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There is a significant interest in the standardized classification of human genetic variants. We used whole-genome sequence data from 10,495 unrelated individuals to contrast population frequency of pathogenic variants to the expected population prevalence of the disease. Analyses included the ACMG-recommended 59 gene-condition sets for incidental findings and 463 genes associated with 265 OrphaNet conditions. A total of 25,505 variants were used to identify patterns of inflation (i.e., excess genetic risk and misclassification). Inflation increases as the level of evidence supporting the pathogenic nature of the variant decreases. We observed up to 11.5% of genetic disorders with inflation in pathogenic variant sets and up to 92.3% for the variant set with conflicting interpretations. This improved to 7.7% and 57.7%, respectively, after filtering for disease-specific allele frequency. The patterns of inflation were replicated using public data from more than 138,000 genomes. The burden of rare variants was a main contributing factor of the observed inflation, indicating collective misclassified rare variants. We also analyzed the dynamics of re-classification of variant pathogenicity in ClinVar over time, which indicates progressive improvement in variant classification. The study shows that databases include a significant proportion of wrongly ascertained variants; however, it underscores the critical role of ClinVar to contrast claims and foster validation across submitters., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The bicuspid aortic valve (AV) is the most common cardiac congenital anomaly and has been found to be a significant risk factor for developing calcific AV disease. However, the mechanisms of disease development remain unclear. In this study we quantified the structure of human normal and bicuspid leaflets in the early disease stage. From these individual leaflet maps average fiber structure maps were generated using a novel spline based technique. Interestingly, we found statistically different and consistent regional structures between the normal and bicuspid valves. The regularity in the observed microstructure was a surprising finding, especially for the pathological BAV leaflets and is an essential cornerstone of any predictive mathematical models of valve disease. In contrast, we determined that isolated valve interstitial cells from BAV leaflets show the same in vitro calcification pathways as those from the normal AV leaflets. This result suggests the VICs are not intrinsically different when isolated, and that external features, such as abnormal microstructure and altered flow may be the primary contributors in the accelerated calcification experienced by BAV patients.

Aims: Aortic valve sclerosis (AVSc) is a hallmark of several cardiovascular conditions ranging from chronic heart failure and myocardial infarction to calcific aortic valve stenosis (AVS). AVSc, present in 25-30% of patients over 65 years of age, is characterized by thickening of the leaflets with marginal effects on the mechanical proprieties of the valve making its presentation asymptomatic. Despite its clinical prevalence, few studies have investigated the pathogenesis of this disease using human AVSc specimens. Here, we investigate in vitro and ex vivo BMP4-mediated transdifferentiation of human valve interstitial cells (VICs) towards an osteogenic-like phenotype in AVSc., Methods and Results: Human specimens from 60 patients were collected at the time of aortic valve replacement (AVS) or through the heart transplant programme (Controls and AVSc). We show that non-calcified leaflets from AVSc patients can be induced to express markers of osteogenic transdifferentiation and biomineralization through the combinatory effect of BMP4 and mechanical stimulation. We show that BMP4 antagonist Noggin attenuates VIC activation and biomineralization. Additionally, patient-derived VICs were induced to transdifferentiate using either cell culture or a Tissue Engineering (TE) Aortic Valve model. We determine that while BMP4 alone is not sufficient to induce osteogenic transdifferentiation of AVSc-derived cells, the combinatory effect of BMP4 and mechanical stretch induces VIC activation towards a phenotype typical of late calcified stage of the disease., Conclusion: This work demonstrates, for the first time using AVSc specimens, that human sclerotic aortic valves can be induced to express marker of osteogenic-like phenotype typical of advanced severe aortic stenosis.

