drinks/week in men and >7 drinks/week in women). In multivariable-adjusted regression models, apoA1 in HDL that contained or lacked apoC3 was differentially associated with liver fat content ( Show less

The ε4 allele of the apolipoprotein E (APOE) gene and lower apolipoprotein E (apoE) protein levels in plasma are risk factors for Alzheimer disease, but the underlying biological mechanisms are not fully understood. Half of plasma apoE circulates on high-density lipoproteins (HDLs). Higher apoE levels in plasma HDL were previously found to be associated with lower coronary heart disease risk, but the coexistence of another apolipoprotein, apoC3, modified this lower risk. To investigate associations between the presence of apoE in different lipoproteins with cognitive function, particularly the risk of dementia. This prospective case-cohort study embedded in the Ginkgo Evaluation of Memory Study (2000-2008) analyzed data from 1351 community-dwelling participants 74 years and older. Of this group, 995 participants were free of dementia at baseline (recruited from September 2000 to June 2002) and 521 participants were diagnosed with incident dementia during follow-up until 2008. Data analysis was performed from January 2018 to December 2019. Enzyme-linked immunosorbent assay-measured concentration of apoE in whole plasma, HDL-depleted plasma (non-HDL), HDL, and HDL subspecies that contain or lack apoC3 or apoJ. Adjusted hazard ratios for risk of dementia and Alzheimer disease during follow-up and adjusted differences (β coefficients) in Alzheimer Disease Assessment-Cognitive Subscale (ADAS-cog) and Modified Mini-Mental State Examination scores at baseline. Among 1351 participants, the median (interquartile range) age was 78 (76-81) years; 639 (47.3%) were women. The median (interquartile range) follow-up time was 5.9 (3.7-6.5) years. Higher whole plasma apoE levels and higher apoE levels in HDL were associated with better cognitive function assessed by ADAS-cog (whole plasma, β coefficient, -0.15; 95% CI, -0.24 to -0.06; HDL, β coefficient, -0.20; 95% CI, -0.30 to -0.10) but were unassociated with dementia or Alzheimer disease risk. When separated by apoC3, a higher apoE level in HDL that lacks apoC3 was associated with better cognitive function (ADAS-cog per SD: β coefficient, 0.17; 95% CI, -0.27 to -0.07; Modified Mini-Mental State Examination score per SD: β coefficient, 0.25; 95% CI, 0.07 to 0.42) and lower risk of dementia (hazard ratio per SD, 0.86; 95% CI, 0.76 to 0.99). In contrast, apoE levels in HDL that contains apoC3 were unassociated with any of these outcomes. In a prospective cohort of older adults with rigorous follow-up of dementia, the apoE level in HDL that lacked apoC3 was associated with better cognitive function and lower dementia risk. This finding suggests that the cardioprotective associations of this novel lipoprotein extend to dementia. Show less

Whether HDL is associated with dementia risk is unclear. In addition to apoA1, other apolipoproteins are found in HDL, creating subspecies of HDL that may have distinct metabolic properties. We measured apoA1, apoC3, and apoJ levels in plasma and apoA1 levels in HDL that contains or lacks apoE, apoJ, or apoC3 using a modified sandwich ELISA in a case-cohort study nested within the Ginkgo Evaluation of Memory Study. We included 995 randomly selected participants and 521 participants who developed dementia during a mean of 5.1 years of follow-up. The level of total apoA1 was not significantly related to dementia risk, regardless of the coexistence of apoC3, apoJ, or apoE. Higher levels of total plasma apoC3 were associated with better cognitive function at baseline (difference in Modified Mini-Mental State Examination scores tertile 3 vs. tertile 1: 0.60; 95% CI: 0.23, 0.98) and a lower dementia risk (adjusted hazard ratio tertile 3 vs. tertile 1: 0.73; 95% CI: 0.55, 0.96). Plasma concentrations of apoA1 in HDL and its apolipoprotein-defined subspecies were not associated with cognitive function at baseline or with the risk of dementia during follow-up. Similar studies in other populations are required to better understand the association between apoC3 and Alzheimer's disease pathology. Show less

