Victor Chang Cardiac Research Institute

Abstract The Trans-Omics for Precision Medicine (TOPMed) programme seeks to elucidate the genetic architecture and biology of heart, lung, blood and sleep disorders, with the ultimate goal of improving diagnosis, treatment and prevention of these diseases. The initial phases of the programme focused on whole-genome sequencing of individuals with rich phenotypic data and diverse backgrounds. Here we describe the TOPMed goals and design as well as the available resources and early insights obtained from the sequence data. The resources include a variant browser, a genotype imputation server, and genomic and phenotypic data that are available through dbGaP (Database of Genotypes and Phenotypes) 1 . In the first 53,831 TOPMed samples, we detected more than 400 million single-nucleotide and insertion or deletion variants after alignment with the reference genome. Additional previously undescribed variants were detected through assembly of unmapped reads and customized analysis in highly variable loci. Among the more than 400 million detected variants, 97% have frequencies of less than 1% and 46% are singletons that are present in only one individual (53% among unrelated individuals). These rare variants provide insights into mutational processes and recent human evolutionary history. The extensive catalogue of genetic variation in TOPMed studies provides unique opportunities for exploring the contributions of rare and noncoding sequence variants to phenotypic variation. Furthermore, combining TOPMed haplotypes with modern imputation methods improves the power and reach of genome-wide association studies to include variants down to a frequency of approximately 0.01%.

AIMS: To calculate robust quantitative estimates on the predictive value of central pressures and derived central haemodynamic indices for cardiovascular (CV) outcomes and all-cause mortality by meta-analysis of longitudinal studies. METHODS AND RESULTS: We meta-analysed 11 longitudinal studies that had employed measures of central haemodynamics and had followed 5648 subjects for a mean follow-up of 45 months. The age- and risk-factor-adjusted pooled relative risk (RR) of total CV events was 1.088 (95% CI 1.040-1.139) for a 10 mmHg increase of central systolic pressure, 1.137 (95% CI 1.063-1.215) for a 10 mmHg increase of central pulse pressure (PP), and 1.318 (95% CI 1.093-1.588) for a 10% absolute increase of central augmentation index (AIx). Furthermore, we found that a 10% increase of central AIx was associated with a RR of 1.384 (95% CI 1.192-1.606) for all-cause mortality. When compared with brachial PP, central PP was associated with marginally but not significantly higher RR of clinical events (P = 0.057). CONCLUSION: Central haemodynamic indexes are independent predictors of future CV events and all-cause mortality. Augmentation index predicts clinical events independently of peripheral pressures, while central PP has a marginally but not significantly (P = 0.057) better predictive ability when compared with peripheral PP.

The modified base 5-methylcytosine (m(5)C) is well studied in DNA, but investigations of its prevalence in cellular RNA have been largely confined to tRNA and rRNA. In animals, the two m(5)C methyltransferases NSUN2 and TRDMT1 are known to modify specific tRNAs and have roles in the control of cell growth and differentiation. To map modified cytosine sites across a human transcriptome, we coupled bisulfite conversion of cellular RNA with next-generation sequencing. We confirmed 21 of the 28 previously known m(5)C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions. Surprisingly, we discovered 10,275 sites in mRNAs and other non-coding RNAs. We observed that distribution of modified cytosines between RNA types was not random; within mRNAs they were enriched in the untranslated regions and near Argonaute binding regions. We also identified five new sites modified by NSUN2, broadening its known substrate range to another tRNA, the RPPH1 subunit of RNase P and two mRNAs. Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function.

SIGNIFICANCE: Oxidative stress is considered to be an important component of various diseases. A vast number of methods have been developed and used in virtually all diseases to measure the extent and nature of oxidative stress, ranging from oxidation of DNA to proteins, lipids, and free amino acids. RECENT ADVANCES: An increased understanding of the biology behind diseases and redox biology has led to more specific and sensitive tools to measure oxidative stress markers, which are very diverse and sometimes very low in abundance. CRITICAL ISSUES: The literature is very heterogeneous. It is often difficult to draw general conclusions on the significance of oxidative stress biomarkers, as only in a limited proportion of diseases have a range of different biomarkers been used, and different biomarkers have been used to study different diseases. In addition, biomarkers are often measured using nonspecific methods, while specific methodologies are often too sophisticated or laborious for routine clinical use. FUTURE DIRECTIONS: Several markers of oxidative stress still represent a viable biomarker opportunity for clinical use. However, positive findings with currently used biomarkers still need to be validated in larger sample sizes and compared with current clinical standards to establish them as clinical diagnostics. It is important to realize that oxidative stress is a nuanced phenomenon that is difficult to characterize, and one biomarker is not necessarily better than others. The vast diversity in oxidative stress between diseases and conditions has to be taken into account when selecting the most appropriate biomarker.

