Rachel Buchan

MicroRNA-223 regulates Glut4 expression and cardiomyocyte glucose metabolism

AIMS MicroRNAs (miRNAs) are important for cardiac function and tissue metabolism. The aim of the present study is to investigate the role(s) of miRNAs in the insulin-resistant heart. METHODS AND RESULTS Left ventricular biopsies were collected from patients with or without type 2 diabetes and from patients with left ventricular dysfunction. Quantitative miRNA expression analyses of 155 miRNAs revealed that miR-223 was consistently upregulated in the insulin-resistant heart. We assessed the effects of miR-223 on glucose metabolism in neonatal rat cardiomyocytes where adenoviral-mediated overexpression of miR-223 increased glucose uptake. Using in silico miRNA target prediction programs, we prioritized candidate miR-223 target genes, but observed no effect of miR-223 on myocyte enhancer factor 2c or insulin-like growth factor 1 receptor, and an unexpected miR-223-induced increase in nuclear factor IA. We next examined the effects of miR-223 on insulin signalling and glucose transport proteins. Neither phosphoinositide 3-kinase (PI3K) signalling nor AMP kinase activity was affected by miR-223 overexpression, whereas glucose transporter 4 (Glut4) protein expression was increased. miR-223 overexpression-induced Glut4 protein expression in cardiomyocytes was necessary and sufficient for increased glucose uptake as demonstrated by siRNA knockdown of Glut4. Loss-of-function studies in vivo, using a synthetic miR-223 inhibitor, confirmed the effect of miR-223 on Glut4. CONCLUSION These data demonstrate a role for miR-223 in Glut4 regulation and glucose metabolism in the heart, reveal the pleiotropic effects of miRNAs across tissues, and show that miRNAs can upregulate target genes in terminally differentiated cardiomyocytes.

A trans-acting locus regulates an anti-viral expression network and type 1 diabetes risk

Combined analyses of gene networks and DNA sequence variation can provide new insights into the aetiology of common diseases that may not be apparent from genome-wide association studies alone. Recent advances in rat genomics are facilitating systems-genetics approaches. Here we report the use of integrated genome-wide approaches across seven rat tissues to identify gene networks and the loci underlying their regulation. We defined an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) enriched for viral response genes, which represents a molecular biomarker for macrophages and which was regulated in multiple tissues by a locus on rat chromosome 15q25. We show that Epstein–Barr virus induced gene 2 (Ebi2, also known as Gpr183), which lies at this locus and controls B lymphocyte migration, is expressed in macrophages and regulates the IDIN. The human orthologous locus on chromosome 13q32 controlled the human equivalent of the IDIN, which was conserved in monocytes. IDIN genes were more likely to associate with susceptibility to type 1 diabetes (T1D)—a macrophage-associated autoimmune disease—than randomly selected immune response genes (P = 8.85 × 10−6). The human locus controlling the IDIN was associated with the risk of T1D at single nucleotide polymorphism rs9585056 (P = 7.0 × 10−10; odds ratio, 1.15), which was one of five single nucleotide polymorphisms in this region associated with EBI2 (GPR183) expression. These data implicate IRF7 network genes and their regulatory locus in the pathogenesis of T1D.

Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease

Truncating variants of the giant protein titin cause dilated cardiomyopathy when they occur toward the protein’s carboxyl terminus and in highly expressed exons. What Happens When Titins Are Trimmed? The most common form of inherited heart failure, dilated cardiomyopathy, can be caused by mutations in a mammoth heart protein, appropriately called titin. Now, Roberts et al. sort out which titin mutations cause disease and why some people can carry certain titin mutations but remain perfectly healthy. In an exhaustive survey of more than 5200 people, with and without cardiomyopathy, the authors sequenced the titin gene and measured its corresponding RNA and protein levels. The alterations in titin were truncating mutations, which cause short nonfunctional versions of the RNA or protein. These defects produced cardiomyopathy when they occurred closer to the protein’s carboxyl terminus and in exons that were abundantly transcribed. The titin-truncating mutations that occur in the general population tended not to have these characteristics and were usually benign. This new detailed understanding of the molecular basis of dilated cardiomyopathy penetrance will promote better disease management and accelerate rational patient stratification. The recent discovery of heterozygous human mutations that truncate full-length titin (TTN, an abundant structural, sensory, and signaling filament in muscle) as a common cause of end-stage dilated cardiomyopathy (DCM) promises new prospects for improving heart failure management. However, realization of this opportunity has been hindered by the burden of TTN-truncating variants (TTNtv) in the general population and uncertainty about their consequences in health or disease. To elucidate the effects of TTNtv, we coupled TTN gene sequencing with cardiac phenotyping in 5267 individuals across the spectrum of cardiac physiology and integrated these data with RNA and protein analyses of human heart tissues. We report diversity of TTN isoform expression in the heart, define the relative inclusion of TTN exons in different isoforms (using the TTN transcript annotations available at http://cardiodb.org/titin), and demonstrate that these data, coupled with the position of the TTNtv, provide a robust strategy to discriminate pathogenic from benign TTNtv. We show that TTNtv is the most common genetic cause of DCM in ambulant patients in the community, identify clinically important manifestations of TTNtv-positive DCM, and define the penetrance and outcomes of TTNtv in the general population. By integrating genetic, transcriptome, and protein analyses, we provide evidence for a length-dependent mechanism of disease. These data inform diagnostic criteria and management strategies for TTNtv-positive DCM patients and for TTNtv that are identified as incidental findings.

Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function.

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

Defining the genetic architecture of hypertrophic cardiomyopathy: re-evaluating the role of non-sarcomeric genes

Abstract Aim Hypertrophic cardiomyopathy (HCM) exhibits genetic heterogeneity that is dominated by variation in eight sarcomeric genes. Genetic variation in a large number of non-sarcomeric genes has also been implicated in HCM but not formally assessed. Here we used very large case and control cohorts to determine the extent to which variation in non-sarcomeric genes contributes to HCM. Methods and results We sequenced known and putative HCM genes in a new large prospective HCM cohort (n = 804) and analysed data alongside the largest published series of clinically genotyped HCM patients (n = 6179), previously published HCM cohorts and reference population samples from the exome aggregation consortium (ExAC, n = 60 706) to assess variation in 31 genes implicated in HCM. We found no significant excess of rare (minor allele frequency < 1:10 000 in ExAC) protein-altering variants over controls for most genes tested and conclude that novel variants in these genes are rarely interpretable, even for genes with previous evidence of co-segregation (e.g. ACTN2). To provide an aid for variant interpretation, we integrated HCM gene sequence data with aggregated pedigree and functional data and suggest a means of assessing gene pathogenicity in HCM using this evidence. Conclusion We show that genetic variation in the majority of non-sarcomeric genes implicated in HCM is not associated with the condition, reinforce the fact that the sarcomeric gene variation is the primary cause of HCM known to date and underscore that the aetiology of HCM is unknown in the majority of patients.

Phenotype and Clinical Outcomes of Titin Cardiomyopathy.

Background Improved understanding of dilated cardiomyopathy (DCM) due to titin truncation (TTNtv) may help guide patient stratification. Objectives The purpose of this study was to establish relationships among TTNtv genotype, cardiac phenotype, and outcomes in DCM. Methods In this prospective, observational cohort study, DCM patients underwent clinical evaluation, late gadolinium enhancement cardiovascular magnetic resonance, TTN sequencing, and adjudicated follow-up blinded to genotype for the primary composite endpoint of cardiovascular death, and major arrhythmic and major heart failure events. Results Of 716 subjects recruited (mean age 53.5 ± 14.3 years; 469 men [65.5%]; 577 [80.6%] New York Heart Association function class I/II), 83 (11.6%) had TTNtv. Patients with TTNtv were younger at enrollment (49.0 years vs. 54.1 years; p = 0.002) and had lower indexed left ventricular mass (5.1 g/m2 reduction; padjusted = 0.03) compared with patients without TTNtv. There was no difference in biventricular ejection fraction between TTNtv+/− groups. Overall, 78 of 604 patients (12.9%) met the primary endpoint (median follow-up 3.9 years; interquartile range: 2.0 to 5.8 years), including 9 of 71 patients with TTNtv (12.7%) and 69 of 533 (12.9%) without. There was no difference in the composite primary outcome of cardiovascular death, heart failure, or arrhythmic events, for patients with or without TTNtv (hazard ratio adjusted for primary endpoint: 0.92 [95% confidence interval: 0.45 to 1.87]; p = 0.82). Conclusions In this large, prospective, genotype-phenotype study of ambulatory DCM patients, we show that prognostic factors for all-cause DCM also predict outcome in TTNtv DCM, and that TTNtv DCM does not appear to be associated with worse medium-term prognosis.