Ankylosing spondylitis (AS) is a common complex inflammatory disease; however, up to now distinct genes with monogenic pattern have not been reported for this disease. In the present study, we report a large Iranian family with several affected members with AS. DNAs of the three affected and two healthy cases were chosen for performing whole-exome sequencing (WES). After several filtering steps, candidate variants in the following genes were detected: RELN, DNMT1, TAF4β, MUC16, DLG2, and FAM208. However, segregation analysis confirmed the association of only one variant, c.7456A>G; p.(Ser2486Gly) in the RELN gene with AS in this family. In addition, in silico predictions supported the probable pathogenicity of this variant. In this study, for the first time, we report a novel variant in the RELN gene, c.7456A>G; p.(Ser2486Gly), which completely co-segregates with AS. This association suggests potential insights into the pathophysiological bases of AS and it could broaden horizons toward new therapeutic strategies. Show less

The aim of this experiment was to investigate the effect of dietary supplementation of crushed high oleic sunflower seeds (HOS) and rumen-protected choline (RPC) on the fatty acid (FA) profile of phospholipids and sphingomyelin and mammary transcription of genes that are important for milk fat synthesis and de novo synthesis of sphingolipids. Twenty-four cows were divided into four groups that either received an unsupplemented diet (Control), the Control diet supplemented with 50 g RPC per day, a diet supplemented with HOS at 10% of dry matter, or RPC and HOS in combination (RPC + HOS). RPC supplementation had no effect on the FA composition of milk or sphingomyelin. Cows receiving RPC and RPC + HOS had increased incorporation of C22:5 (n-3) into phospholipids. Milk FA proportion of C18:0 and C18:1 isomers was increased in cows receiving HOS (HOS and RPC + HOS). Sphingomyelin proportion of C22:0 was increased in cows receiving HOS and RPC + HOS, at the expense of C23:0. HOS supplementation further increased the proportion of unsaturated fatty acids (UFA) in milk phospholipids. HOS supplementation increased mammary transcription of UDP-glucose ceramide glycosyltransferase (UGCG), sterol response element-binding protein cleavage-activating protein (SCAP) and peroxisome proliferation-activated receptor Gamma subunit C 1b (PPARGC1b), and reduced transcription of insulin induced gene 1 (INSIG1) and fatty acid-binding protein 3 (FABP3). Dietary supplementation of RPC increased mammary transcription of fatty acid desaturase 1 (FADS1) and longevity assurance gene 2 (LASS2), and reduced transcription of sphingomyelin synthase (SGMS). The results show that the FA profile of milk phospholipids is sensitive to dietary lipid supplementation and, to a minor degree, RPC supplementation. Furthermore, transcription of genes that are important for milk fat synthesis and sphingolipids synthesis is affected by dietary supplementation of RPC and HOS. Show less

Activation of endothelial β-catenin signaling by neural cell-derived Norrin or Wnt ligands is vital for the vascularization of the retina and brain. Mutations in members of the Norrin/β-catenin pathway contribute to inherited blinding disorders because of defective vascular development and dysfunctional blood-retina barrier. Despite a vital role for endothelial β-catenin signaling in central nervous system health and disease, its contribution to central nervous system angiogenesis and its interactions with downstream signaling cascades remains incompletely understood. Approach and Results: Here, using genetically modified mouse models, we show that impaired endothelial β-catenin signaling caused hypovascularization of the postnatal retina and brain because of deficient endothelial cell proliferation and sprouting. Mosaic genetic analysis demonstrated that endothelial β-catenin promotes but is not required for tip cell formation. In addition, pharmacological treatment revealed that angiogenesis under conditions of inhibited Notch signaling depends upon endothelial β-catenin. Importantly, impaired endothelial β-catenin signaling abrogated the expression of the VEGFR (vascular endothelial growth factor receptor)-2 and VEGFR3 in brain microvessels but not in the lung endothelium. Our study identifies molecular crosstalk between the Wnt/β-catenin and the Notch and VEGF-A signaling pathways and strongly suggest that endothelial β-catenin signaling supports central nervous system angiogenesis by promoting endothelial cell sprouting, tip cell formation, and VEGF-A/VEGFR2 signaling. Show less

Objective- The Wnt/β-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/β-catenin signaling by induced overexpression of Axin1, an inhibitor of β-catenin signaling, specifically in endothelial cells ( Axin1 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

