Novel Mechanisms Of Disease Research Articles

SARS-CoV-2 S Protein Reduces Cytoprotective Defenses and Promotes Human Endothelial Cell Senescence.

Premature vascular aging and endothelial cell senescence are major risk factors for cardiovascular diseases and atherothrombotic disturbances, which are main complications of both acute and long COVID-19. The S protein of SARS-CoV2, which acts as the receptor binding protein for the viral infection, is able to induce endothelial cells inflammation and it has been found as an isolated element in the circulation and in human tissues reservoirs months after infection. Here, we investigated whether the S protein is able to directly induce endothelial cell senescence and deciphered some of the mechanisms involved. In primary cultures of human umbilical vein endothelial cells (HUVEC), SARS-CoV-2 S protein enhanced in a concentration-dependent manner the cellular content of senescence and DNA damage response markers (senescence-associated-β galactosidase, γH2AX), as well as growth-arrest effectors (p53, p21, p16). In parallel, the S protein reduced the availability of cytoprotective proteins, such as the anti-aging protein klotho, Nrf2 or heme oxygenase-1, and caused functional harm by impairing ex vivo endothelial-dependent vasorelaxation in murine microvessels. These effects were prevented by the pharmacological inhibition of the NLRP3 inflammasome with MCC950. Furthermore, the supplementation with either recombinant klotho or angiotensin-(1-7), equally protected against the pro-senescence, pro-inflammatory and pro-oxidant action of the S protein. Globally, this study proposes novel mechanisms of disease in the context of COVID-19 and its vascular sequelae and provides pharmacological clues in order to prevent such complications.

Multiomic Analysis and CRISPR Perturbation Screens Identify Endothelial Cell Programs and Novel Therapeutic Targets for Coronary Artery Disease.

Endothelial cells (EC) are an important mediator of atherosclerosis and vascular disease. Their exposure to atherogenic risk factors such as hypertension and serum cholesterol leads to endothelial dysfunction and many disease-associated processes. Identifying which of these multiple EC functions is causally related to disease risk has been challenging. There is evidence from in vivo models and human sequencing studies that dysregulation of nitric oxide production directly affects risk of coronary artery disease. Human genetics can help prioritize the other EC functions with causal relationships because germline mutations are acquired at birth and serve as a randomized test of which pathways affect disease risk. Though several coronary artery disease risk variants have been linked to EC function, this process has been slow and laborious. Unbiased analyses of EC dysfunction using multiomic approaches promise to identify the causal genetic mechanisms responsible for vascular disease. Here, we review the data from genomic, epigenomic, and transcriptomic studies that prioritize EC-specific causal pathways. New methods that CRISPR (clustered regularly interspaced short palindromic repeats) perturbation technology with genomic, epigenomic, and transcriptomic analysis promise to speed up the characterization of disease-associated genetic variation. We summarize several recent studies in ECs which use high-throughput genetic perturbation to identify disease-relevant pathways and novel mechanisms of disease. These genetically validated pathways can accelerate the identification of drug targets for the prevention and treatment of atherosclerosis.

Meeting the Challenges of Myocarditis: New Opportunities for Prevention, Detection, and Intervention-A Report from the 2021 National Heart, Lung, and Blood Institute Workshop.

The National Heart, Lung, and Blood Institute (NHLBI) convened a workshop of international experts to discuss new research opportunities for the prevention, detection, and intervention of myocarditis in May 2021. These experts reviewed the current state of science and identified key gaps and opportunities in basic, diagnostic, translational, and therapeutic frontiers to guide future research in myocarditis. In addition to addressing community-acquired myocarditis, the workshop also focused on emerging causes of myocarditis including immune checkpoint inhibitors and SARS-CoV-2 related myocardial injuries and considered the use of systems biology and artificial intelligence methodologies to define workflows to identify novel mechanisms of disease and new therapeutic targets. A new priority is the investigation of the relationship between social determinants of health (SDoH), including race and economic status, and inflammatory response and outcomes in myocarditis. The result is a proposal for the reclassification of myocarditis that integrates the latest knowledge of immunological pathogenesis to refine estimates of prognosis and target pathway-specific treatments.

Long-read human genome sequencing and its applications.

Over the past decade, long-read, single-molecule DNA sequencing technologies have emerged as powerful players in genomics. With the ability to generate reads tens to thousands of kilobases in length with an accuracy approaching that of short-read sequencing technologies, these platforms have proven their ability to resolve some of the most challenging regions of the human genome, detect previously inaccessible structural variants and generate some of the first telomere-to-telomere assemblies of whole chromosomes. Long-read sequencing technologies will soon permit the routine assembly of diploid genomes, which will revolutionize genomics by revealing the full spectrum of human genetic variation, resolving some of the missing heritability and leading to the discovery of novel mechanisms of disease.

