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Background: Heart failure is a hallmark of severe hypertrophic cardiomyopathy and dilated cardiomyopathy (DCM). Several mutations in the β-MYH7 gene lead to hypertrophic cardiomyopathy. Recently, causative mutations in the β-MYH7 gene have also been detected in DCM from different populations. Methods: Here, we sequenced the β-MYH7 gene in 137 Indian DCM patients and 167 ethnically matched healthy controls to detect the frequency of mutations and their association. Results: Our study revealed 27 variations, of which 7 mutations (8.0%) were detected exclusively in Indian DCM patients for the first time. These included 4 missense mutations—Arg723His, Phe510Leu, His358Leu, and Ser384Tyr (2.9%); a frameshift mutation—Asn676_T-del (1.5%); and 2 splice-site mutations (IVS17+2T) T>G and (IVS19-1G) G>A (3.6%). Remarkably, all 4 missense mutations altered evolutionarily conserved amino acids. All 4 missense mutations were predicted to be pathogenic by 2 bioinformatics tools—polymorphism phenotyping v2 (PolyPhen-2) and sorting intolerant from tolerant (SIFT). In addition, the 4 homology models of β-MYH7—p.Leu358, p.Tyr384, p.Leu510, and p.His723—displayed root-mean-square deviations of ∼2.55 Å, ∼1.24 Å, ∼3.36 Å, and ∼3.86 Å, respectively. Conclusions: In the present study, we detected numerous novel, unique, and rare mutations in the β-MYH7 gene exclusively in Indian DCM patients (8.0%). Here, we demonstrated how each mutant (missense) uniquely disrupts a critical network of non-bonding interactions at the mutation site (molecular level) and may contribute to development of dilated cardiomyopathy (DCM). Therefore, our findings may provide insight into the understanding of the molecular bases of disease and into diagnosis along with promoting novel therapeutic strategies (through personalized medicine). Résumé: Introduction: L’insuffisance cardiaque est une caractéristique de la cardiomyopathie hypertrophique grave et de la cardiomyopathie dilatée (CMD). Plusieurs mutations dans le gène β-MYH7 conduisent à la cardiomyopathie hypertrophique. Récemment, les mutations causales dans le gène β-MYH7 ont également été détectées au sein de différentes populations atteintes de CMD. Méthodes: Ici, nous avons séquencé le gène β-MYH7 de 137 patients indiens atteints de CMD et de 167 témoins sains appariés selon l’origine ethnique pour détecter la fréquence des mutations et leur association. Résultats: L’étude nous a permis de révéler 27 variations, dont sept mutations (8,0 %) étaient exclusivement détectées chez les patients indiens atteints de CMD pour la première fois. Parmi ces mutations, nous avons observé quatre mutations faux-sens—Arg723His, Phe510Leu, His358Leu et Ser384Tyr (2,9 %), une mutation par déphasage—Asn676_T-del (1,5 %) et deux mutations des sites d’épissage (IVS17+2T) T>G et (IVS19-1G) G>A (3,6 %). Étonnamment, les quatre mutations faux-sens changeaient les acides aminés évolutivement conservés. Selon deux outils bioinformatiques—PolyPhen-2 (de l’anglais, polymorphism phenotyping v2) et SIFT (de l’anglais, sorting intolerant from tolerant), les quatre mutations faux-sens devaient être pathogènes. De plus, les quatre modélisations de β-MYH7 par homologie—p.Leu358, p.Tyr384, p.Leu510 et p.His723—affichaient de façon respective des écarts quadratiques moyens de ∼2,55 Å, ∼1,24 Å, ∼3,36 Å et ∼3,86 Å. Conclusions: Dans la présente étude, nous avons détecté de nombreuses nouvelles mutations, uniques et rares, dans le gène β-MYH7, exclusivement chez les patients indiens atteints de CMD (8,0 %). Ici, nous avons démontré comment chaque mutant (faux-sens) perturbe de manière unique un réseau essentiel d’interactions non liantes au site de mutation (moléculaire) et peut contribuer à la survenue de la CMD. Par conséquent, les conclusions de notre étude peuvent donner un aperçu des bases moléculaires de la maladie et du diagnostic tout en favorisant la promotion de nouvelles stratégies thérapeutiques (par la médecine personnalisée).

BackgroundHypertrophic cardiomyopathy (HCM) is a genetic heart muscle disease with preserved or increased ejection fraction in the absence of secondary causes. Mutations in the sarcomeric protein-encoding genes predominantly cause HCM. However, relatively little is known about the genetic impact of signalling proteins on HCM.Methods and resultsHere, using exome and targeted sequencing methods, we analysed two independent cohorts comprising 401 Indian patients with HCM and 3521 Indian controls. We identified novel variants in ribosomal protein S6 kinase beta-1 (RPS6KB1 or S6K1) gene in two unrelated Indian families as a potential candidate gene for HCM. The two unrelated HCM families had the same heterozygous missense S6K1 variant (p.G47W). In a replication association study, we identified two S6K1 heterozygotes variants (p.Q49K and p.Y62H) in the UK Biobank cardiomyopathy cohort (n=190) compared with matched controls (n=16 479). These variants are neither detected in region-specific controls nor in the human population genome data. Additionally, we observed an S6K1 variant (p.P445S) in an Arab patient with HCM. Functional consequences were evaluated using representative S6K1 mutated proteins compared with wild type in cellular models. The mutated proteins activated the S6K1 and hyperphosphorylated the rpS6 and ERK1/2 signalling cascades, suggesting a gain-of-function effect.ConclusionsOur study demonstrates for the first time that the variants in the S6K1 gene are associated with HCM, and early detection of the S6K1 variant carriers can help to identify family members at risk and subsequent preventive measures. Further screening in patients with HCM with different ethnic populations will establish the specificity and frequency of S6K1 gene variants.