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer mortality in American women. Normal ovarian physiology is intricately connected to small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) which govern processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. We hypothesized that common germline variation in genes encoding small GTPases is associated with EOC risk. We investigated 322 variants in 88 small GTPase genes in germline DNA of 18,736 EOC patients and 26,138 controls of European ancestry using a custom genotype array and logistic regression fitting log-additive models. Functional annotation was used to identify biofeatures and expression quantitative trait loci that intersect with risk variants. One variant, ARHGEF10L (Rho guanine nucleotide exchange factor 10 like) rs2256787, was associated with increased endometrioid EOC risk (OR = 1.33, p = 4.46 x 10-6). Other variants of interest included another in ARHGEF10L, rs10788679, which was associated with invasive serous EOC risk (OR = 1.07, p = 0.00026) and two variants in AKAP6 (A-kinase anchoring protein 6) which were associated with risk of invasive EOC (rs1955513, OR = 0.90, p = 0.00033; rs927062, OR = 0.94, p = 0.00059). Functional annotation revealed that the two ARHGEF10L variants were located in super-enhancer regions and that AKAP6 rs927062 was associated with expression of GTPase gene ARHGAP5 (Rho GTPase activating protein 5). Inherited variants in ARHGEF10L and AKAP6, with potential transcriptional regulatory function and association with EOC risk, warrant investigation in independent EOC study populations. Show less

Subspecies of HDL contain apolipoprotein E (apoE) and/or apoCIII. Both proteins have properties that could affect HDL metabolism. The relation between HDL metabolism and risk of coronary heart disease (CHD) is not well understood. Eighteen participants were given a bolus infusion of [D3]L-leucine to label endogenous proteins on HDL. HDL was separated into subspecies containing apoE and/or apoCIII and then into 4 sizes. Metabolic rates for apoA-I in HDL subspecies and sizes were determined by interactive modeling. The concentrations of apoE in HDL that contain or lack apoCIII were measured in a prospective study in Denmark including 1,949 incident CHD cases during 9 years. HDL containing apoE but not apoCIII is disproportionately secreted into the circulation, actively expands while circulating, and is quickly cleared. These are key metabolic steps in reverse cholesterol transport, which may protect against atherosclerosis. ApoCIII on HDL strongly attenuates these metabolic actions of HDL apoE. In the epidemiological study, the relation between HDL apoE concentration and CHD significantly differed depending on whether apoCIII was present. HDL apoE was associated significantly with lower risk of CHD only in the HDL subspecies lacking apoCIII. ApoE and apoCIII on HDL interact to affect metabolism and CHD. ApoE promotes metabolic steps in reverse cholesterol transport and is associated with lower risk of CHD. ApoCIII, when coexisting with apoE on HDL, abolishes these benefits. Therefore, differences in metabolism of HDL subspecies pertaining to reverse cholesterol transport are reflected in differences in association with CHD. Clinicaltrials.gov NCT01399632. This work was supported by NIH grant R01HL095964 to FMS and by a grant to the Harvard Clinical and Translational Science Center (8UL1TR0001750) from the National Center for Advancing Translational Science. Show less

Polyunsaturated fatty acids (PUFAs) are associated with a lower risk of multiple diseases. Fatty acid desaturase 1 gene (FADS1) polymorphisms and dietary PUFA intake are both established determinants of circulating PUFA proportions. We explored the joint effects of FADS1 polymorphisms and dietary PUFA intake on circulating PUFA proportions. We studied 2288 participants from a nested case-control study of coronary artery disease among participants who provided blood samples in the Nurses' Health Study and the Health Professionals Follow-Up Study. Dietary PUFA intake was obtained from semiquantitative food-frequency questionnaires. FADS1 rs174546 was genotyped by using the Affymetrix 6.0 platform, and circulating PUFA proportions were measured with gas-liquid chromatography. Linear regression models were used to examine the associations between rs174546 and circulating proportions of each fatty acid. Gene-diet interactions were tested by including a cross-product term of dietary intake of each PUFA by rs174546 genotype in the linear regression models. After adjustment for sex and ancestry, each copy of the C allele of rs174546 was associated with higher circulating proportions of arachidonic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) and lower proportions of linoleic acid and α-linolenic acid. The magnitude of positive association between higher consumption of dietary EPA or DHA and circulating proportions of EPA increased with each copy of the rs174546_T allele (P-interaction = 0.01 and 0.007, respectively). Each 1-SD increment in EPA intake was associated with an average 3.7% increase in circulating EPA proportions among participants with the rs174546_CC genotype and an average 7.8% increase among participants with the TT genotype. Carriers of the T allele at FADS1 rs174546 may need higher doses of dietary EPA and DHA to achieve the same circulating proportions of EPA as carriers of the C allele. The implications of these findings on disease risk and dietary guidelines require further study. Show less