Background and Aim of the Study: Aortic valve sclerosis (AVSc), the early asymptomatic stage of calcific aortic valve disease (CAVD), is characterized by a progressive thickening of the aortic cusps without obstruction of the left ventricular outflow. In spite of its high prevalence, there are no molecular markers to characterize the early stages of CAVD before it progresses to a severe, symptomatic stage of aortic valve stenosis (AVS). The study aim was to identify any correlation between circulating biomarkers and transesophageal echocardiography (TEE) evaluation., Methods: A total of 330 patients with various degrees of CAVD was enrolled into the study. Blood was collected from each patient prior to surgery, and analyzed using ELISA kits following the manufacturers' instructions., Results: Significantly higher plasma osteopontin (OPN) levels were observed in AVSc patients (72.7 +/- 1.8 ng/ml; p < 0.001) and AVS patients (64.3 +/- 5.1 ng/ml; p < 0.001) when compared to controls (30.3 +/- 1.8 ng/ml). Parathyroid hormone (PTH) levels in AVSc and AVS patients (164.1 +/- 16.5 and 134.3 +/- 14.6 pg/ml; p < 0.001 and p = 0.04, respectively) were also significantly higher than in controls (61.8 +/- 4.92 pg/ml). Upon further analysis, plasma levels of OPN (p < 0.001) and PTH (p < 0.001) were found to be significantly higher in asymptomatic AVSc patients, even before calcium deposition was detected on TEE evaluation. Fetuin-A levels were lower at all stages of CAVD when compared to controls (p < 0.001 and p < or = 0.05, respectively), but were comparable among the patient groups. NT-proBNP levels were significantly higher in AVS patients than in controls (p < or = 0.01)., Conclusion: Serum levels of OPN, PTH, and fetuin-A showed a significant association with different stages of CAVD, with variations in their levels occurring before calcium nodules are visualized during TEE evaluation. The study results may help not only to provide a better understanding of the progression of CAVD but also to develop new tools that can be used to stage these patients.

Objective: Accumulation of reactive oxygen species (ROS) and remodeling of the microstructure of the cusp characterize aortic valve sclerosis, the early phase of calcific aortic valve disease. These events are associated with activation of valvular interstitial cells (VICs) toward an osteogenic-like phenotype. Because ROS cause DNA damage and transcriptional activation we investigated the relationship between ROS, DNA damage response, and transdifferentiation of VICs., Methods and Results: Human aortic valve cusps and patient-matched VICs were collected from 39 patients both with and without calcific aortic valve disease. VICs were exposed to hydrogen peroxide (0.1-1 mmol/L) after cell transduction with extracellular superoxide dismutase/catalase adenoviruses and characterized for DNA-damage response, osteogenic transdifferentiation, and calcification. ROS induce relocalization of phosphorylated γH2AX, MRE11, and XRCC1 proteins with expression of osteogenic signaling molecule RUNX2 via AKT. We report a sustained activation of γH2AX in aortic valve sclerosis-derived VICs suggesting their impaired ability to repair DNA damage. Adenovirus superoxide dismutase/catalase transduction decreases ROS-induced DNA damage and VIC transdifferentiation in aortic valve sclerosis-derived cells. Finally, adenoviral transduction with catalase reverts ROS-mediated calcification and cellular transdifferentiation., Conclusions: We conclude that the ROS-induced DNA damage response is dysfunctional in early asymptomatic stages of calcific aortic valve disease. We unveiled an association among ROS, DNA-damage response, and cellular transdifferentiation, reversible by antioxidant enzymes delivery.

Myxomatous mitral valve prolapse (MVP) is the most common cardiac valvular abnormality in industrialized countries and a leading cause of mitral valve surgery for isolated mitral regurgitation. The key role of valvular interstitial cells (VICs) during mitral valve development and homeostasis has been recently suggested, however little is known about the molecular pathways leading to MVP. We aim to characterize bone morphogenetic protein 4 (BMP4) as a cellular regulator of mitral VIC activation towards a pathologic synthetic phenotype and to analyze the cellular phenotypic changes and extracellular matrix (ECM) reorganization associated with the development of myxomatous MVP. Microarray analysis showed significant up regulation of BMP4-mediated signaling molecules in myxomatous MVP when compared to controls. Histological analysis and cellular characterization suggest that during myxomatous MVP development, healthy quiescent mitral VICs undergo a phenotypic activation via up regulation of BMP4-mediated pathway. In vitro hBMP4 treatment of isolated human mitral VICs mimics the cellular activation and ECM remodeling as seen in MVP tissues. The present study characterizes the cell biology of mitral VICs in physiological and pathological conditions and provides insights into the molecular and cellular mechanisms mediated by BMP4 during MVP. The ability to test and control the plasticity of VICs using different molecules may help in developing new diagnostic and therapeutic strategies for myxomatous MVP., (Copyright © 2011 Wiley Periodicals, Inc.)

Context: Calcific Aortic Valve Disease (CAVD) is an active pathological process leading to biomineralization of the aortic cusps. We characterized circulating and tissue Osteopontin (OPN) as a biomarker for CAVD., Objectives: Here we investigate the post-translational modifications of circulating OPN and correlate the phosphorylation status with the ability to prevent calcification., Methods: Circulating OPN levels were estimated in CAVD patients (n = 51) and controls (n = 56). In a subgroup of 27 subjects, OPN was purified and the phosphorylation status analyzed., Results: Plasma OPN levels were significantly elevated in CAVD patients as compared to the controls and correlates with the aortic valve calcium score. Our study demonstrates that phospho-threonine levels of OPN purified from controls were higher when compared to CAVD subjects, whereas phospho-serine and phospho-tyrosine levels were comparable between the two groups., Conclusion: The dephosphorylation of circulating OPN correlates with severe valvular calcification in patients with CAVD.