Systemic sclerosis (SSc) is a fibrotic disease that initiates in the skin and progresses to internal organs, leading to a poor prognosis. Unraveling the etiology of a chronic, multifactorial disease such as SSc has been aided by various animal models that recapitulate certain aspects of the human pathology. We found that the transcription factor Snail is overexpressed in the epidermis of SSc patients and a transgenic mouse recapitulating this expression pattern is sufficient to induce many clinical features of the human disease. Using this mouse model as a discovery platform, we have uncovered a critical role for the matricellular protein Mindin (Spondin-2) in fibrogenesis. Mindin is produced by Snail transgenic skin keratinocytes and aids fibrogenesis by inducing early inflammatory cytokine production and collagen secretion in resident dermal fibroblasts. Given the dispensability of Mindin in normal tissue physiology, targeting this protein holds promise as an effective therapy for fibrosis.

Myosin binding protein C3 (MYBPC3) is a thick filament contractile protein that interacts with myosin, titin and actin and regulates cardiac muscle contraction. Genetic variations in the MYBPC3 gene are known causal factors for cardiomyopathy and heart failure. Previously, we identified a recurrent MYBPC3 deletion (25 base pairs) among South Asians associated with cardiomyopathy and heart failure. Here, we generated an induced pluripotent stem cell (iPSC) line using peripheral blood mononuclear cells (PBMC) from an Indian harboring MYBPC3 deletion. This iPSC line displayed embryonic stem cell morphology, expressed pluripotency markers, differentiated into three germ layers and exhibited normal karyotype.

The coordinated contraction of myocytes drives the heart to beat and circulate blood. Due to the limited spatial resolution of whole heart imaging and the piecemeal nature of high-magnification studies, a confirmed model of myofiber geometry does not yet exist. Using microscopy and computer vision we report the first three-dimensional reconstruction of myofibers across entire mouse ventricular walls at the micron scale, representing a gain of three orders of magnitude in spatial resolution over the existing models. Our analysis reveals prominent longitudinal bands of fibers that are orthogonal to the well-known circumferential ones. Our discovery impacts present understanding of heart wall mechanics and electrical function, with fundamental implications for the study of diseases related to myofiber disorganization.

Significance India is the world’s largest fruit producer and second most populous country. Pollinators are therefore important for India’s food security. India also contains 9 of the world’s 10 most polluted cities, but the impact of air pollution on plant and animal systems is largely unknown. We performed a multiyear study in the megacity of Bangalore to correlate the mechanistic effects of air pollution on a major Indian pollinator, the Giant Asian honey bee, Apis dorsata. Wild honey bees and lab-reared Drosophila exposed to air pollution exhibited differences in survival, behavior, heart rate, blood cell count, and/or the expression of genes related to stress, immunity, and metabolism. Our study indicates the urgency for more studies on wild systems to better inform international air quality guidelines., While the impact of air pollution on human health is well studied, mechanistic impacts of air pollution on wild systems, including those providing essential ecosystem services, are largely unknown, but directly impact our health and well-being. India is the world’s largest fruit producer, second most populous country, and contains 9 of the world’s 10 most polluted cities. Here, we sampled Giant Asian honey bees, Apis dorsata, at locations with varying air pollution levels in Bangalore, India. We observed significant correlations between increased respirable suspended particulate matter (RSPM) deposition and changes in bee survival, flower visitation, heart rate, hemocyte levels, and expression of genes related to lipid metabolism, stress, and immunity. Lab-reared Drosophila melanogaster exposed to these same sites also exhibited similar molecular and physiological differences. Our study offers a quantitative analysis on the current impacts of air pollution on insects, and indicates the urgency for more nonhuman studies to accurately assess the effects of pollution on our natural world.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by ventricular enlargement, diastolic dysfunction, and increased risk for sudden cardiac death. Sarcomeric genetic defects are the predominant known cause of HCM. In particular, mutations in the myosin-binding protein C gene (MYBPC3) are associated with ~ 40% of all HCM cases in which a genetic basis has been established. A decade ago, our group reported a 25–base pair deletion in intron 32 of MYBPC3 (MYBPC3Δ25bp) that is uniquely prevalent in South Asians and is associated with autosomal dominant cardiomyopathy. Although our studies suggest that this deletion results in left ventricular dysfunction, cardiomyopathies, and heart failure, the precise mechanism by which this variant predisposes to heart disease remains unclear. Increasingly appreciated, however, is the contribution of secondary risk factors, additional mutations, and lifestyle choices in augmenting or modifying the HCM phenotype in MYBPC3Δ25bp carriers. Therefore, the goal of this review article is to summarize the current research dedicated to understanding the molecular pathophysiology of HCM in South Asians with the MYBPC3Δ25bp variant. An emphasis is to review the latest techniques currently applied to explore the MYBPC3Δ25bp pathogenesis and to provide a foundation for developing new diagnostic strategies and advances in therapeutics.