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disease, which goes along with increased risk for sudden cardiac death (SCD). Despite the knowledge about the different causal genes, the relationship between individual genotypes and phenotypes is incomplete. We retrieved PubMed/Medline literatures on genotype-phenotype associations in patients with HCM and mutations in MYBPC3, MYH7, TNNT2, and TNNI3. Altogether, 51 studies with 7675 HCM patients were included in our meta-analysis. The average frequency of mutations in MYBPC3 (20%) and MYH7 (14%) was higher than TNNT2 and TNNI3 (2% each). The mean age of HCM onset for MYH7 mutation positive patients was the beginning of the fourth decade, significantly earlier than patients without sarcomeric mutations. A high male proportion was observed in TNNT2 (69%), MYBPC3 (62%) and mutation negative group (64%). Cardiac conduction disease, ventricular arrhythmia and heart transplantation (HTx) rate were higher in HCM patients with MYH7 mutations in comparison to MYBPC3 (p < 0.05). Furthermore, SCD was significantly higher in patients with sarcomeric mutations (p < 0.01). A pooled dataset and a comprehensive genotype-phenotype analysis show that the age at disease onset of HCM patients with MYH7 is earlier and leads to a more severe phenotype than in patient without such mutations. Furthermore, patients with sarcomeric mutations are more susceptible to SCD. The present study further supports the clinical interpretation of sarcomeric mutations in HCM patients. 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

Physical activity promotes specific adaptations in most tissues including skeletal muscle. Acute exercise activates numerous signaling cascades including pathways involving mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK)1/2, which returns to pre-exercise level after exercise. The expression of MAPK phosphatases (MKPs) in human skeletal muscle and their regulation by exercise have not been investigated before. In this study, we used mRNA sequencing to monitor regulation of MKPs in human skeletal muscle after acute cycling. In addition, primary human myotubes were used to gain more insights into the regulation of MKPs. The two ERK1/2-specific MKPs, dual specificity phosphatase 5 (DUSP5) and DUSP6, were the most regulated MKPs in skeletal muscle after acute exercise. Show less

MUFAs are unsaturated FAs with one double bond and are derived from endogenous synthesis and dietary intake. Accumulating evidence has suggested that plasma and erythrocyte MUFA levels are associated with cardiometabolic disorders, including CVD, T2D, and metabolic syndrome (MS). Previous genome-wide association studies (GWASs) have identified seven loci for plasma and erythrocyte palmitoleic and oleic acid levels in populations of European origin. To identify additional MUFA-associated loci and the potential functional variant at each locus, we performed ethnic-specific GWAS meta-analyses and trans-ethnic meta-analyses in more than 15,000 participants of Chinese and European ancestry. We identified novel genome-wide significant associations for vaccenic acid at Show less

Several common genetic variants have been associated with either ventricular fibrillation (VF) or sudden cardiac death (SCD). However, replication efforts have been limited. Therefore, we aimed to analyze whether such variants may contribute to VF caused by first ST-elevation myocardial infarction (STEMI). We analyzed 27 single nucleotide polymorphisms (SNP) previously associated with SCD/VF in other cohorts, and examined whether these SNPs were associated with VF caused by first STEMI in the GEnetic causes of Ventricular Arrhythmias in patients with first ST-elevation Myocardial Infarction (GEVAMI) study on ethnical Danes. The GEVAMI study is a prospective case-control study involving 257 cases (STEMI with VF) and 537 controls (STEMI without VF). Of the 27 candidate SNPs, one SNP (rs11720524) located in intron 1 of SCN5A which was previously associated with SCD was significantly associated with VF caused by first STEMI. The major C-allele of rs11720524 was present in 64% of the cases and the C/C genotype was significantly associated with VF with an odds ratio (OR) of 1.87 (95% CI: 1.12-3.12; P = 0.017). After controlling for clinical differences between cases and controls such as age, sex, family history of sudden death, alcohol consumption, previous atrial fibrillation, statin use, angina, culprit artery, and thrombolysis in myocardial infarction (TIMI) flow, the C/C genotype of rs11720524 was still significantly associated with VF with an OR of 1.9 (95% CI: 1.05-3.43; P = 0.032). Marginal associations with VF were also found for rs9388451 in HEY2 gene. The CC genotype showed an insignificant risk for VF with OR = 1.50 (95% CI: 0.96-2.40; P = 0.070). One common intronic variant in SCN5A suggested an association with VF caused by first STEMI. Further studies into the functional abnormalities associated with the noncoding variant in SCN5A may lead to important insights into predisposition to VF during STEMI. Show less

