Browsing by Author "Aldred, Micheala A."
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Item Clinical Characteristics and Transplant-Free Survival Across the Spectrum of Pulmonary Vascular Disease(Elsevier, 2022) Hemnes, Anna R.; Leopold, Jane A.; Radeva, Milena K.; Beck, Gerald J.; Abidov, Aiden; Aldred, Micheala A.; Barnard, John; Rosenzweig, Erika B.; Borlaug, Barry A.; Chung, Wendy K.; Comhair, Suzy A. A.; Desai, Ankit A.; Dubrock, Hilary M.; Erzurum, Serpil C.; Finet, J. Emanuel; Frantz, Robert P.; Garcia, Joe G. N.; Geraci, Mark W.; Gray, Michael P.; Grunig, Gabriele; Hassoun, Paul M.; Highland, Kristin B.; Hill, Nicholas S.; Hu, Bo; Kwon, Deborah H.; Jacob, Miriam S.; Jellis, Christine L.; Larive, A. Brett; Lempel, Jason K.; Maron, Bradley A.; Mathai, Stephen C.; McCarthy, Kevin; Mehra, Reena; Nawabit, Rawan; Newman, John H.; Olman, Mitchell A.; Park, Margaret M.; Ramos, Jose A.; Renapurkar, Rahul D.; Rischard, Franz P.; Sherer, Susan G.; Tang, W. H. Wilson; Thomas, James D.; Vanderpool, Rebecca R.; Waxman, Aaron B.; Wilcox, Jennifer D.; Yuan, Jason X-J; Horn, Evelyn M.; PVDOMICS Study Group; Medicine, School of MedicineBackground: PVDOMICS (Pulmonary Vascular Disease Phenomics) is a precision medicine initiative to characterize pulmonary vascular disease (PVD) using deep phenotyping. PVDOMICS tests the hypothesis that integration of clinical metrics with omic measures will enhance understanding of PVD and facilitate an updated PVD classification. Objectives: The purpose of this study was to describe clinical characteristics and transplant-free survival in the PVDOMICS cohort. Methods: Subjects with World Symposium Pulmonary Hypertension (WSPH) group 1-5 PH, disease comparators with similar underlying diseases and mild or no PH and healthy control subjects enrolled in a cross-sectional study. PH groups, comparators were compared using standard statistical tests including log-rank tests for comparing time to transplant or death. Results: A total of 1,193 subjects were included. Multiple WSPH groups were identified in 38.9% of PH subjects. Nocturnal desaturation was more frequently observed in groups 1, 3, and 4 PH vs comparators. A total of 50.2% of group 1 PH subjects had ground glass opacities on chest computed tomography. Diffusing capacity for carbon monoxide was significantly lower in groups 1-3 PH than their respective comparators. Right atrial volume index was higher in WSPH groups 1-4 than comparators. A total of 110 participants had a mean pulmonary artery pressure of 21-24 mm Hg. Transplant-free survival was poorest in group 3 PH. Conclusions: PVDOMICS enrolled subjects across the spectrum of PVD, including mild and mixed etiology PH. Novel findings include low diffusing capacity for carbon monoxide and enlarged right atrial volume index as shared features of groups 1-3 and 1-4 PH, respectively; unexpected, frequent presence of ground glass opacities on computed tomography; and sleep alterations in group 1 PH, and poorest survival in group 3 PH. PVDOMICS will facilitate a new understanding of PVD and refine the current PVD classification.Item Comparison of whole genome sequencing and targeted sequencing for mitochondrial DNA(Elsevier, 2021) Chen, Ruoying; Aldred, Micheala A.; Xu, Weiling; Zein, Joe; Bazeley, Peter; Comhai, Suzy A. A.; Meyers, Deborah A.; Bleecker, Eugene R.; Liu, Chunyu; Erzurum, Serpil C.; Hu, Bo; NHLBI Severe Asthma Research Program (SARP); Medicine, School of MedicineMitochondrial dysfunction has emerged to be associated with a broad spectrum of diseases, and there is an increasing demand for accurate detection of mitochondrial DNA (mtDNA) variants. Whole genome sequencing (WGS) has been the dominant sequencing approach to identify genetic variants in recent decades, but most studies focus on variants on the nuclear genome. Whole genome sequencing is also costly and time consuming. Sequencing specifically targeted for mtDNA is commonly used in the diagnostic settings and has lower costs. However, there is a lack of pairwise comparisons between these two sequencing approaches for calling mtDNA variants on a population basis. In this study, we compared WGS and mtDNA-targeted sequencing (targeted-seq) in analyzing mitochondrial DNA from 1499 participants recruited