Cardiomyopathy (CMP) is a heritable disorder. Over 50% of cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole-genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine percent of cases harbored pathogenic coding variants in known CMP genes, and 5% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen percent harbored high-risk regulatory variants in promoters and enhancers of CMP genes (odds ratio 2.25, p = 6.70 × 10−7 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (odds ratio 6.7–58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early onset CMP. This project was supported by the Ted Rogers Centre for Heart Research (SM, JE), the Canadian Institutes of Health Research (PJT 175034) (SM, JE) and by the Canadian Institutes of Health Research (ENP 161429), under the frame of ERA PerMed (SM). SM holds the Heart and Stroke Foundation of Canada & Robert M Freedom Chair in Cardiovascular Science. SWS holds the GlaxoSmithKline Endowed Chair in Genome Sciences at the Hospital for Sick Children and the University of Toronto. PGM holds a Canada Research Chair Tier 2 in Non-coding Disease Mechanisms. PGM acknowledges the support of the Government of Canada’s New Frontiers in Research Fund (NFRF), [NFRFE-2018-01305]. EO holds the Bitove Family Professorship of Adult Congenital Heart Disease. MM holds a Ramon y Cajal grant from the Spanish Ministry of Science and Innovation (RYC-2017-22249). WO is supported by funding from Fundació La Marató (321/C/2019). JB is funded by a Frans Van de Werf fellowship for clinical cardiovascular research, and by a senior clinical investigator fellowship of the FWO Flanders. KM was a National Science Foundation Graduate Research Fellow under grant no. DGE1144152 during the majority of the project. CS is the recipient of a National Health and Medical Research Council (NHMRC) Practitioner Fellowship (1154992). JI is the recipient of an NHMRC Career Development Fellowship (1162929). RDB is the recipient of a New South Wales Health Cardiovascular Disease Senior Scientist Grant. PSD is supported by the DBT/Wellcome Trust- Indian Alliance. We acknowledge the Labatt Family Heart Centre Biobank at the Hospital for Sick Children for access to DNA samples, and The Centre for Applied Genomics at the Hospital for Sick Children for performing WGS. We thank Xiucheng Cui and Emanuela Pannia for performing the zebrafish experiments at the SickKids Zebrafish Genetics and Disease Models Core (CRISPR-Cas9 and gRNA syntheses, zebrafish embryo microinjections, gRNA PCR validation, qRT-PCR, cardiac imaging). This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support. We thank members of the ICGC/PCAWG working groups for generating the variant calls used in our case-control burden analyses. Peer Reviewed "Article signat per 38 autors/es: Robert Lesurf, Abdelrahman Said, Oyediran Akinrinade, Jeroen Breckpot, Kathleen Delfosse, Ting Liu, Roderick Yao, Gabrielle Persad, Fintan McKenna, Ramil R. Noche, Winona Oliveros, Kaia Mattioli, Shreya Shah, Anastasia Miron, Qian Yang, Guoliang Meng, Michelle Chan Seng Yue, Wilson W. L. Sung, Bhooma Thiruvahindrapuram, Jane Lougheed, Erwin Oechslin, Tapas Mondal, Lynn Bergin, John Smythe, Shashank Jayappa, Vinay J. Rao, Jayaprakash Shenthar, Perundurai S. Dhandapany, Christopher Semsarian, Robert G. Weintraub, Richard D. Bagnall, Jodie Ingles, Genomics England Research Consortium, Marta Melé, Philipp G. Maass, James Ellis, Stephen W. Scherer & Seema Mital"

Fibrosis is a result of chronically activated fibroblasts leading to the overproduction of extracellular matrix (ECM), causing tissue hardening and loss of organ function. Systemic sclerosis (SSc) is a fibrotic skin disease marked by inflammation, autoimmunity and vasculopathy along with progressive fibrosis of the skin and internal organs. A major bottleneck in understanding the etiology of SSc has been the lack of a holistic animal model that can mimic the human SSc disease. We found that the transcription factor Snail is overexpressed in the epidermis of SSc patients and a transgenic mouse recapitulating this expression pattern is sufficient to induce hallmark clinical features of the human disease. Using this mouse model as a discovery platform, we have uncovered a critical role for the matricellular protein Mindin in fibrogenesis. Mindin is produced by Snail transgenic skin keratinocytes and aids fibrogenesis by inducing inflammatory cytokine and collagen production in resident dermal fibroblasts. Given the dispensability of Mindin in normal tissue physiology, targeting this protein holds promise as an effective therapy for fibrosis.

Rationale: Myosin binding protein-C ( MYBPC ) is a sarcomeric protein which regulates the force of contraction in striated muscles. Mutations in the MYBPC family of genes, slow skeletal( MYBPC1 ), fast skeletal ( MYBPC2 ) and cardiac ( MYBPC3 ) can result in cardiac and skeletal myopathies, yet their evolutionary pattern, pathogenicity and impact on MYBPC structure remain to be elucidated. Objective: To assess conserved and epigenetic patterns of MYBPC family mutations. Methods and Results: Leveraging a machine learning approach, the Genome Aggregation Database (gnomAD) provided 12353, 6227 and 5279 variants in MYBPC1 , MYBPC2 and MYBPC3 genes, respectively, followed by analysis with the Ensembl variant effector predictor. Missense variants comprised 62-68% of total variants in which the first and second nucleotide (nt) positions in the codons were highly altered, with G/A substitution nt substitution the most predominant. Arginine was the most mutated amino acid, important because most disease-causing mutations in MYBPC are arginine in origin. Domains C5 and C6of MYBPC were found to be hotspots for most mutations in the MYBPC family. A high percentage of truncated mutations in MYBPC3 cause cardiomyopathies. Arginine and glutamate were the top hits in MYBPC1 and MYBPC3, respectively, and tryptophan and tyrosine were the most common among the three paralogs changing to premature stop codons and causing protein truncations at the carboxy terminus. Conclusion: A heterogeneous epigenetic pattern was identified among the three MYBPC paralogs. Genomic databases using machine learning approaches can be used to diagnose and design therapeutics to treat muscle disorders caused by MYBPC mutations.

Rationale: Hypertrophic cardiomyopathy (HCM) is common inheritable heart disease. HCM is highly associated with arrhythmias and/or sudden death. Studies show that molecular defects in calcium handling impairing the cardiomyocyte contractility is a predominant cause. However, the pathophysiology underlying HCM with arrhythmias is not well understood, hindering the identification of novel therapies. Objective: To investigate the pathophysiological consequences of compound variants, consisting of Histidine Rich Calcium Binding Protein gene ( HRC S96A ) and an intronic 25bp deletion in cardiac myosin binding protein-C ( MYBPC3 Δ25bp ). Methods and Results: Clinical data revealed that co-segregation of HRC S96A and MYBPC3 ΔInt32 results in cardiac arrhythmia, heart failure, and sudden cardiac death in South Asians. To determine the cellular/molecular trigger underlying the pathophysiology of this dual variant, we used humanized, knock-in, heterozygous mouse models, including HRC S81A (equivalent to HRC S96A ) MYBPC3 Δ25bp , HRC S81A / MYBPC3 Δ25bp (double variant, DV), and wild-type controls. Echocardiography revealed a significant decrease in the percentage of ejection fraction and fractional shortening in DV mice, as well as the presence of diastolic dysfunction, at 12 weeks of age, compared to single-variant and wild-type mice. Electrocardiogram tracing of DV mice showed the presence of stress-induced arrhythmias, such as ventricular tachycardia after caffeine and epinephrine administration. Using isolated cardiomyocytes in vitro , Calcium transient experiments indicated a significant decrease in fractional shortening, Ca 2+ transient amplitude, and a higher number of after-contractions in cardiomyocytes from DV mice. DV mouse hearts showed increased phosphorylation of CaMKII and SR Ca 2+ leak by cardiomyocytes. Inclusion of the CaMKII inhibitor KN-93 rescued the increases in SR Ca 2+ leak and in aftercontractions. Conclusion: Impaired Ca 2+ -handling, owing to the HRC S96A variant, aggravates SR Ca 2+ leak and aftercontractions in MYBPC3 Δ25bp cardiomyocytes, subsequently triggering cardiac arrhythmias and sudden death in vivo .