Routine genetic testing in Dilated Cardiomyopathy (DCM) has recently become reality using Next-Generation Sequencing. Several studies have explored the relationship between genotypes and clinical phenotypes to support risk estimation and therapeutic decisions, however, most studies are small or restricted to a few genes. This study provides to our knowledge the first systematic meta-analysis on genotype-phenotype associations in DCM. We retrieved PubMed/Medline literature on genotype-phenotype associations in patients with DCM and mutations in LMNA, PLN, RBM20, MYBPC3, MYH7, TNNT2 and TNNI3. We summarized and extensively reviewed all studies that passed selection criteria and performed a meta-analysis on key phenotypic parameters. Together, 48 studies with 8097 patients were included. Furthermore, we reviewed recent studies investigating genotype-phenotype associations in DCM patients with TTN mutations. The average frequency of mutations in the investigated genes was between 1 and 5 %. The mean age of DCM onset was the beginning of the fifth decade for all genes. Heart transplantation (HTx) rate was highest in LMNA mutation carriers (27 %), while RBM20 mutation carriers were transplanted at a markedly younger age (mean 28.5 years). While 73 % of DCM patients with LMNA mutations showed cardiac conduction diseases, low voltage was the reported ECG hallmark in PLN mutation carriers. The frequency of ventricular arrhythmia in DCM patients with LMNA (50 %) and PLN (43 %) mutations was significantly higher. The penetrance of DCM phenotype in subjects with TTN truncating variants increased with age and reached 100 % by age of 70. A pooled analysis of available genotype-phenotype data shows a higher prevalence of sudden cardiac death (SCD), cardiac transplantation, or ventricular arrhythmias in LMNA and PLN mutation carriers compared to sarcomeric gene mutations. This study will further support the clinical interpretation of genetic findings. Show less

Women with epithelial ovarian cancer (EOC) are usually treated with platinum/taxane therapy after cytoreductive surgery but there is considerable inter-individual variation in response. To identify germline single-nucleotide polymorphisms (SNPs) that contribute to variations in individual responses to chemotherapy, we carried out a multi-phase genome-wide association study (GWAS) in 1,244 women diagnosed with serous EOC who were treated with the same first-line chemotherapy, carboplatin and paclitaxel. We identified two SNPs (rs7874043 and rs72700653) in TTC39B (best P=7x10-5, HR=1.90, for rs7874043) associated with progression-free survival (PFS). Functional analyses show that both SNPs lie in a putative regulatory element (PRE) that physically interacts with the promoters of PSIP1, CCDC171 and an alternative promoter of TTC39B. The C allele of rs7874043 is associated with poor PFS and showed increased binding of the Sp1 transcription factor, which is critical for chromatin interactions with PSIP1. Silencing of PSIP1 significantly impaired DNA damage-induced Rad51 nuclear foci and reduced cell viability in ovarian cancer lines. PSIP1 (PC4 and SFRS1 Interacting Protein 1) is known to protect cells from stress-induced apoptosis, and high expression is associated with poor PFS in EOC patients. We therefore suggest that the minor allele of rs7874043 confers poor PFS by increasing PSIP1 expression. Show less

To report optical coherence tomography findings obtained in two patients with juvenile neuronal ceroid lipofuscinosis. Two case reports. Two 7-year-old girls presented with decreased visual acuity, clumsiness, night blindness, and behavioral problems. Optical coherence tomography showed an overall reduction in thickness of the central retina, as well as the outer and the inner retinal layers. The degenerative retinal changes were the same, despite different mutations in the CLN3 gene. In these rare cases of juvenile neuronal ceroid lipofuscinosis, optical coherence tomography enabled unambiguous detection of prominent morphologic abnormalities of the retina at the patient's first presentation. The advanced stage of photoreceptor degeneration seen in our patients shows that a diagnosis can potentially be made much earlier. Show less