Six years\u2019 experience with LipidSeq: clinical and research learnings from a hybrid, targeted sequencing panel for dyslipidemias

BackgroundIn 2013, our laboratory designed a targeted sequencing panel, “LipidSeq”, to study the genetic determinants of dyslipidemia and metabolic disorders. Over the last 6 years, we have analyzed 3262 patient samples obtained from our own Lipid Genetics Clinic and international colleagues. Here, we highlight our findings and discuss research benefits and clinical implications of our panel.MethodsLipidSeq targets 69 genes and 185 single-nucleotide polymorphisms (SNPs) either causally related or associated with dyslipidemia and metabolic disorders. This design allows us to simultaneously evaluate monogenic—caused by rare single-nucleotide variants (SNVs) or copy-number variants (CNVs)—and polygenic forms of dyslipidemia. Polygenic determinants were assessed using three polygenic scores, one each for low-density lipoprotein cholesterol, triglyceride, and high-density lipoprotein cholesterol.ResultsAmong 3262 patient samples evaluated, the majority had hypertriglyceridemia (40.1%) and familial hypercholesterolemia (28.3%). Across all samples, we identified 24,931 unique SNVs, including 2205 rare variants predicted disruptive to protein function, and 77 unique CNVs. Considering our own 1466 clinic patients, LipidSeq results have helped in diagnosis and improving treatment options.ConclusionsOur LipidSeq design based on ontology of lipid disorders has enabled robust detection of variants underlying monogenic and polygenic dyslipidemias. In more than 50 publications related to LipidSeq, we have described novel variants, the polygenic nature of many dyslipidemias—some previously thought to be primarily monogenic—and have uncovered novel mechanisms of disease. We further demonstrate several tangible clinical benefits of its use.

624: MicroRNA expression profile in human visceral and subcutaneous adipose tissues during pregnancy: Implications for metabolic adaptations to normal gestation and putative novel mechanism of disease in gestational diabetes mellitus

624: MicroRNA expression profile in human visceral and subcutaneous adipose tissues during pregnancy: Implications for metabolic adaptations to normal gestation and putative novel mechanism of disease in gestational diabetes mellitus

Novel Mechanism of Disease: Altered RNA metabolism due a dysfunctional RNA binding protein contributes to neurodegeneration in MS (P2.362)

Novel Mechanism of Disease: Altered RNA metabolism due a dysfunctional RNA binding protein contributes to neurodegeneration in MS (P2.362)

NETosis-associated serum biomarkers are reduced in type 1 diabetes in association with neutrophil count.

As the immune pathways involved in the pathogenesis of type 1 diabetes (T1D) are not fully understood, biomarkers implicating novel mechanisms of disease are of great interest and call for independent evaluation. Recently, it was reported that individuals with T1D display dramatic elevations in circulating components of neutrophil extracellular traps (NETs), indicating a potential role for NETosis in T1D. Our aim was to evaluate further the potential of NET-associated proteins as novel circulating biomarkers in T1D. We tested serum from subjects with T1D (n = 44) with a median age of 26·5 years and a median duration of 2·2 years, along with 38 age-matched controls. T1D subjects did not show elevations in either neutrophil elastase (NE) or proteinase 3 (PR3), as reported previously. In fact, both NE and PR3 levels were reduced significantly in T1D subjects, particularly in subjects within 3 years of diagnosis, consistent with the known reduction in neutrophil counts in recent-onset T1D. Indeed, levels of both NE and PR3 correlated with absolute neutrophil counts. Therefore, while not ruling out potential local or transient spikes in NETosis activity, the levels of these serum markers do not support a role for systemically elevated NETosis in the T1D population we studied. Rather, a modest reduction in these markers may reflect other important aspects of disease activity associated with reduced neutrophil numbers.

Prospects and Challenges of Induced Pluripotent Stem Cells in Equine Health.