Abstract Common single-nucleotide polymorphisms (SNPs) are predicted to collectively explain 40–50% of phenotypic variation in human height, but identifying the specific variants and associated regions requires huge sample sizes 1 . Here, using data from a genome-wide association study of 5.4 million individuals of diverse ancestries, we show that 12,111 independent SNPs that are significantly associated with height account for nearly all of the common SNP-based heritability. These SNPs are clustered within 7,209 non-overlapping genomic segments with a mean size of around 90 kb, covering about 21% of the genome. The density of independent associations varies across the genome and the regions of increased density are enriched for biologically relevant genes. In out-of-sample estimation and prediction, the 12,111 SNPs (or all SNPs in the HapMap 3 panel 2 ) account for 40% (45%) of phenotypic variance in populations of European ancestry but only around 10–20% (14–24%) in populations of other ancestries. Effect sizes, associated regions and gene prioritization are similar across ancestries, indicating that reduced prediction accuracy is likely to be explained by linkage disequilibrium and differences in allele frequency within associated regions. Finally, we show that the relevant biological pathways are detectable with smaller sample sizes than are needed to implicate causal genes and variants. Overall, this study provides a comprehensive map of specific genomic regions that contain the vast majority of common height-associated variants. Although this map is saturated for populations of European ancestry, further research is needed to achieve equivalent saturation in other ancestries.

Atrial fibrillation (AF) affects more than 33 million individuals worldwide1 and has a complex heritability2. We conducted the largest meta-analysis of genome-wide association studies (GWAS) for AF to date, consisting of more than half a million individuals, including 65,446 with AF. In total, we identified 97 loci significantly associated with AF, including 67 that were novel in a combined-ancestry analysis, and 3 that were novel in a European-specific analysis. We sought to identify AF-associated genes at the GWAS loci by performing RNA-sequencing and expression quantitative trait locus analyses in 101 left atrial samples, the most relevant tissue for AF. We also performed transcriptome-wide analyses that identified 57 AF-associated genes, 42 of which overlap with GWAS loci. The identified loci implicate genes enriched within cardiac developmental, electrophysiological, contractile and structural pathways. These results extend our understanding of the biological pathways underlying AF and may facilitate the development of therapeutics for AF. This large, multi-ethnic genome-wide association study identifies 97 loci significantly associated with atrial fibrillation. These loci are enriched for genes involved in cardiac development, electrophysiology, structure and contractile function.

The human ether-a-go-go related gene (hERG) encodes the pore-forming subunit of the rapid component of the delayed rectifier K(+) channel, Kv11.1, which are expressed in the heart, various brain regions, smooth muscle cells, endocrine cells, and a wide range of tumor cell lines. However, it is the role that Kv11.1 channels play in the heart that has been best characterized, for two main reasons. First, it is the gene product involved in chromosome 7-associated long QT syndrome (LQTS), an inherited disorder associated with a markedly increased risk of ventricular arrhythmias and sudden cardiac death. Second, blockade of Kv11.1, by a wide range of prescription medications, causes drug-induced QT prolongation with an increase in risk of sudden cardiac arrest. In the first part of this review, the properties of Kv11.1 channels, including biogenesis, trafficking, gating, and pharmacology are discussed, while the second part focuses on the pathophysiology of Kv11.1 channels.

MicroRNAs (miRNAs) repress translation of target mRNAs by interaction with partially mismatched sequences in their 3' UTR. The mechanism by which they act on translation has remained largely obscure. We examined the translation of mRNAs containing four partially mismatched miRNA-binding sites in the 3' UTR in HeLa cells cotransfected with a cognate miRNA. The mRNAs were prepared by in vitro transcription and were engineered to employ different modes of translation initiation. We find that the 5' cap structure and the 3' poly(A) tail are each necessary but not sufficient for full miRNA-mediated repression of mRNA translation. Replacing the cap structure with an internal ribosome entry site from either the cricket paralysis virus or the encephalomyocarditis virus impairs miRNA-mediated repression. Collectively, these results demonstrate that miRNAs interfere with the initiation step of translation and implicate the cap-binding protein eukaryotic initiation factor 4E as a molecular target.