The goals are to understand the primary genetic mechanisms that cause Sick Sinus Syndrome and to identify potential modifiers that may result in intrafamilial variability within a multigenerational family. The proband is a 63-year-old male with a family history of individuals (>10) with sinus node dysfunction, ventricular arrhythmia, cardiomyopathy, heart failure, and sudden death. We used exome sequencing of a single individual to identify a novel LMNA mutation and demonstrated the importance of Sanger validation and family studies when evaluating candidates. After initial single-gene studies were negative, we conducted exome sequencing for the proband which produced 9 gigabases of sequencing data. Bioinformatics analysis showed 94% of the reads mapped to the reference and identified 128,563 unique variants with 108,795 (85%) located in 16,319 genes of 19,056 target genes. We discovered multiple variants in known arrhythmia, cardiomyopathy, or ion channel associated genes that may serve as potential modifiers in disease expression. To identify candidate mutations, we focused on ~2,000 variants located in 237 genes of 283 known arrhythmia, cardiomyopathy, or ion channel associated genes. We filtered the candidates to 41 variants in 33 genes using zygosity, protein impact, database searches, and clinical association. Only 21 of 41 (51%) variants were validated by Sanger sequencing. We selected nine confirmed variants with minor allele frequencies <1% for family studies. The results identified LMNA c.357-2A>G, a novel heterozygous splice-site mutation as the primary mutation with rare or novel variants in HCN4, MYBPC3, PKP4, TMPO, TTN, DMPK and KCNJ10 as potential modifiers and a mechanism consistent with haploinsufficiency. Show less

The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius. Show less

In both animals and plants, messenger (m)RNA export has been shown to contribute to immune response regulation. The Arabidopsis nuclear protein MOS11, along with the nucleoporins MOS3/Nup96/SAR3 and Nup160/SAR1 are components of the mRNA export machinery and contribute to immunity mediated by nucleotide binding leucine-rich repeat immune receptors (NLR). The human MOS11 ortholog CIP29 is part of a small protein complex with three additional members: the RNA helicase DDX39, ALY, and TAF15b. We systematically assessed the biological roles of the Arabidopsis homologs of these proteins in toll interleukin 1 receptor-type NLR (TNL)-mediated immunity using reverse genetics. Although mutations in ALY and DDX39 did not result in obvious defects, taf15b mutation partially suppressed the autoimmune phenotypes of a gain-of-function TNL mutant, snc1. An additive effect on snc1 suppression was observed in mos11-1 taf15b snc1 triple mutant plants, suggesting that MOS11 and TAF15b have independent functions. TAF15b-GFP fusion protein, which fully complemented taf15b mutant phenotypes, localized to nuclei similarly to MOS11. However, it was also targeted to cytosolic granules identified as processing bodies. In addition, we observed no change in SNC1 mRNA levels, whereas less SNC1 protein accumulated in taf15b mutant, suggesting that TAF15b contributes to SNC1 homeostasis through posttranscriptional mechanisms. In summary, this study highlights the importance of posttranscriptional RNA processing mediated by TAF15b in the regulation of TNL-mediated immunity. Show less

Circulating trans fatty acids (TFAs), which cannot be synthesized by humans, are linked to adverse health outcomes. Although TFAs are obtained from diet, little is known about subsequent influences (e.g., relating to incorporation, metabolism, or intercompetition with other fatty acids) that could alter circulating concentrations and possibly modulate or mediate impacts on health. The objective was to elucidate novel biologic pathways that may influence circulating TFAs by evaluating associations between common genetic variation and TFA biomarkers. We performed meta-analyses using 7 cohorts of European-ancestry participants (n = 8013) having measured genome-wide variation in single-nucleotide polymorphisms (SNPs) and circulating TFA biomarkers (erythrocyte or plasma phospholipids), including trans-16:1n-7, total trans-18:1, trans/cis-18:2, cis/trans-18:2, and trans/trans-18:2. We further evaluated SNPs with genome-wide significant associations among African Americans (n = 1082), Chinese Americans (n = 669), and Hispanic Americans (n = 657) from 2 of these cohorts. Among European-ancestry participants, 31 SNPs in or near the fatty acid desaturase (FADS) 1 and 2 cluster were associated with cis/trans-18:2; a top hit was rs174548 (β = 0.0035, P = 4.90 × 10(-15)), an SNP previously associated with circulating n-3 and n-6 polyunsaturated fatty acid concentrations. No significant association was identified for other TFAs. rs174548 in FADS1/2 was also associated with cis/trans-18:2 in Hispanic Americans (β = 0.0053, P = 1.05 × 10(-6)) and Chinese Americans (β = 0.0028, P = 0.002) but not African Americans (β = 0.0009, P = 0.34); however, in African Americans, fine mapping identified a top hit in FADS2 associated with cis/trans-18:2 (rs174579: β = 0.0118, P = 4.05 × 10(-5)). The association between rs174548 and cis/trans-18:2 remained significant after further adjustment for individual circulating n-3 and n-6 fatty acids, except arachidonic acid. After adjustment for arachidonic acid concentrations, the association between rs174548 and cis/trans-18:2 was nearly eliminated in European-ancestry participants (β-coefficient reduced by 86%), with similar reductions in Hispanic Americans and Chinese Americans. Our findings provide novel evidence for genetic regulation of cis/trans-18:2 by the FADS1/2 cluster and suggest that this regulation may be influenced/mediated by concentrations of arachidonic acid, an n-6 polyunsaturated fat. Show less