Background: Calcific aortic valve disease (CAVD) is the most common cause of acquired valve disease. Initial phases of CAVD include thickening of the cusps, whereas advanced stages are associated with biomineralization and reduction of the aortic valve area. These conditions are known as aortic valve sclerosis (AVSc) and aortic valve stenosis (AVS), respectively. Because of its asymptomatic presentation, little is known about the molecular determinants of AVSc. The aim of this study was to correlate plasma and tissue osteopontin (OPN) levels with echocardiographic evaluation for the identification of asymptomatic patients at risk for CAVD. In addition, our aim was to analyze the differential expression and biological function of OPN splicing variants as biomarkers of early and late stages of CAVD., Methods: From January 2010 to February 2011, 310 patients were enrolled in the study. Patients were divided into 3 groups based on transesophageal echocardiographic (TEE) evaluation: controls (56 patients), AVSc (90 patients), and AVS (164 patients). Plasma and tissue OPN levels were measured by immunohistochemical evaluation, enzyme-linked immunosorbent assay (ELISA), and real-time quantitative polymerase chain reaction (qPCR)., Results: Patients with AVSc and AVS have higher OPN levels compared with controls. OPN levels are elevated in asymptomatic patients with AVSc with no appearance of calcification during TEE evaluation. OPN splicing variants OPN-a, OPN-b, and OPN-c are differentially expressed during CAVD progression and are able to inhibit biomineralization in a cell-based biomineralization assay., Conclusions: The analysis of the differential expression of OPN splicing variants during CAVD may help in developing diagnostic and risk stratification tools to follow the progression of asymptomatic aortic valve degeneration., (Copyright © 2012 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Calcific aortic stenosis (CAS) is a pathological condition of the aortic valve characterized by dystrophic calcification of the valve leaflets. Despite the high prevalence and mortality associated with CAS, little is known about its pathogenetic mechanisms. Characterized by progressive dystrophic calcification of the valve leaflets, the early stages of aortic valve degeneration are similar to the active inflammatory process of atherosclerosis including endothelial disruption, inflammatory cell infiltration, lipid deposition, neo-vascularization and calcification. In the vascular system, the endothelium is an important regulator of physiological and pathological conditions; however, the contribution of endothelial dysfunction to valvular degeneration at the cellular and molecular level has received little attention. Endothelial cell (EC) activation and neo-vascularization of the cusps characterizes all stages of aortic valvular degeneration from aortic sclerosis to aortic stenosis. Here we reported the role of osteopontin (OPN) in the regulation of EC activation in vitro and in excised tissue from CAS patients and controls. OPN is an important pro-angiogenic factor in several pathologies. High levels of OPN have been demonstrated in both tissue and plasma of patients with aortic valve sclerosis and stenosis. The characterization of valvular ECs as a cellular target for OPN will help us uncover the pathogenesis of aortic valve degeneration and stenosis, opening new perspectives for the prevention and therapy of this prevalent disease., (Copyright © 2010 Wiley-Liss, Inc.)

Calcific aortic valve disease (CAVD) is the most common acquired valvular disorder in developed countries. CAVD ranges from mild thickening of the valve, known as aortic valve sclerosis (AVSc), to severe impairment of the valve motion, which is termed aortic valve stenosis (AVS). The prevalence of CAVD is nearing epidemic status: its preceding stage, in which there is aortic sclerosis without obstruction of the left ventricular outflow, is present in almost 30% of adults aged over 65 years. As there is no existing medical therapy to treat or slow the progression of CAVD, surgery for advanced disease represents the only available treatment. Aortic valve replacement is the second most frequently performed cardiac surgical procedure after coronary artery bypass grafting, and consequently CAVD represents a major societal and economic burden. The pathophysiological development of CAVD is incompletely defined. At the present time, the major methods for its diagnosis are clinical examination, echocardiography, and cardiac catheterization. Yet, due to the multiple biological pathways leading to CAVD, there are many potential biomarkers that might be suitable for deriving clinically useful information regarding the presence, severity, progression, and prognosis of CAVD. Although at the present time the available data do not permit recommendations for clinicians, they do support a paradigm of screening patients based on multiple biomarkers to provide the information necessary to optimize future therapeutic interventions. This review summarizes the results of several studies investigating the value of potential biomarkers that have been used to predict the severity, progression, and prognosis of CAVD.