Pluripotent stem cells (PSCs) hold, through the capacity to differentiate into virtually all body cell types, unprecedented promise for human and animal medicine. PSCs are naturally found in the early embryo, and in rodents and humans they can be robustly harvested and grown in culture in the form of embryonic stem cells (ESCs); however, the availability of ESCs from horses is limited. ES-like cells named induced pluripotent stem cells (iPSCs) can be derived in vitro by transcription factor-mediated reprogramming of adult cells. As such, iPSCs can be generated in a patient-specific manner providing unmatched potential for tissue transplantation and in vitro disease modeling. In humans, clinical trials using iPSC-derived cells are already taking place and the use of in vitro iPSC models has identified novel mechanisms of disease and therapeutic targets. Although to a more limited extent, iPSCs have also been generated from horses, a species in which, after humans, these cells are likely to hold the greatest potential in regenerative medicine. Before a clinical use can be envisioned, however, significant challenges will need to be addressed in relation to the robust derivation, long-term culture, differentiation, and clinical safety of equine iPSCs. Toward this objective, recent studies have reported significant improvement in culture conditions and the successful derivation for the first time of functional cell types from equine iPSCs. Given the wide range of exciting applications they could have, it is hoped future research will make the biomedical promise of iPSCs a reality not only for humans but also horses.

Genetic variants associated with vein graft stenosis after coronary artery bypass grafting.

Vein graft stenosis after coronary artery bypass grafting (CABG) is common. Identifying genes associated with vein graft stenosis after CABG could reveal novel mechanisms of disease and discriminate patients at risk for graft failure. We hypothesized that genome-wide association would identify these genes. We performed a genome-wide association study on a subset of patients presenting for cardiac catheterization for concern of ischemic heart disease, who also underwent CABG and subsequent coronary angiography after CABG for clinical indications (n = 521). Cases were defined as individuals with ≥50% stenosis in any vein graft on any cardiac catheterization, and controls were defined as those who did not have vein graft stenosis on any subsequent cardiac catheterization. Multivariable logistic regression was used to assess the association between single nucleotide polymorphisms (SNPs) and vein graft stenosis. Sixty-nine percent of patients had vein graft failure after CABG. Seven SNPs were significantly associated with vein graft stenosis, including intronic SNPs in the genes PALLD (Rs6854137, P = 3.77 × 10(-6)), ARID1B (Rs184074, P = 5.97 × 10(-6)), and TMEM123 (Rs11225247, P = 8.25 × 10(-6)); and intergenic SNPs near the genes ABCA13 (Rs10232860, P = 4.54 × 10(-6)), RMI2 (Rs9921338, P = 6.15 × 10(-6)), PRM2 (Rs7198849, P = 7.27 × 10(-6)), and TNFSF4 (Rs17346536, P = 9.33 × 10(-6)). We have identified novel genetic variants that may predispose to risk of vein graft failure after CABG, many within biologically plausible pathways. These polymorphisms merit further investigation, as they could assist in stratifying patients with multi-vessel coronary artery disease, which could lead to alterations in management and revascularization strategy.

Genomic editing tools to model human diseases with isogenic pluripotent stem cells.

Patient-specific induced pluripotent stem cells (iPSCs) are considered a versatile resource in the field of biomedicine. As iPSCs are generated on an individual basis, iPSCs may be the optimal cellular material to use for disease modeling, drug discovery, and the development of patient-specific cellular therapies. Recently, to gain an in-depth understanding of human pathologies, patient-specific iPSCs have been used to model human diseases with some iPSC-derived cells recapitulating pathological phenotypes in vitro. However, complex multigenic diseases generally have not resulted in concise conclusions regarding the underlying mechanisms of disease, in large part due to genetic variations between disease-state and control iPSCs. To circumvent this, the use of genomic editing tools to generate perfect isogenic controls is gaining momentum. To date, DNA binding domain-based zinc finger nucleases and transcription activator-like effector nucleases have been utilized to create genetically defined conditions in patient-specific iPSCs, with some examples leading to the successful identification of novel mechanisms of disease. As the feasibility and utility of genomic editing tools in iPSCs improve, along with the introduction of the clustered regularly interspaced short palindromic repeat system, understanding the features and limitations of genomic editing tools and their applications to iPSC technology is critical to expending the field of human disease modeling.

A.P.9: Elucidation of the mechanism of disease in BAG3-related myofibrillar myopathy