Coffee, a major dietary source of caffeine, is among the most widely consumed beverages in the world and has received considerable attention regarding health risks and benefits. We conducted a genome-wide (GW) meta-analysis of predominately regular-type coffee consumption (cups per day) among up to 91,462 coffee consumers of European ancestry with top single-nucleotide polymorphisms (SNPs) followed-up in ~30 062 and 7964 coffee consumers of European and African-American ancestry, respectively. Studies from both stages were combined in a trans-ethnic meta-analysis. Confirmed loci were examined for putative functional and biological relevance. Eight loci, including six novel loci, met GW significance (log10Bayes factor (BF)>5.64) with per-allele effect sizes of 0.03-0.14 cups per day. Six are located in or near genes potentially involved in pharmacokinetics (ABCG2, AHR, POR and CYP1A2) and pharmacodynamics (BDNF and SLC6A4) of caffeine. Two map to GCKR and MLXIPL genes related to metabolic traits but lacking known roles in coffee consumption. Enhancer and promoter histone marks populate the regions of many confirmed loci and several potential regulatory SNPs are highly correlated with the lead SNP of each. SNP alleles near GCKR, MLXIPL, BDNF and CYP1A2 that were associated with higher coffee consumption have previously been associated with smoking initiation, higher adiposity and fasting insulin and glucose but lower blood pressure and favorable lipid, inflammatory and liver enzyme profiles (P<5 × 10(-8)).Our genetic findings among European and African-American adults reinforce the role of caffeine in mediating habitual coffee consumption and may point to molecular mechanisms underlying inter-individual variability in pharmacological and health effects of coffee. Show less

Food ingestion decreases bone resorption, and glucose-dependent insulinotropic polypeptide (GIP) may mediate this effect. Mice overexpressing GIP have increased osteoblast activity and are rescued from age-related bone loss, whereas GIPR knockout mice have decreased cortical bone mass and compromised bone quality. Carriers of the functional variant GIPR Glu354Gln (rs1800437) have higher plasma glucose 2 hours after glucose ingestion, suggesting that the variant encoding GIPR 354Gln decreases the effect of GIP. The objective of the study was to investigate the effect of GIPR Glu354Gln on bone mineral density (BMD) and fracture risk. This was a prospective, comprehensive, cohort study (number NCT00252408). A total of 1686 perimenopausal women were included. Dual-energy X-ray absorptiometry was performed at baseline and after 10 years. Incident fractures were recorded during the follow-up and were obtained from the Danish National Patient Registry, giving a total follow-up time of a minimum 16 years. After 10 years, women with the minor frequency C allele of rs1800437 (354Gln) had significantly lower BMD at the femoral neck compared with carriers of the major G-allele (CC: 0.755 ± 0.015 g/cm(2) vs CG: 747 ± 0.005 g/cm(2); GG: 0.766 ± 0.004 g/cm(2), P < .001). Correspondingly, total hip BMD was significantly lower among C allele carriers (CC: 0.881 ± 0.016 g/cm(2); CG: 0.884 ± 0.005 g/cm(2); and GG: 0.906 ± 0.004 g/cm(2), P < .001). Finally, women homozygous for the variant C allele had an increased risk (hazard ratio 1.6, confidence interval 1.0-2.6, P < .05) of nonvertebral fractures. This study demonstrates an association between a functional GIPR polymorphism Glu354Gln (rs1800437) and BMD and fracture risk. These findings further establish GIP to be involved in the regulation of bone density. Show less