Truncating Variants in Titin Independently Predict Early Arrhythmias in Patients With Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) has a population prevalence of ∼1 in 500 and is associated with prognostically adverse arrhythmias at initial disease presentation in up to one-third of patients [(1)][1]. While increasing age, male sex, and impaired ventricular function are established arrhythmic risk

Genetic Etiology for Alcohol-Induced Cardiac Toxicity

Background Alcoholic cardiomyopathy (ACM) is defined by a dilated and impaired left ventricle due to chronic excess alcohol consumption. It is largely unknown which factors determine cardiac toxicity on exposure to alcohol. Objectives This study sought to evaluate the role of variation in cardiomyopathy-associated genes in the pathophysiology of ACM, and to examine the effects of alcohol intake and genotype on dilated cardiomyopathy (DCM) severity. Methods The authors characterized 141 ACM cases, 716 DCM cases, and 445 healthy volunteers. The authors compared the prevalence of rare, protein-altering variants in 9 genes associated with inherited DCM. They evaluated the effect of genotype and alcohol consumption on phenotype in DCM. Results Variants in well-characterized DCM-causing genes were more prevalent in patients with ACM than control subjects (13.5% vs. 2.9%; p = 1.2 ×10−5), but similar between patients with ACM and DCM (19.4%; p = 0.12) and with a predominant burden of titin truncating variants (TTNtv) (9.9%). Separately, we identified an interaction between TTN genotype and excess alcohol consumption in a cohort of DCM patients not meeting ACM criteria. On multivariate analysis, DCM patients with a TTNtv who consumed excess alcohol had an 8.7% absolute reduction in ejection fraction (95% confidence interval: −2.3% to −15.1%; p < 0.007) compared with those without TTNtv and excess alcohol consumption. The presence of TTNtv did not predict phenotype, outcome, or functional recovery on treatment in ACM patients. Conclusions TTNtv represent a prevalent genetic predisposition for ACM, and are also associated with a worse left ventricular ejection fraction in DCM patients who consume alcohol above recommended levels. Familial evaluation and genetic testing should be considered in patients presenting with ACM.

95 Identification Of Likely Pathogenic Variants In Patients With Bicuspid Aortic Valve: Correlation Of Complex Genotype With A More Severe Aortic Phenotype