Idiopathic dilated cardiomyopathy (DCM) is a structural heart disease with strong genetic background. The aim of this study was to assess the role of mitochondrial DNA (mtDNA) variations and haplogroups in Indian DCM patients. Whole mtDNA analysis of 221 DCM patients revealed 48 novel, 42 disease-associated and 97 private variations. The frequency of reported variations associated with hearing impairment, DEAF, SNHL and LHON are significantly high in DCM patients than controls. Haplogroups H and HV were over represented in DCM than controls. Functional analysis of two private variations (m.8812A>G & m.10320G>A) showed decrease in mitochondrial functions, suggesting the role of mtDNA variations in DCM.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by ventricular enlargement, diastolic dysfunction, and increased risk for sudden cardiac death. Sarcomeric genetic defects are the predominant known cause of HCM. In particular, mutations in the myosin-binding protein C gene (MYBPC3) are associated with ~ 40% of all HCM cases in which a genetic basis has been established. A decade ago, our group reported a 25–base pair deletion in intron 32 of MYBPC3 (MYBPC3(Δ25bp)) that is uniquely prevalent in South Asians and is associated with autosomal dominant cardiomyopathy. Although our studies suggest that this deletion results in left ventricular dysfunction, cardiomyopathies, and heart failure, the precise mechanism by which this variant predisposes to heart disease remains unclear. Increasingly appreciated, however, is the contribution of secondary risk factors, additional mutations, and lifestyle choices in augmenting or modifying the HCM phenotype in MYBPC3(Δ25bp) carriers. Therefore, the goal of this review article is to summarize the current research dedicated to understanding the molecular pathophysiology of HCM in South Asians with the MYBPC3(Δ25bp) variant. An emphasis is to review the latest techniques currently applied to explore the MYBPC3(Δ25bp) pathogenesis and to provide a foundation for developing new diagnostic strategies and advances in therapeutics.

Gene variants in adiponectin receptor 1 cause pathological enlarged hearts., Hypertrophic cardiomyopathy (HCM) is a heterogeneous genetic heart muscle disease characterized by hypertrophy with preserved or increased ejection fraction in the absence of secondary causes. However, recent studies have demonstrated that a substantial proportion of individuals with HCM also have comorbid diabetes mellitus (~10%). Whether genetic variants may contribute a combined phenotype of HCM and diabetes mellitus is not known. Here, using next-generation sequencing methods, we identified novel and ultrarare variants in adiponectin receptor 1 (ADIPOR1) as risk factors for HCM. Biochemical studies showed that ADIPOR1 variants dysregulate glucose and lipid metabolism and cause cardiac hypertrophy through the p38/mammalian target of rapamycin and/or extracellular signal–regulated kinase pathways. A transgenic mouse model expressing an ADIPOR1 variant displayed cardiomyopathy that recapitulated the cellular findings, and these features were rescued by rapamycin. Our results provide the first evidence that ADIPOR1 variants can cause HCM and provide new insights into ADIPOR1 regulation.

In their Letter, Negri et al. (1) point out four concerns with our recent publication (2). The first is that the study is correlative, and the next two that we did not measure the toxicity of air pollution directly, nor how the particulate matter (PM) measured on the surface of the cuticle and internal organs could be impacting health. Finally, the authors are concerned with the identity of the elements we found using scanning electron microscopy coupled with X-ray spectroscopy (SEM-EDX). First, some specific responses. The assertion that we did not investigate the contact between PM and the internal organs is erroneous and is already shown in figure 2 (crop and midgut … [↵][1]1To whom correspondence may be addressed. Email: shannon{at}nice.ncbs.res.in or geetha{at}nice.ncbs.res.in. [1]: #xref-corresp-1-1

Habitual sleep duration is increasingly being recognized as an important risk factor for stroke. We sought to describe the association between sleep duration and stroke in a cohort of individuals with diabetes.Data from the National Health Interview Survey for the years 2004-2013 were used. Only those answering "yes" to the question "Have you EVER been told by a doctor or other health professional that you have diabetes or sugar diabetes?" were included in the analysis. Sleep duration was categorized as short (≤6 hours), normal (7-8 hours), or long (≥9 hours). Self-reported diagnosis of stroke was the main outcome of interest.A total number of 26,364 self-reported diabetic individuals provided data for analysis. Stroke was reported in 9.1% of short sleepers, 16.1% of long sleepers, and 8.3% of normative sleepers (P .05). In the unadjusted model, short and long sleepers had an increased odds of stroke compared to normal sleepers (odds ratio [OR] = 1.12, 95% confidence interval [CI]: 1.02-1.23, P = .01; and OR = 2.18, 95% CI: 1.96-2.42, P = .01; respectively), but the association between short sleep and stroke became nonsignificant after multivariate adjustment (OR = 1.15, 95% CI: .95-1.40, P = .16) except in white participants. The association between long sleep duration and stroke persisted (OR = 1.46, 95% CI: 1.16-1.84, P = .01), especially in males (OR = 1.62, 95% CI: 1.14-2.28) and in white participants (OR = 1.97, 95% CI: 1.47-2.65).In diabetic patients, abnormal sleep duration was associated with increased risk of stroke, and this association varied among different sex and ethnic groups.