LC/MS quantification of multiple plasma proteins that differ by several orders of magnitude in concentration from a single sample is challenging. We present a strategy that allows the simultaneous determination of the concentration and turnover kinetics of higher and lower abundant proteins from a single digestion mixture. Our attention was directed at a cluster of proteins that interact to affect the absorption and interorgan lipid trafficking. We demonstrate that apos involved in TG metabolism such as apoC2, C3, E, and A4 (micromolar concentration), and apoB48 and apoA5 (single-digit nanomolar concentration) can be quantified from a single digestion mixture. A high degree of correlation between LC/MS and immunobased measurements for apoC2, C3, E, and B48 was observed. Moreover, apoA5 fractional synthesis rate was measured in humans for the first time. Finally, the method can be directly applied to studies involving nonhuman primates because peptide sequences used in the method are conserved between humans and nonhuman primates. Show less

Functional variants that contribute to genomewide association study (GWAS) signals are difficult to identify. MicroRNAs could contribute to some of these gene-trait relationships. We compiled a set of GWAS trait gene SNPs that were predicted to affect microRNA regulation of mRNA. Trait associations were tested in a sample of 6725 European-American (EA) and African-American (AA) subjects that were interviewed using the polydiagnostic SSADDA to diagnose major psychiatric disorders. A predicted miR-330-3p target site SNP (rs41305272) in mitogen-activated protein kinase kinase 5 (MAP2K5) mRNA was in LD (d' = 1.0, r(2) = 0.02) with a reported GWAS-identified variant for restless legs syndrome (RLS), a disorder frequently comorbid with anxiety and depression, possibly because of a shared pathophysiology. We examined the SNP's association with mood and anxiety-related disorders. Rs41305272 was associated with agoraphobia (Ag) in EAs (odds ratio [OR] = 1.95, P = 0.007; 195 cases) and AAs (OR = 3.2, P = 0.03; 148 cases) and major depressive disorder (MDD) in AAs (OR = 2.64, P = 0.01; 427 cases), but not EAs (465 cases). Rs41305272*T carrier frequency was correlated with the number of anxiety and depressive disorders diagnosed per subject. RLS was not evaluated in our subjects. Predicted miR-330-3p target genes were enriched in pathways relevant to psychiatric disorders. These findings suggest that microRNA target site information may be useful in the analysis of GWAS signals for complex traits. MiR-330-3p and MAP2K5 are potentially important contributors to mood and anxiety-related traits. With support from additional studies, these findings could add to the large number of risk genes identified through association to medical disorders that have primary psychiatric effects. Show less

Recently, a number of patients have been described with structural rearrangements at 3q13.31, delineating a novel microdeletion syndrome with common clinical features including developmental delay and other neurodevelopmental disorders (NDD). A smallest region of overlapping deletions (SRO) involved five RefSeq genes, including the transcription factor gene ZBTB20 and the dopamine receptor gene DRD3, considered as candidate genes for the syndrome. We used array comparative genomic hybridization and next-generation mate-pair sequencing to identify key structural rearrangements involving ZBTB20 in two patients with NDD. In a patient with developmental delay, attention-deficit hyperactivity disorder, psychosis, Tourette's syndrome and autistic traits, a de novo balanced t(3;18) translocation truncated ZBTB20. The other breakpoint did not disrupt any gene. In a second patient with developmental delay and autism, we detected the first microdeletion at 3q13.31, which truncated ZBTB20 but did not involve DRD3 or the other genes within the previously defined SRO. Zbtb20 directly represses 346 genes in the developing murine brain. Of the 342 human orthologous ZBTB20 candidate target genes, we found 68 associated with NDD. Using chromatin immunoprecipitation and quantitative PCR, we validated the in vivo binding of Zbtb20 in evolutionary conserved regions in six of these genes (Cntn4, Gad1, Nrxn1, Nrxn3, Scn2a, Snap25). Our study links dosage imbalance of ZBTB20 to a range of neurodevelopmental, cognitive and psychiatric disorders, likely mediated by dysregulation of multiple ZBTB20 target genes, and provides new knowledge on the genetic background of the NDD seen in the 3q13.31 microdeletion syndrome. Show less