Background: The polycomb group (PcG) protein BMI1 is an important regulator of development. Additionally, aberrant expression of BMI1 has been linked to cancer stem cell phenotype and oncogenesis. In particular, its overexpression has been found in several human malignancies including breast cancer. Despite its established role in stem cell maintenance, cancer and development, at present not much is known about the functional domains of BMI1 oncoprotein. In the present study, we carried out a deletion analysis of BMI1 to identify its negative regulatory domain., Results: We report that deletion of the C-terminal domain of BMI1, which is rich in proline-serine (PS) residues and previously described as PEST-like domain, increased the stability of BMI1, and promoted its pro-oncogenic activities in human mammary epithelial cells (HMECs). Specifically, overexpression of a PS region deleted mutant of BMI1 increased proliferation of HMECs and promoted an epithelial-mesenchymal transition (EMT) phenotype in the HMECs. Furthermore, when compared to the wild type BMI1, exogenous expression of the mutant BMI1 led to a significant downregulation of p16INK4a and an efficient bypass of cellular senescence in human diploid fibroblasts., Conclusions: In summary, our data suggest that the PS domain of BMI1 is involved in its stability and that it negatively regulates function of BMI1 oncoprotein. Our results also suggest that the PS domain of BMI1 could be targeted for the treatment of proliferative disorders such as cancer and aging.

Elevated expression of Bmi-1 is associated with many cancers, including breast cancer. Here, we examined the oncogenic potential of Bmi-1 in MCF10A cells, a spontaneously immortalized, nontransformed strain of human mammary epithelial cells (HMEC). Bmi-1 overexpression alone in MCF10A cells did not result in oncogenic transformation. However, Bmi-1 co-overexpression with activated H-Ras (RasG12V) resulted in efficient transformation of MCF10A cells in vitro. Although early-passage H-Ras-expressing MCF10A cells were not transformed, late-passage H-Ras-expressing cells exhibited features of transformation in vitro. Early- and late-passage H-Ras-expressing cells also differed in levels of expression of H-Ras and Ki-67, a marker of proliferation. Subsets of early-passage H-Ras-expressing cells exhibited high Ras expression and were negative for Ki-67, whereas most late-passage H-Ras-expressing cells expressed low levels of Ras and were Ki-67 positive. Injection of late-passage H-Ras-expressing cells in severe combined immunodeficient mice formed carcinomas with leiomatous, hemangiomatous, and mast cell components; these tumors were quite distinct from those induced by late-passage cells co-overexpressing Bmi-1 and H-Ras, which formed poorly differentiated carcinomas with spindle cell features. Bmi-1 and H-Ras co-overexpression in MCF10A cells also induced features of epithelial-to-mesenchymal transition. Importantly, Bmi-1 inhibited senescence and permitted proliferation of cells expressing high levels of Ras. Examination of various growth-regulatory pathways suggested that Bmi-1 overexpression together with H-Ras promotes HMEC transformation and breast oncogenesis by deregulation of multiple growth-regulatory pathways by p16(INK4a)-independent mechanisms.

Glutathione, an antioxidant plays an important role in phase-II detoxification of carcinogens. The levels of reduced glutathione are maintained by glutathione-depleting as well as replenishing enzymes such as glutathione-s-transferase (GST) and glutathione reductase (GR), respectively. Pre and post treatment changes in GST and GR activities in head and neck cancer patients were analysed. Serum GST and GR were analysed from untreated head and neck cancer patients (PT) (n=146), controls with habit of tobacco (VHT) (n=25) as well as without (no) habit of tobacco (NHT) (n=25) and patients with oral precancerous conditions (OPC) (n=50). The cancer patients were followed-up after initiation of anticancer therapy. Follow-up blood samples were collected. Serum GST and GR activities were estimated by highly sensitive and specific spectrophotometric methods. Untreated cancer patients showed elevated mean serum GST and GR activities as compared to NHT. Patients with OPC had declined mean GST activity as compared to WHT and untreated cancer patients. Paired t-test revealed that complete responders (CR) showed significantly elevated GST levels and declined GR activities (p < 0.001) as compared to those in PT. No correlation was found between stage of the disease and GST, GR activity. Paired t-test showed significant decreased in GR activity in nonresponders (NR) treated with radiotherapy (p=0.01). The study suggested that analysis of glutathione and glutathione-depleting enzymes can be helpful for treatment monitoring of head and neck cancer patients.