Background:Associations of genetic variants within certain fibril-forming genes have previously been observed with anterior cruciate ligament (ACL) injuries. Evidence suggests a significant role of angiogenesis-associated cytokines in remodeling the ligament fibril matrix after mechanical loading and maintaining structural and functional integrity of the ligament. Functional polymorphisms within the vascular endothelial growth factor A (VEGFA) gene have emerged as plausible candidates owing to their role in the regulation of angiogenic responses.Hypothesis:VEGFA promoter polymorphisms rs699947 and rs35569394 are associated with ACL injury risk among athletes.Study Design:Cross-sectional study; Level of evidence, 3.Methods:A total of 90 Indian athletes with radiologically confirmed or surgically proven isolated ACL tears and 76 matched-control athletes were selected for the present cross-sectional genetic association study. Oral mouthwash samples were collected from all the case and control athletes and genotyped for VEGFA rs699947 and rs35569394 using the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method.Results:The A allele (rs699947) was significantly overrepresented in the ACL group (C vs A allele: odds ratio [OR], 1.68 [95% CI, 1.08-2.60]; P = .021) (CC vs CA + AA: OR, 2.69 [95% CI, 1.37-5.26]; P = .004). There was a greater frequency of the AA genotype in the ACL group in comparison with the control group (OR, 3.38 [95% CI, 1.23-9.28]; P = .016) when only male athletes were compared. Likewise, there was a greater frequency of the I allele (rs35569394) in the ACL group (D vs I allele: OR, 1.64 [95% CI, 1.06-2.55]; P = .025) (DD vs ID + II: OR, 2.61 [95% CI, 1.31-5.21]; P = .006). The A-I haplotype was overrepresented in the ACL group compared with the control group (OR, 1.68 [95% CI, 1.08-2.60]; χ2= 5.320; P = .021), and both the polymorphisms were found to be in complete linkage disequilibrium ( r2= 0.929; logarithm of the odds score = 63.74; D′ = 1.0). Female athletes did not show any difference in genotype or allele frequency.Conclusion:This is the first study to investigate the association of VEGFA promoter polymorphisms in ACL tears among Indian athletes. Increased frequencies of the A allele (rs699947) and I allele (rs35569394) were observed in the ACL group. These results suggest that sequence variants in the VEGF gene are associated with ACL injury risk among athletes. Further research with long-term follow-ups measuring VEGF expression levels during recovery is warranted to establish its role in ACL injuries and healing.

Height is a heritable and highly heterogeneous trait. Short stature affects 3% of the population and in most cases is genetic in origin. After excluding known causes, 67% of affected individuals remain without diagnosis. To identify novel candidate genes for short stature, we performed exome sequencing in 254 unrelated families with short stature of unknown cause and identified variants in 63 candidate genes in 92 (36%) independent families. Based on systematic characterization of variants and functional analysis including expression in chondrocytes, we classified 13 genes as strong candidates. Whereas variants in at least two families were detected for all 13 candidates, two genes had variants in 6 (UBR4) and 8 (LAMA5) families, respectively. To facilitate their characterization, we established a clustered network of 1025 known growth and short stature genes, which yielded 29 significantly enriched clusters, including skeletal system development, appendage development, metabolic processes, and ciliopathy. Eleven of the candidate genes mapped to 21 of these clusters, including CPZ, EDEM3, FBRS, IFT81, KCND1, PLXNA3, RASA3, SLC7A8, UBR4, USP45, and ZFHX3. Fifty additional growth-related candidates we identified await confirmation in other affected families. Our study identifies Mendelian forms of growth retardation as an important component of idiopathic short stature.

Cardiorenal syndrome is defined by primary heart failure conditions influencing or leading to renal injury or dysfunction. Dilated cardiomyopathy (DCM) is a major co-existing form of heart failure (HF) with renal diseases. Myocardin (MYOCD), a cardiac-specific co-activator of serum response factor (SRF), is increased in DCM mice and patient cardiac tissues and plays a crucial role in the pathophysiology of DCM. Inhibiting the increased MYOCD has shown to be partially rescuing the DCM mice phenotype. However, expression levels of MYOCD in the cardiac tissues of the cardiorenal syndromic patients and the beneficial effect of inhibiting MYOCD in a cardiorenal syndrome model remains to be explored.Here, we analyzed the expression levels of MYOCD in the DCM patients with and without renal diseases. We also explored, whether cardiac specific silencing of MYOCD expression could ameliorate the cardiac remodeling and improve cardiac function in a renal artery ligated rat model (RAL). We observed an increase in MYOCD levels in the endomyocardial biopsies of DCM patients associated with renal failure compared to DCM alone. Silencing of MYOCD in RAL rats by a cardiac homing peptide conjugated MYOCD siRNA resulted in attenuation of cardiac hypertrophy, fibrosis and restoration of the left ventricular functions.Our data suggest hyper-activation of MYOCD in the pathogenesis of the cardiorenal failure cases. Also, MYOCD silencing showed beneficial effects by rescuing cardiac hypertrophy, fibrosis, size and function in a cardiorenal rat model.

Hypertrophic cardiomyopathy (HCM) is a heterogenous disease predominantly caused by sarcomeric genes. However, 40-50% cases etiology are not known. RAF1 mutations cause syndromic and isolated childhood cardiomyopathies. Functional mutations in untranslated regions (UTRs) of RAF-1 gene are rare, and their role in cardiomyopathy is unexplored. UTRs are important sites for interaction of epigenetic regulators such as microRNAs (miR). miRs have exhibited as crucial regulators of the cardiac remodeling process. We identified a novel mutation in RAF-1 3’-UTR (an important site for binding of miR-19a-3p/19b-3p) in large family members associated with HCM. Whole exome sequencing revealed that these family members are negative for mutations in known cardiomyopathy associated genes. miR-19a/b are known to play a crucial role in cardiac hypertrophy. The aim of this study is to determine the role of miR-19a-3p/19b-3p in regulating RAF-1 expression and delineating the molecular and functional consequences of identified RAF-1 3’-UTR mutation. Our results show that the overexpression of miR-19b-3p, leads to downregulation of RAF-1 expression and regulation. Notably, miR-19b-3p was found to be pro-hypertrophic as its overexpression resulted in increasing hypertrophic markers including ANP and β-MHC expression. Moreover, findings from experiments using RAF1 +/+ and RAF1 -/- mouse embryo fibroblasts (MEFs) confirms the role of miR-19b-3p interaction and regulation of RAF-1 signaling axis. Interestingly, we also observed many autophagy markers (Atg3, Atg 12-5 complex, and LC3 II) are dysregulated by miR-19-b-3p overexpression. Our study uncovers a novel mechanism through which miR-19b-3p regulates autophagy by targeting RAF-1.