Mutations in the co-chaperone Bcl2-associated athanogene 3 (BAG3) have been shown to cause myofibrillar myopathy (MFM). MFM is characterised at the cellular level by the formation of protein aggregates and structural failure of the Z-disk. In contrast to the other MFM causing proteins BAG3 has no structural role in the Z-disk but is involved in the regulation of autophagy and the degradation of misfolded proteins. Protein aggregation in BAG3-related MFM has therefore been proposed to result from a reduction in autophagic activity. To investigate the mechanism of disease in BAG3-related MFM we expressed the human wildtype BAG3 or the dominant myofibrillar myopathy causing mutant form BAG3^P209L in zebrafish muscle. The expression of BAG3^P209L resulted in protein aggregation and we performed time-lapse imaging to examine aggregate formation in vivo and examined their composition. To investigate the role of impaired autophagy in BAG related MFM we stimulated or inhibited autophagy in BAG3^wt or BAG3^P209L expressing embryos and examined its effect on protein aggregation. Our studies revealed that autophagy is active in BAG3^P209L expressing fish and that inhibition of autophagy is not sufficient to induce protein aggregation, suggesting that the pathology is not due to impaired autophagic activity. We did not observe fibre failure in the BAG3^P209L model fish and therefore examined the effect of knocking down BAG3 expression. Loss of BAG3 function resulted in fibre failure but not the formation of protein aggregates. Together our knockdown and overexpressing models demonstrate contraction-dependent fiber failure and the formation of protein aggregates. Furthermore, our experimental analysis of the transgenic and knockdown embryos demonstrates that protein aggregation is not due to an inhibition of autophagy and suggests a novel mechanism of disease with the cellular pathology caused by a combination of toxic gain of function and BAG3 insufficiency.

Amniotic fluid RNA gene expression profiling provides insights into the phenotype of Turner syndrome.

Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p < 0.0001); SHOX was not differentially expressed. One of the most highly represented organ systems was the hematologic/immune system, distinguishing the Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.

Beyond the genome: epigenetic mechanisms in lung remodeling.

The lung develops from a very simple outpouching of the foregut into a highly complex, finely structured organ with multiple specialized cell types that are required for its normal physiological function. During both the development of the lung and its remodeling in the context of disease or response to injury, gene expression must be activated and silenced in a coordinated manner to achieve the tremendous phenotypic heterogeneity of cell types required for homeostasis and pathogenesis. Epigenetic mechanisms, consisting of DNA base modifications such as methylation, alteration of histones resulting in chromatin modification, and the action of noncoding RNA, control the regulation of information "beyond the genome" required for both lung modeling and remodeling. Epigenetic regulation is subject to modification by environmental stimuli, such as oxidative stress, infection, and aging, and is thus critically important in chronic remodeling disorders such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), bronchopulmonary dysplasia (BPD), and pulmonary hypertension (PH). Technological advances have made it possible to evaluate genome-wide epigenetic changes (epigenomics) in diseases of lung remodeling, clarifying existing pathophysiological paradigms and uncovering novel mechanisms of disease. Many of these represent new therapeutic targets. Advances in epigenomic technology will accelerate our understanding of lung development and remodeling, and lead to novel treatments for chronic lung diseases.

Aberrant ZNF423 impedes B cell differentiation and is linked to adverse outcome of ETV6-RUNX1 negative B precursor acute lymphoblastic leukemia

Differentiation arrest is a hallmark of acute leukemia. Genomic alterations in B cell differentiation factors such as PAX5, IKZF1, and EBF-1 have been identified in more than half of all cases of childhood B precursor acute lymphoblastic leukemia (ALL). Here, we describe a perturbed epigenetic and transcriptional regulation of ZNF423 in ALL as a novel mechanism interfering with B cell differentiation. Hypomethylation of ZNF423 regulatory sequences and BMP2 signaling result in transactivation of ZNF423α and a novel ZNF423β-isoform encoding a nucleosome remodeling and histone deacetylase complex-interacting domain. Aberrant ZNF423 inhibits the transactivation of EBF-1 target genes and leads to B cell maturation arrest in vivo. Importantly, ZNF423 expression is associated with poor outcome of ETV6-RUNX1-negative B precursor ALL patients. Our work demonstrates that ALL is more than a genetic disease and that epigenetics may uncover novel mechanisms of disease with prognostic implications.

VMA21 deficiency prevents vacuolar ATPase assembly and causes autophagic vacuolar myopathy

X-linked Myopathy with Excessive Autophagy (XMEA) is a childhood onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p, VMA21 is an essential assembly chaperone of the vacuolar ATPase (V-ATPase), the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids which leads to downregulation of the mTORC1 pathway, and consequent increased macroautophagy resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge, and vacuolate the cell. Our results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy.

Diagnosis, novel mechanisms of disease and promising agents

The results of various studies from 2010 have underlined the importance of the careful diagnostic assessment of GERD. Recent studies have also provided further evidence of the complex pathogenesis of GERD, and promising agents targeting the underlying factors in the pathogenesis of this condition are in the pipeline, especially for treatment-refractory GERD.

