Vidu Garg

Mutations in NOTCH1 cause aortic valve disease

Calcification of the aortic valve is the third leading cause of heart disease in adults. The incidence increases with age, and it is often associated with a bicuspid aortic valve present in 1–2% of the population. Despite the frequency, neither the mechanisms of valve calcification nor the developmental origin of a two, rather than three, leaflet aortic valve is known. Here, we show that mutations in the signalling and transcriptional regulator NOTCH1 cause a spectrum of developmental aortic valve anomalies and severe valve calcification in non-syndromic autosomal-dominant human pedigrees. Consistent with the valve calcification phenotype, Notch1 transcripts were most abundant in the developing aortic valve of mice, and Notch1 repressed the activity of Runx2, a central transcriptional regulator of osteoblast cell fate. The hairy-related family of transcriptional repressors (Hrt), which are activated by Notch1 signalling, physically interacted with Runx2 and repressed Runx2 transcriptional activity independent of histone deacetylase activity. These results suggest that NOTCH1 mutations cause an early developmental defect in the aortic valve and a later de-repression of calcium deposition that causes progressive aortic valve disease.

Inhibitory Role of Notch1 in Calcific Aortic Valve Disease

Aortic valve calcification is the most common form of valvular heart disease, but the mechanisms of calcific aortic valve disease (CAVD) are unknown. NOTCH1 mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood. The aim of this study was to investigate the molecular changes that occur with inhibition of Notch signaling in the aortic valve. Notch signaling pathway members are expressed in adult aortic valve cusps, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs). We found significant downregulation of Sox9 along with several cartilage-specific genes that were direct targets of the transcription factor, Sox9. Loss of Sox9 expression has been published to be associated with aortic valve calcification. Utilizing an in vitro porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process. Finally, the addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition. In conclusion, loss of Notch signaling contributes to aortic valve calcification via a Sox9-dependent mechanism.

A unified test of linkage analysis and rare-variant association for analysis of pedigree sequence data

High-throughput sequencing of related individuals has become an important tool for studying human disease. However, owing to technical complexity and lack of available tools, most pedigree-based sequencing studies rely on an ad hoc combination of suboptimal analyses. Here we present pedigree-VAAST (pVAAST), a disease-gene identification tool designed for high-throughput sequence data in pedigrees. pVAAST uses a sequence-based model to perform variant and gene-based linkage analysis. Linkage information is then combined with functional prediction and rare variant case-control association information in a unified statistical framework. pVAAST outperformed linkage and rare-variant association tests in simulations and identified disease-causing genes from whole-genome sequence data in three human pedigrees with dominant, recessive and de novo inheritance patterns. The approach is robust to incomplete penetrance and locus heterogeneity and is applicable to a wide variety of genetic traits. pVAAST maintains high power across studies of monogenic, high-penetrance phenotypes in a single pedigree to highly polygenic, common phenotypes involving hundreds of pedigrees.

Chromosomal Haplotypes by Genetic Phasing of Human Families

Assignment of alleles to haplotypes for nearly all the variants on all chromosomes can be performed by genetic analysis of a nuclear family with three or more children. Whole-genome sequence data enable deterministic phasing of nearly all sequenced alleles by permitting assignment of recombinations to precise chromosomal positions and specific meioses. We demonstrate this process of genetic phasing on two families each with four children. We generate haplotypes for all of the children and their parents; these haplotypes span all genotyped positions, including rare variants. Misassignments of phase between variants (switch errors) are nearly absent. Our algorithm can also produce multimegabase haplotypes for nuclear families with just two children and can handle families with missing individuals. We implement our algorithm in a suite of software scripts (Haploscribe). Haplotypes and family genome sequences will become increasingly important for personalized medicine and for fundamental biology.

MicroRNA miR145 Regulates TGFBR2 Expression and Matrix Synthesis in Vascular Smooth Muscle Cells

Rationale: MicroRNA miR145 has been implicated in vascular smooth muscle cell differentiation, but its mechanisms of action and downstream targets have not been fully defined. Objective: Here, we sought to explore and define the mechanisms of miR145 function in smooth muscle cells. Methods and Results: Using a combination of cell culture assays and in vivo mouse models to modulate miR145, we characterized its downstream actions on smooth muscle phenotypes. Our results show that the miR-143/145 gene cluster is induced in smooth muscle cells by coculture with endothelial cells. Endothelial cell–induced expression of miR-143/145 is augmented by Notch signaling and accordingly expression is reduced in Notch receptor–deficient cells. Screens to identify miR145-regulated genes revealed that the transforming growth factor (TGF)-&bgr; pathway has a significantly high number of putative target genes, and we show that TGF&bgr; receptor II is a direct target of miR145. Extracellular matrix genes that are regulated by TGF&bgr; receptor II were attenuated by miR145 overexpression, and miR145 mutant mice exhibit an increase in extracellular matrix synthesis. Furthermore, activation of TGF&bgr; signaling via angiotensin II infusion revealed a pronounced fibrotic response in the absence of miR145. Conclusions: These data demonstrate a specific role for miR145 in the regulation of matrix gene expression in smooth muscle cells and suggest that miR145 acts to suppress TGF&bgr;-dependent extracellular matrix accumulation and fibrosis, while promoting TGF&bgr;-induced smooth muscle cell differentiation. Our findings offer evidence to explain how TGF&bgr; signaling exhibits distinct downstream actions via its regulation by a specific microRNA.