Dilated cardiomyopathy (DCM) is a highly heterogeneous trait with sarcomeric gene mutations predominating. The cause of a substantial percentage of DCMs remains unknown, and no gene-specific therapy is available. On the basis of resequencing of 513 DCM cases and 1,150 matched controls from various cohorts of distinct ancestry, we discovered rare, functional RAF1 mutations in 3 of the cohorts (South Indian, North Indian and Japanese). The prevalence of RAF1 mutations was ~9% in childhood-onset DCM cases in these three cohorts. Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner. Constitutive expression of these mutants in zebrafish embryos resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin. These findings provide new mechanistic insights and potential therapeutic targets for RAF1-associated DCM and further expand the clinical spectrum of RAF1-related human disorders.

The study was carried to determine the association of angiotensin converting enzyme (ACE) insertion/deletion (I/D) polymorphism with the risk of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM).A total of 174 patients diagnosed with cardiomyopathy (118 with HCM, 51 with DCM, and 5 with RCM) and 164 ethnically, age- and gender-matched controls were included in the study. ACE I/D genotyping was performed by PCR. In total, 25.86% of the patients were in New York Heart Association (NYHA) class III and IV at presentation. A total of 67.24% patients had dyspnea, 56.89% had angina pectoris, and 25.28% of the patients had at least one event of syncope. Frequency of occurrence of the disease was more in male patients compared to female patients (P0.05). After adjustment for age, sex, body mass index (BMI), and smoking habit, the prevalence of ACE DD genotype, and ACE 'D' allele was significantly higher in patients as compared to controls and was associated with increased risk (DD: OR 2.11, 95% CI 1.27-3.52, P0.05; 'D': OR 1.91, 95% CI 1.08-3.35, P0.05). The mean septal thickness was higher for DD and ID genotypes (20.40 +/- 3.73 mm and 21.82 +/- 5.35 mm, respectively) when compared with II genotype (18.63 +/- 6.69 mm) in HCM patients, however, the differences were not significant statistically (P0.05). The DCM patients with ID genotype showed significantly decreased left ventricular ejection fraction (LVEF) at enrolment (26.50 +/- 8.04%) (P = 0.04).Our results suggest that D allele of ACE I/D polymorphism significantly influences the HCM and DCM phenotypes.

The importance of the oncogene Ras in cardiac hypertrophy is well appreciated. The hypertrophic effects of the constitutively active mutant Ras-Val12 are revealed by clinical syndromes due to the Ras mutations and experimental studies. We examined the possible anti-hypertrophic effect of Ras inhibition in vitro using rat neonatal cardiomyocytes (NRCM) and in vivo in the setting of pressure-overload left ventricular (LV) hypertrophy (POH) in rats. Ras functions were modulated via adenovirus directed gene transfer of active mutant Ras-Val12 or dominant negative mutant N17-DN-Ras (DN-Ras). Ras-Val12 expression in vitro activates NFAT resulting in pro-hypertrophic and cardio-toxic effects on NRCM beating and Z-line organization. In contrast, the DN-Ras was antihypertrophic on NRCM, inhibited NFAT and exerted cardio-protective effects attested by preserved NRCM beating and Z line structure. Additional experiments with silencing H-Ras gene strategy corroborated the antihypertrophic effects of siRNA-H-Ras on NRCM. In vivo, with the POH model, both Ras mutants were associated with similar hypertrophy two weeks after simultaneous induction of POH and Ras-mutant gene transfer. However, LV diameters were higher and LV fractional shortening lower in the Ras-Val12 group compared to control and DN-Ras. Moreover, DN-Ras reduced the cross-sectional area of cardiomyocytes in vivo, and decreased the expression of markers of pathologic cardiac hypertrophy. In isolated adult cardiomyocytes after 2weeks of POH and Ras-mutant gene transfer, DN-Ras improved sarcomere shortening and calcium transients compared to Ras-Val12. Overall, DN-Ras promotes a more physiological form of hypertrophy, suggesting an interesting therapeutic target for pathological cardiac hypertrophy.

Cardiac isoform of alpha-2 macroglobulin [cardiac alpha2M] has been shown to be an early marker in cardiac hypertrophy and left ventricular mass in humans. We, here, for the first time report its presence in myocardial infarcted humans and tried to explore the possibility of using this protein as a novel diagnostic marker for myocardial infarcted diabetic patients. A total of 260 samples were analyzed in this study for the presence of cardiac alpha2M. These include 55 patients of diabetic with post myocardial infarction [PMI], 45 diabetic patients without PMI, 60 patients of PMI alone and 100 controls without any ailments. Levels of cardiac alpha2M present in the sera of diabetic patients with PMI are significantly higher than that of normal human sera and diabetic patients without PMI but not with PMI alone group, suggesting this protein as a marker for PMI itself. However, our results reveal that cardiac alpha2M could be a valuable marker for the diagnosis of myocardial infarcted diabetic patients and differentiating them from diabetic patients without myocardial infarction by sandwich ELISA.

Mutations in sarcomeric genes are the leading cause for cardiomyopathies. However, not many genetic studies have been carried out on Indian cardiomyopathy patients. We performed sequence analyses of a thin filament sarcomeric gene, α-tropomyosin (TPM1), in 101 hypertrophic cardiomyopathy (HCM) patients and 147 dilated cardiomyopathy (DCM) patients against 207 ethnically matched healthy controls, revealing 13 single nucleotide polymorphisms (SNPs). Of these, one mutant, S215L, was identified in two unrelated HCM cases-patient #1, aged 44, and patient #2, aged 65-and was cosegregating with disease in these families as an autosomal dominant trait. In contrast, S215L was completely absent in 147 DCM and 207 controls. Patient #1 showed a more severe disease phenotype, with poor prognosis and a family history of sudden cardiac death, than patient #2. Therefore, these two patients and the family members positive for S215L were further screened for variations in MYH7, MYBPC3, TNNT2, TNNI3, MYL2, MYL3, and ACTC. Interestingly, two novel thick filaments, D896N (homozygous) and I524K (heterozygous) mutations, in the MYH7 gene were identified exclusively in patient #1 and his family members. Thus, we strongly suggest that the coexistence of these digenic mutations is rare, but leads to severe hypertrophy in a South Indian familial hypertrophic cardiomyopathy (FHCM).