Study protocol: a randomised controlled trial investigating the effect of exercise training on peripheral blood gene expression in patients with stable angina

BackgroundExercise training has been shown to reduce angina and promote collateral vessel development in patients with coronary artery disease. However, the mechanism whereby exercise exerts these beneficial effects is unclear. There has been increasing interest in the use of whole genome peripheral blood gene expression in a wide range of conditions to attempt to identify both novel mechanisms of disease and transcriptional biomarkers. This protocol describes a study in which we will assess the effect of a structured exercise programme on peripheral blood gene expression in patients with stable angina, and correlate this with changes in angina level, anxiety, depression, and exercise capacity.Methods/DesignSixty patients with stable angina will be recruited and randomised 1:1 to exercise training or conventional care. Patients randomised to exercise training will attend an exercise physiology laboratory up to three times weekly for supervised aerobic interval training sessions of one hour in total duration. Patients will undergo assessments of angina, anxiety, depression, and peripheral blood gene expression at baseline, after six and twelve weeks of training, and twelve weeks after formal exercise training ceases.DiscussionThis study will provide comprehensive data on the effect of exercise training on peripheral blood gene expression in patients with angina. By correlating this with improvement in angina status we will identify candidate peripheral blood transcriptional markers predictive of improvements in angina level in response to exercise training.Trial RegistrationClinicaltrials.gov identifier: NCT01147952

Oxidative biology in psychiatric disorders: Novel mechanism of disease and novel therapeutic opportunities

Oxidative biology in psychiatric disorders: Novel mechanism of disease and novel therapeutic opportunities

C3-Mediated Extravascular Hemolysis as Additional Mechanism of Disease in Paroxysmal Nocturnal Hemoglobinuria (PNH) Patients Treated by the Complent Inhibitor Eculizumab

PNH is a hematological disorder characterized by complement-mediated intravascular hemolysis due to lack on RBCs of the complement regulators CD55 and CD59 and subsequent activation of the membrane attack complex (MAC). Eculizumab (EC) is an anti-complement fraction 5 antibody which abolishes intravascular hemolysis in PNH patients, leading to reduction of transfusion requirement and of anemia. Although the effector complement pathway is completely blocked by eculizumab in all patients, response to this agent in terms of transfusion requirement and hemoglobin level varies considerably. We have investigated the notion that patients with suboptimal hematological response may suffer from residual hemolysis mediated by mechanisms other than intravascular hemolysis via MAC. After the initial observation of a positive C3d Coombs test in some PNH patients on EC, we extensively studies C3 coating by flow cytometry in 56 PNH patients (41 of them while receiving EC). We found that in all cases on EC treatment a significant proportion of RBCs were coated with complement fraction 3 (C3); by converse, in 28 untreated PNH patients we found no evidence of C3 on red cells. C3 coating was strictly limited to CD59-neg RBCs, as CD59+/C3+ RBCs (as those seen in cold agglutinine disease patients, positive control) were never found. C3 coating was quite different among EC-treated patients, and correlated with the PNH RBC population. The percentage of C3+ cells within the PNH RBC population (the only subjected to C3 coating) was quite different in individual patients (0.5–61.3%, median 22.6%) and substantially preserved over time. We compared the level of C3 coating with the hematological response: all the 41 EC-treated patients showed marked LDH reduction with a substantial improvement of anemia, leading to transfusion independence in 34/41 patients (83%) and stable resolution of anemia in 14 (34%, defined optimal responders). The optimal responders showed a lower percentage of C3+ cells in comparison to suboptimal responders (20.9±19.0 vs 32.2±17.8; p=0.04). Indeed, patients with lower C3 coating (below the median value of 22.6%) showed a significantly higher rate of optimal response (51% in comparison to 15% of those with coating above the median, p=0.01). C3 coating also correlated with the absolute reticulocyte count (p=0.03), clearly suggesting that C3 coating was associated with ongoing residual extravascular hemolysis in vivo, and with pre-treatment LDH level (p=0.001). To confirm the presence of extravascular hemolysis, in 3 index patients with suboptimal response reduced RBC half-life was demonstrated in vivo by 51Cr RBC survival study, which showed reduced RBC half-life and excess uptake in liver and spleen. One of these patients underwent video-laparoscopic splenectomy, which led to transfusion independence and significant increase in Hb level. These data demonstrate that C3 coating of PNH RBCs is a common phenomenon in PNH patients on EC; in addition, we provide evidence that this leads to C3-mediated extravascular hemolysis through the reticulo-endothelial system. This novel mechanism of disease may account for residual hemolysis and suboptimal clinical benefit in some EC treated PNH, paving the way for additional therapeutic strategies to optimize the hematological response to this agent.