Chemotactic cytokines (chemokines) attract immune cells, although their original evolutionary role may relate more closely with embryonic development. We noted differential expression of the chemokine receptor CXCR7 (RDC-1) on marginal zone B cells, a cell type associated with autoimmune diseases. We generated Cxcr7(-/-) mice but found that CXCR7 deficiency had little effect on B cell composition. However, most Cxcr7(-/-) mice died at birth with ventricular septal defects and semilunar heart valve malformation. Conditional deletion of Cxcr7 in endothelium, using Tie2-Cre transgenic mice, recapitulated this phenotype. Gene profiling of Cxcr7(-/-) heart valve leaflets revealed a defect in the expression of factors essential for valve formation, vessel protection, or endothelial cell growth and survival. We confirmed that the principal chemokine ligand for CXCR7 was CXCL12/SDF-1, which also binds CXCR4. CXCL12 did not induce signaling through CXCR7; however, CXCR7 formed functional heterodimers with CXCR4 and enhanced CXCL12-induced signaling. Our results reveal a specialized role for CXCR7 in endothelial biology and valve development and highlight the distinct developmental role of evolutionary conserved chemokine receptors such as CXCR7 and CXCR4.

Endothelial to mesenchymal transition (EndMT) is a process whereby an endothelial cell undergoes a series of molecular events that lead to a change in phenotype toward a mesenchymal cell (e.g., myofibroblast, smooth muscle cell). EndMT plays a fundamental role during development, and mounting evidence indicates that EndMT is involved in adult cardiovascular diseases (CVDs), including atherosclerosis, pulmonary hypertension, valvular disease, and fibroelastosis. Therefore, the targeting of EndMT may hold therapeutic promise for treating CVD. However, the field faces a number of challenges, including the lack of a precise functional and molecular definition, a lack of understanding of the causative pathological role of EndMT in CVDs (versus being a "bystander-phenomenon"), and a lack of robust human data corroborating the extent and causality of EndMT in adult CVDs. Here, we review this emerging but exciting field, and propose a framework for its systematic advancement at the molecular and translational levels.

Most existing dimensionality reduction and clustering packages for single-cell RNA-seq (scRNA-seq) data deal with dropouts by heavy modeling and computational machinery. Here, we introduce CIDR (Clustering through Imputation and Dimensionality Reduction), an ultrafast algorithm that uses a novel yet very simple implicit imputation approach to alleviate the impact of dropouts in scRNA-seq data in a principled manner. Using a range of simulated and real data, we show that CIDR improves the standard principal component analysis and outperforms the state-of-the-art methods, namely t-SNE, ZIFA, and RaceID, in terms of clustering accuracy. CIDR typically completes within seconds when processing a data set of hundreds of cells and minutes for a data set of thousands of cells. CIDR can be downloaded at https://github.com/VCCRI/CIDR .

Besides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (Pdgfra-GFP+) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery. Clustering of >30,000 single cells identified >30 populations representing nine cell lineages, including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature. We also uncovered novel myofibroblast subtypes expressing either pro-fibrotic or anti-fibrotic signatures. Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury, and provide an entry point for deeper analysis of cardiac homeostasis, inflammation, fibrosis, repair and regeneration.

The conversion of mechanical force to chemical signals is critical for many biological processes, including the senses of touch, pain, and hearing. Mechanosensitive ion channels play a key role in sensing the mechanical stimuli experienced by various cell types and are present in organisms from bacteria to mammals. Bacterial mechanosensitive channels are characterized thoroughly, but less is known about their counterparts in vertebrates. Piezos have been recently established as ion channels required for mechanotransduction in disparate cell types in vitro and in vivo. Overexpression of Piezos in heterologous cells gives rise to large mechanically activated currents; however, it is unclear whether Piezos are inherently mechanosensitive or rely on alternate cellular components to sense mechanical stimuli. Here, we show that mechanical perturbations of the lipid bilayer alone are sufficient to activate Piezo channels, illustrating their innate ability as molecular force transducers.

Mechanosensitive ion channels are force-transducing enzymes that couple mechanical stimuli to ion flux. Understanding the gating mechanism of mechanosensitive channels is challenging because the stimulus seen by the channel reflects forces shared between the membrane, cytoskeleton and extracellular matrix. Here we examine whether the mechanosensitive channel PIEZO1 is activated by force-transmission through the bilayer. To achieve this, we generate HEK293 cell membrane blebs largely free of cytoskeleton. Using the bacterial channel MscL, we calibrate the bilayer tension demonstrating that activation of MscL in blebs is identical to that in reconstituted bilayers. Utilizing a novel PIEZO1-GFP fusion, we then show PIEZO1 is activated by bilayer tension in bleb membranes, gating at lower pressures indicative of removal of the cortical cytoskeleton and the mechanoprotection it provides. Thus, PIEZO1 channels must sense force directly transmitted through the bilayer.