Cardiomyopathy is a major cause of heart failure and sudden cardiac death; several mutations in sarcomeric protein genes have been associated with this disease. Our aim in the present study is to investigate the genetic variations in Troponin T (cTnT) gene and its association with dilated cardiomyopathy (DCM) in south-Indian patients. Analyses of all the exons and exon-intron boundaries of cTnT in 147 DCM and in 207 healthy controls had revealed a total of 15 SNPs and a 5 bp INDEL; of which, polymorphic SNPs were compared with the HapMap population data. Interestingly, a novel R144W mutation, that substitutes polar-neutral tryptophan for a highly conserved basic arginine in cTnT, altering the charge drastically, was identified in a DCM, with a family history of sudden-cardiac death (SCD). This mutation was found within the tropomyosin (TPM1) binding domain, and was evolutionarily conserved across species, therefore it is expected to have a significant impact on the structure and function of the protein. Family studies had revealed that the R144W is co-segregating with disease in the family as an autosomal dominant trait, but it was completely absent in 207 healthy controls and in 162 previously studied HCM patients. Further screening of the proband and three of his family members (positive for R144W mutant) with eight other genes β-MYH7, MYBPC3, TPM1, TNNI3, TTN, ACTC, MYL2 and MYL3, did not reveal any disease causing mutation, proposing the absence of compound heterozygosity. Therefore, we strongly suggest that the novel R144W unique/private mutant identified in this study is associated with FDCM. This is furthermore signifying the unique genetic architecture of Indian population.

Adiponectin, an adipocyte-derived cytokine, is known to influence cardiac remodeling and to suppress pathological cardiac growth and diabetes-induced disorders. Disruption of adiponectin or its receptor AdipoR1 has significant effects on cardiomyocyte hypertrophy and myocardial metabolism signal transduction. We recently identified a novel mutation in the AdipoR1 gene among 126 hypertrophic cardiomyopathy patients, including two family members with diabetes. Our screening identified a G>A transition at 146th position that replaced the amino acid valine (GUG) into methionine (AUG) (V146M). The consequences of this mutation on cardiac function and metabolism are not known. Here, using adenoviral gene delivery system, we found that Ad.V146M increased cell size, protein synthesis, induced expression of hypertrophic gene markers and increased serine phosphorylation of insulin receptor substrate 1 (IRS1) compared to control Ad.βGal or wild type AdipoR1 (Ad.AR1) in cultured cardiomyocytes. We also discovered that p38 and mTOR were activated by AdipoR1 mutant, but not wild-type. Pharmacological inhibition of p38 (SB203580) and mTOR (rapamycin) reversed the Ad.V146M-induced hypertrophic responses. Adiponectin is also known to promote the uptake and oxidation of fatty acid and glucose through AR1 in cultured myocytes. Recent studies suggest that p38 acts as an essential mediator in regulating adiponectin-induced glucose uptake and fatty acid oxidation in C2C12 myocytes, and the serine phosphorylation of IRS1 at S636/639 is known to be mediated by mTOR pathway. AdipoR1 mutant significantly deceased glucose uptake, facilitated palmitate uptake and repressed insulin responses. Interestingly, inhibition of p38 and mTOR pathways significantly attenuated the effects of Ad.V146M on the expression of genes involved in lipid oxidation (peroxisome proliferator-activated receptor α, PPARα; carnitine palmitoyltransferase 1, CPT1) or glucose utilization (phosphofructokinase, muscle, PFK-M), respectively. Intriguingly, these findings were confirmed in vivo in cardiac-specific transgenic mice overexpressing AdipoR1 mutant. Taken together, these results suggest that a mutation in the AdipoR1 may contribute to the development of cardiomyopathy via alterations in mTOR/p38 pathways and cardiac metabolism.

This study investigated the possible role of the cardiac isoform of alpha 2-macroglobulin (CA2M) as an early diagnostic marker for HIV-associated cardiovascular manifestations. A total of 349 samples were analysed by Western blot and quantified by sandwich enzyme-linked immunosorbent assay. The levels of CA2M present in sera of HIV-associated cardiac diseases were significantly higher than those of HIV without cardiac involvement and healthy sera. CA2M may act as a novel diagnostic marker to identify cardiac manifestations in HIV/AIDS patients.

BACKGROUND Earlier we showed cardiac isoform of alpha-2 macroglobulin (CA2M) to be an early marker of cardiac hypertrophy. DESIGN In this study, we tried to explore the possibility of using this protein as a marker for diagnosis of cardiac diseases. METHODS A total of 593 samples were analyzed for the presence of CA2M using sandwich enzyme-linked immunosorbent assay. RESULTS Samples include various cardiac diseases (230), non-cardiac ailments (263) and controls (100). CONCLUSION Levels of CA2M in cardiac diseases were significantly higher than other sample groups but moderately elevated in leprosy. This protein could be considered for diagnosis of cardiac diseases as a serum marker.