Introduction The common heritable condition of Bicuspid Aortic Valve (BAV) is phenotypically heterogeneous, with valve dysfunction and aortopathy the major complications. We report overrepresentation of rare, likely pathogenic variants in target genes in a large cohort of 176 patients with BAV. We also describe a more severe aortic phenotype in patients with more than one known or likely pathogenic variant, supporting a multi-hit hypothesis for development of complications of BAV. Methods We recruited 176 patients with BAV without known syndromic basis from two large tertiary referral centres. Phenotyping was performed with routine clinical MRI and/or echocardiography. We identified 63 genes of interest with known or suspected links to BAV or to aortic /aortic valve (AV) pathology. We used genomic DNA from our patients for NGS of these target genes. Control populations were provided by the Exome Variant Server (EVS; 6503 samples) and 1000 genomes project. We analysed called variants in silico , usingSIFT, Polyphen2, Grantham scoring and phastCONS, and categorised variants into the following groups based on likely pathogenicity using a combination of in silico tools: known links with disease, likely, possible, and unlikely pathogenicity. Results 10 patients (5.7% of our cohort) had variants previously associated with aortic or AV pathology or with abnormalities of smooth muscle function; 3 in GATA5, 2 in FBN1, 2 in MYH11, 1 in NOTCH1 and 1 in COL3A1. In silico analysis identified 45 further instances of 31 likely pathogenic, rare variants in 33 patients (a further 19% of our cohort), in 11 different genes: GATA5 (see Table 1), NOTCH1 (see Figure 1), MYH11, PLOD3, FBN1, MMP9, NKX2–5, JAG1, ACE, ENG, PDIA2 and KCNJ2. Abstract 95 Figure 1 3D structure of NOTCH1 showing position of likely pathogenic variant Val2119Glu in intracellular signalling domain The combined prevalence of these known or likely pathogenic variants in our cohort was significantly greater than in the EVS control populations (p < 0.0001). 10 patients had known or likely pathogenic variants in more than one gene of interest. These 10 patients had a significantly higher prevalence of significant aortopathy and/or coarctation of the aorta than the rest of our cohort (6/10 vs 20/176; p = 0.0006). Abstract 95 Table 1 Classification of previously identified (red) / likely (orange) pathogenic variants found in our cohort in GATA5: example of analysis Base change c.8A >G c.56C >G c.1173G >T c.698T >C Amino acid change p.Gln3Arg p.Ser19Trp p.Trp391Cys p.Leu233Pro dbSNP ID rs113068438 rs200383755 Novel rs116164480 No. of alleles in our cohort 3 1 1 1 Minor Allele Frequency (from EVS) 0.00328 0 0 0.00108 Polyphen 2 score 0.891 (possibly damaging) 1 (probably damaging) 1 (probably damaging) 0.723 (possibly damaging) SIFT score 0.003 (damaging) 0.001 (damaging) 0.000 (damaging) 0.039 (damaging) Grantham difference 43 177 214 98 phastCONS score 0.997 - - 1 Previous evidence of pathogenicity Previously associated with BAV; variant in highly conserved transcriptional activation domain (Padang et al. J Mol Cell Cardiol 2012) Previously associated with pathogenicity (Padang et al J Mol Cell Cardiol 2012) Conclusions We identified known or likely pathogenic genetic variants in nearly a quarter of our BAV cohort. Some of these (eg the c.698T >C variant in GATA5) may be common polymorphisms, wrongly classified by in silico tools. However, many are undoubtedly genuine determinants of phenotype, as evidenced by their overrepresentation in our cohort, and the finding of a more severe aortic phenotype in patients with more than one presumed pathogenic variant. Further research, including our own control population and family studies, is planned. This is also the first study, to our knowledge, to correlate BAV aortopathy with more complex genotypes, and lends support to a multi-hit genetic hypothesis for development of aortic complications of BAV.

Genetic modifiers to the PLN L39X mutation in a patient with DCM and sustained ventricular tachycardia

[first paragraph of article] Idiopathic dilated cardiomyopathy (DCM) is a leading cause of heart failure characterized by an enlarged ventricular cavity leading to systolic dysfunction. DCM patients have a considerable annual mortality rate of 5–10%, with half of them being sudden unexpected deaths due to ventricular tachycardia (VT) or ventricular fibrillation (VF). Although a multifactorial disease, DCM appears to be inheritable in approximately 70% of cases. Causative gene mutations have been identified in a broad range of genes coding for proteins with a variety of function, such as cytoskeletal, sarcomeric or ion homeostasis related. Among the latter category, several mutations have been identified in Ca 2+ handling proteins in familial and sporadic DCM cases. An increasing body of evidence indicates that abnormal intracellular Ca 2+ handling underlies contractile dysfunction and contributes to ventricular arrhythmogenesis in failing myocardium. A prime example is phospholamban (PLN), which is directly involved in the uptake of Ca 2+ by the sarcoplasmic reticulum (SR), on a beat-to-beat basis, thereby regulating cardiac contraction and relaxation. PLN mutations have been directly associated with the development of dilated cardiomyopathy and heart failure in patients and animal models. However, modifier genes are thought to influence the clinical outcome both in PLN cases, as well as DCM cases in general. We herein describe a DCM case which illustrates the complex genetic contribution to disease development and progression.