BACKGROUND: Each of the cardiomyopathies, classically categorized as hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy, has a signature genetic theme. Hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy are largely understood as genetic diseases of sarcomere or desmosome proteins, respectively. In contrast, >250 genes spanning >10 gene ontologies have been implicated in DCM, representing a complex and diverse genetic architecture. To clarify this, a systematic curation of evidence to establish the relationship of genes with DCM was conducted. METHODS: An international panel with clinical and scientific expertise in DCM genetics evaluated evidence supporting monogenic relationships of genes with idiopathic DCM. The panel used the Clinical Genome Resource semiquantitative gene-disease clinical validity classification framework with modifications for DCM genetics to classify genes into categories on the basis of the strength of currently available evidence. Representation of DCM genes on clinically available genetic testing panels was evaluated. RESULTS: ) including 2 additional ontologies were classified as moderate evidence; these genes are likely to emerge as strong or definitive with additional evidence. Of these 19 genes, 6 were similarly classified for hypertrophic cardiomyopathy and 3 for arrhythmogenic right ventricular cardiomyopathy. Of the remaining 32 genes (63%), 25 (49%) had limited evidence, 4 (8%) were disputed, 2 (4%) had no disease relationship, and 1 (2%) was supported by animal model data only. Of the 16 evaluated clinical genetic testing panels, most definitive genes were included, but panels also included numerous genes with minimal human evidence. CONCLUSIONS: In the curation of 51 genes, 19 had high evidence (12 definitive/strong, 7 moderate). It is notable that these 19 genes explain only a minority of cases, leaving the remainder of DCM genetic architecture incompletely addressed. Clinical genetic testing panels include most high-evidence genes; however, genes lacking robust evidence are also commonly included. We recommend that high-evidence DCM genes be used for clinical practice and that caution be exercised in the interpretation of variants in variable-evidence DCM genes.

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.

National Heart Foundation of Australia and Cardiac Society of Australia and New Zealand: Guidelines for the Prevention, Detection, and Management of Heart Failure in Australia 2018

Summary paragraph The Trans-Omics for Precision Medicine (TOPMed) program seeks to elucidate the genetic architecture and disease biology of heart, lung, blood, and sleep disorders, with the ultimate goal of improving diagnosis, treatment, and prevention. The initial phases of the program focus on whole genome sequencing of individuals with rich phenotypic data and diverse backgrounds. Here, we describe TOPMed goals and design as well as resources and early insights from the sequence data. The resources include a variant browser, a genotype imputation panel, and sharing of genomic and phenotypic data via dbGaP. In 53,581 TOPMed samples, >400 million single-nucleotide and insertion/deletion variants were detected by alignment with the reference genome. Additional novel variants are detectable through assembly of unmapped reads and customized analysis in highly variable loci. Among the >400 million variants detected, 97% have frequency <1% and 46% are singletons. These rare variants provide insights into mutational processes and recent human evolutionary history. The nearly complete catalog of genetic variation in TOPMed studies provides unique opportunities for exploring the contributions of rare and non-coding sequence variants to phenotypic variation. Furthermore, combining TOPMed haplotypes with modern imputation methods improves the power and extends the reach of nearly all genome-wide association studies to include variants down to ~0.01% in frequency.

Heparin-induced thrombocytopenia/thrombosis (HIT) is a serious immune reaction to heparins, characterized by thrombocytopenia and often severe thrombosis with high morbidity and mortality. HIT is mediated by IgG antibodies against heparin/platelet factor 4 antigenic complexes. These complexes are thought to activate platelets leading to thrombocytopenia and thrombosis. Here we show that HIT immune complexes induce NETosis via interaction with FcγRIIa on neutrophils and through neutrophil-platelet association. HIT immune complexes induce formation of thrombi containing neutrophils, extracellular DNA, citrullinated histone H3 and platelets in a microfluidics system and in vivo, while neutrophil depletion abolishes thrombus formation. Absence of PAD4 or PAD4 inhibition with GSK484 abrogates thrombus formation but not thrombocytopenia, suggesting they are induced by separate mechanisms. NETs markers and neutrophils undergoing NETosis are present in HIT patients. Our findings demonstrating the involvement of NETosis in thrombosis will modify the current concept of HIT pathogenesis and may lead to new therapeutic strategies.

Persons who have hypermethylation of one allele of MLH1 in somatic cells throughout the body (a germ-line epimutation) have a predisposition for the development of cancer in a pattern typical of hereditary nonpolyposis colorectal cancer. By studying the families of two such persons, we found evidence that the epimutation was transmitted from a mother to her son but was erased in his spermatozoa. The affected maternal allele was inherited by three other siblings from these two families, but in those offspring the allele had reverted to the normal active state. These findings demonstrate a novel pattern of inheritance of cancer susceptibility and are consistent with transgenerational epigenetic inheritance.