We sought to determine the frequency of the genetic variations in the Troponin T (TNNT2) gene and its association in Indian cardiomyopathy patients. Sequencing of the entire TNNT2 gene in 162 hypertrophic cardiomyopathy (HCM) patients, along with 179 healthy controls, revealed a total of 15 variants. These included an A28V missense mutation, a novel single-nucleotide polymorphism (SNP) (g.7239;G→A) predicted to disturb the splicing significantly, three SNPs, rs3729547 (C→T), rs3729843 (G→A), rs3729842 (C→T), which were in high linkage disequilibrium, and a 5 bp polymorphism that skipped exon 4 during splicing, which was found to be significantly higher in HCM patients (del/del genotype, p=0.00011; deletion allele, p=0.00008). Further studies on the 5 bp polymorphism in 2092 randomly selected individuals belonging to 39 ethnic and endogamous populations from 19 states of India, and representing the major linguistic Indian families, revealed that the South and the Northwest Indians have a high frequency of 5 bp deletions. The missense mutations in TNNT2 are responsible for 15%-20% of familial HCM by impairing the function of the heart muscle. However, other than the 5 bp polymorphism, our comprehensive study on the Indian HCM patients have lowered the occurrence and overall prevalence of supposedly more aggressive and worst disease causing percentage of missense mutations in TNNT2 dramatically.

RAS activation is implicated in physiologic and pathologic cardiac hypertrophy. Cross-talk between the Ras and calcineurin pathways, the latter also having been implicated in cardiac hypertrophy, has been suspected for pathologic hypertrophy. Our recent discovery that germ-line mutations in RAF1, which encodes a downstream RAS effector, cause Noonan and LEOPARD syndromes with a high prevalence of hypertrophic cardiomyopathy provided an opportunity to elaborate the role of RAF1 in cardiomyocyte biology. Here, we characterize the role of RAF1 signaling in cardiomyocyte hypertrophy with an aim of identifying potential therapeutic targets. We modeled hypertrophic cardiomyopathy by infecting neonatal and adult rat cardiomyocytes (NRCMs and ARCMs, respectively) with adenoviruses encoding wild-type RAF1 and three Noonan/LEOPARD syndrome-associated RAF1 mutants (S257L, D486N or L613V). These RAF1 proteins, except D486N, engendered cardiomyocyte hypertrophy. Surprisingly, these effects were independent and dependent of mitogen activated protein kinases in NRCMs and ARCMs, respectively. Inhibiting Mek1/2 in RAF1 overexpressing cells blocked hypertrophy in ARCMs but not in NRCMs. Further, we found that endogenous and heterologously expressed RAF1 complexed with calcineurin, and RAF1 mutants causing hypertrophy signaled via nuclear factor of activated T cells (Nfat) in both cell types. The involvement of calcineurin was also reflected by down regulation of Serca2a and dysregulation of calcium signaling in NRCMs. Furthermore, treatment with the calcineurin inhibitor cyclosporine blocked hypertrophy in NRCMs and ARCMs overexpressing RAF1. Thus, we have identified calcineurin as a novel interaction partner for RAF1 and established a mechanistic link and possible therapeutic target for pathological cardiomyocyte hypertrophy induced by mutant RAF1. This article is part of a Special Issue entitled 'Possible Editorial'.

The chromosomal region 9p21 has been reported to be associated with myocardial infarction, coronary artery disease (CAD), diabetes, and many other related multifactorial diseases in humans. Although the genome-wide association studies have identified a limited number of single-nucleotide polymorphisms (SNPs) at 9p21 for CAD risk, the role of flanking SNPs has not been studied so far. Therefore, in the present work, we studied the role of flanking SNPs with respect to that of the previously identified SNPs rs10757278 and rs2383207 at 9p21 among the Indian subjects found to have CAD (n = 414) along with age- and sex-matched control subjects (n = 408). Our study replicated the association of genome-wide association studies that had identified SNPs rs2383207 (p = 4.7 × 10(-5)) and rs10757278 (p = 5.5 × 10(-5)) among Indians with CAD. Further, we evaluated nine additional SNPs, of which two SNPs flanking rs2383207 (rs1537375 [p = 2.4 × 10(-5)] and rs1537374 [p = 5.6 × 10(-5)]) were also strongly associated with CAD. The haplotypes constructed using four risk SNPs revealed that the haplotypes with combinations of rs10757278 showed CAD risks, whereas the minor alleles of rs2383207, rs1537375, and rs1537374 in combinations reduce the CAD risks substantially. Our study demonstrates that the variation in the chromosomal region 9p21 is involved in modifying progression toward CAD among Indians and the risk may be variable, contributed by the SNPs that are flanking previously identified SNPs.

Mutations in PTPN11 gene was responsible for approximately 50% of the Noonan syndrome (NS), however, we did not find any mutation in PTPN11 in any of seven NS patients analysed. Whereas, the complete mtDNA sequencing revealed 146 mutations, of which five, including one heteroplasmic (A11144R; Thr-->Ala) non-synonymous mutation, were novel and exclusively observed in NS patients. Interestingly all the seven probands and their maternal relatives were clustered under a major haplogroup R and its novel sub-haplogroups (R7b1b, R30a1, R30c, T2b7, U9a1) exclusive in NS, therefore we strongly suggest that these haplogroups may influence NS in South Indian populations.

We report a case of Noonan syndrome with compound mutations in a sarcomeric contractile protein gene and several novel mutations in mitochondrial genes. Our case forms the first report, which emphasizes the importance of mtDNA mutations in Noonan syndrome and extends the scope for mitochondrial related syndromes.

Only a small fraction of large genomes such as that of the human contains the functional regions such as the exons, promoters, and polyA sites. A platform technique for selective enrichment of functional genomic regions will enable several next-generation sequencing applications that include the discovery of causal mutations for disease and drug response. Here, we describe a powerful platform technique, termed “functional genomic fingerprinting” (FGF), for the multiplexed genomewide isolation and analysis of targeted regions such as the exome, promoterome, or exon splice enhancers. The technique employs a fixed part of a uniquely designed Fixed-Randomized primer, while the randomized part contains all the possible sequence permutations. The Fixed-Randomized primers bind with full sequence complementarity at multiple sites where the fixed sequence (such as the splice signals) occurs within the genome, and multiplex amplify many regions bounded by the fixed sequences (e.g., exons). Notably, validation of this technique using cardiac myosin binding protein-C (MYBPC3) gene as an example strongly supports the application and efficacy of this method. Further, assisted by genomewide computational analyses of such sequences, the FGF technique may provide a unique platform for high-throughput sample production and analysis of targeted genomic regions by the next-generation sequencing techniques, with powerful applications in discovering disease and drug response genes.