Percutaneous Patent Ductus Arteriosus (PDA) Closure in Very Preterm Infants: Feasibility and Complications

Background Percutaneous closure of patent ductus arteriosus (PDA) in term neonates is established, but data regarding outcomes in infants born very preterm (<32 weeks of gestation) are minimal, and no published criteria exist establishing a minimal weight of 4 kg as a suitable cutoff. We sought to analyze outcomes of percutaneous PDA occlusion in infants born very preterm and referred for PDA closure at weights <4 kg. Methods and Results Retrospective analysis (January 2005–January 2014) was done at a single pediatric center. Procedural successes and adverse events were recorded. Markers of respiratory status (need for mechanical ventilation) were determined, with comparisons made before and after catheterization. A total of 52 very preterm infants with a median procedural weight of 2.9 kg (range 1.2–3.9 kg) underwent attempted PDA closure. Twenty‐five percent (13/52) of infants were <2.5 kg. Successful device placement was achieved in 46/52 (88%) of infants. An adverse event occurred in 33% of cases, with an acute arterial injury the most common complication. We observed no association between weight at time of procedure and the risk of an adverse event. No deaths were attributable to the PDA closure. Compared to precatheterization trends, percutaneous PDA closure resulted in improved respiratory status, including less exposure to mechanical ventilation (mixed effects logistic model, P<0.01). Conclusions Among infants born very preterm, percutaneous PDA closure at weights <4 kg is generally safe and may improve respiratory health, but risk of arterial injury is noteworthy. Randomized clinical trials are needed to assess clinically relevant differences in outcomes following percutaneous PDA closure versus alternative (surgical ligation) management strategies.

Utilization of Whole Exome Sequencing to Identify Causative Mutations in Familial Congenital Heart Disease

Background—Congenital heart disease (CHD) is the most common type of birth defect with family- and population-based studies supporting a strong genetic cause for CHD. The goal of this study was to determine whether a whole exome sequencing (WES) approach could identify pathogenic-segregating variants in multiplex CHD families. Methods and Results—WES was performed on 9 kindreds with familial CHD, 4 with atrial septal defects, 2 with patent ductus arteriosus, 2 with tetralogy of Fallot, and 1 with pulmonary valve dysplasia. Rare variants (<1% minor allele frequency) that segregated with disease were identified by WES, and variants in 69 CHD candidate genes were further analyzed. These selected variants were subjected to in silico analysis to predict pathogenicity and resulted in the discovery of likely pathogenic mutations in 3 of 9 (33%) families. A GATA4 mutation in the transactivation domain, p.G115W, was identified in familial atrial septal defects and demonstrated decreased transactivation ability in vitro. A p.I263V mutation in TLL1 was identified in an atrial septal defects kindred and is predicted to affect the enzymatic functionality of TLL1. A disease-segregating splice donor site mutation in MYH11 (c.4599+1delG) was identified in familial patent ductus arteriosus and found to disrupt normal splicing of MYH11 mRNA in the affected individual. Conclusions—Our findings demonstrate the clinical utility of WES to identify causative mutations in familial CHD and demonstrate the successful use of a CHD candidate gene list to allow for a more streamlined approach enabling rapid prioritization and identification of likely pathogenic variants from large WES data sets. Clinical Trial Registration—URL: https://clinicaltrials.gov; Unique Identifier: NCT0112048.

Enhancing Literacy in Cardiovascular Genetics: A Scientific Statement From the American Heart Association.

Advances in genomics are enhancing our understanding of the genetic basis of cardiovascular diseases, both congenital and acquired, and stroke. These advances include finding genes that cause or increase the risk for childhood and adult-onset diseases, finding genes that influence how patients respond to medications, and the development of genetics-guided therapies for diseases. However, the ability of cardiovascular and stroke clinicians to fully understand and apply this knowledge to the care of their patients has lagged. This statement addresses what the specialist caring for patients with cardiovascular diseases and stroke should know about genetics; how they can gain this knowledge; how they can keep up-to-date with advances in genetics, genomics, and pharmacogenetics; and how they can apply this knowledge to improve the care of patients and families with cardiovascular diseases and stroke.

UTILITY OF GENETIC TESTING IN INFANTS WITH ISOLATED CONGENITAL HEART DISEASE

Background: Recent advances in technology, and a greater demonstration of the genetic contributors to congenital heart disease (CHD), have resulted in an expansion of genetic screening among infants with CHD. However, the utility of this screening strategy has not been well studied. We aimed to

Beyond genetics: focusing on maternal environment for congenital heart disease prevention

Commentary on: Liu S, Joseph KS, Lisonkova S, et al. Association between maternal chronic conditions and congenital heart defects: a population-based cohort study. Circulation 2013;128:583–9.[OpenUrl][1][Abstract/FREE Full Text][2] Congenital heart disease (CHD) is the most common birth defect, accounting for about one-quarter of birth defects resulting in mortality.1 Aetiology for the majority of CHD remains unknown, but is thought to be multifactorial with genetic and environmental (non-genetic) contributors. Advances in molecular biology and genetic technologies have aided identification of genetic contributors for CHD. Increasing numbers of genomic abnormalities have been discovered in individuals with CHD using next-generation sequencing and array-based methodologies.2 While great strides have been made, genetic aetiologies are identified in only a subset of CHD.2 Epidemiological studies have demonstrated associations … [1]: {openurl}?query=rft.jtitle%253DCirculation%26rft_id%253Dinfo%253Adoi%252F10.1161%252FCIRCULATIONAHA.112.001054%26rft_id%253Dinfo%253Apmid%252F23812182%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [2]: /lookup/ijlink?linkType=ABST&journalCode=circulationaha&resid=128/6/583&atom=%2Febmed%2F19%2F2%2Fe8.atom