into the Severe Asthma Research Program (SARP). Our study reveals that targeted-sequencing and WGS have comparable capacity to determine genotypes and to call haplogroups and homoplasmies on mtDNA. However, there exists a large variability in calling heteroplasmies, especially for low-frequency heteroplasmies, which indicates that investigators should be cautious about heteroplasmies acquired from different sequencing methods. Further research is highly desired to improve variant detection methods for mitochondrial DNA.Item Deciphering the Role of Mitochondrial Dysfunction in Pulmonary Arterial Hypertension(2024-06) Balachandar, Srimmitha; Aldred, Micheala A.; Graham, Brett H.; Zhang, Jie; Geraci, Mark W.; Machado, Roberto F.Pulmonary arterial hypertension (PAH) is a life-threatening vasculopathy caused by remodeling of pulmonary arterioles. It is unknown as to why some people are at more risk of developing PAH compared to others. Notably, while germline pathogenic variants in PAH genes are a strong driver of disease susceptibility, less than half of mutation carriers actually develop the disease, suggesting the need for additional triggers. Our previous studies have shown increased DNA damage and total reactive oxygen species (ROS) in cells from PAH patients and unaffected relatives, indicating a potential genetic component, leading to our hypothesis: Mitochondrial dysfunction is an independent genetically determined modifier of PAH susceptibility. Untargeted metabolomics (Metabolon) revealed abnormalities in the antioxidants, glutamate, urea, amino acid, galactose, and phospholipid metabolism pathways in the PAH Lymphoblastoid cells (LCLs) compared to controls. Intriguingly, the healthy relatives also had altered phospholipids, suggesting that it occurs independent of the disease. ROS analysis on LCLs from patients, their relatives and unrelated controls showed that the PAH LCLs had significantly higher levels of all ROS species compared to controls, with the highest in heritable PAH cells. LCLs from relatives clustered into two groups, one with increased mitochondrial (mt) ROS and hydrogen peroxide, the other comparable to controls. Seahorse assays showed that the LCLs with increased mtROS had reduced spare respiratory capacity indicative of dysfunctional electron transport chain (ETC); but no glycolytic switch. Cybrid models generated using the high and low ROS LCLs (H and L-donors) on a 143B nuclear background showed that the H-donors had mt respiration similar to L-donors, suggesting a functional ETC. However, these cells had significantly elevated mtROS, with reduced SOD2 protein (potentially a consequence of increased degradation), passed on from the parental LCLs to the recipient cybrids. PAH is a complex disease, and mutation status alone doesn’t determine disease susceptibility. LCLs from patients recapitulate some of the metabolomic abnormalities in lung vascular cells. Oxidative stress in LCLs extends to some unaffected relatives, suggesting this is an independent genetic trait that modifies PAH risk. Our study highlights the importance of identifying potential modifiers and the second hits in the pathogenesis of PAH.Item Diagnosis and Treatment of Right Heart Failure in Pulmonary Vascular Diseases: A National Heart, Lung, and Blood Institute Workshop(American Heart Association, 2021) Leopold, Jane A.; Kawut, Steven M.; Aldred, Micheala A.; Archer, Stephen L.; Benza, Ray L.; Bristow, Michael R.; Brittain, Evan L.; Chesler, Naomi; DeMan, Frances S.; Erzurum, Serpil C.; Gladwin, Mark T.; Hassoun, Paul M.; Hemnes, Anna R.; Lahm, Tim; Lima, Joao A. C.; Loscalzo, Joseph; Maron, Bradley A.; Mercer Rosa, Laura; Newman, John H.; Redline, Susan; Rich, Stuart; Rischard, Franz; Sugeng, Lissa; Tang, W. H. Wilson; Tedford, Ryan J.; Tsai, Emily J.; Ventetuolo, Corey E.; Zhou, YouYang; Aggarwal, Neil R.; Xiao, Lei; Medicine, School of MedicineRight ventricular dysfunction is a hallmark of advanced pulmonary vascular, lung parenchymal, and left heart disease, yet the underlying mechanisms that govern (mal)adaptation remain incompletely characterized. Owing to the knowledge gaps in our understanding of the right ventricle (RV) in health and disease, the National Heart, Lung, and Blood Institute (NHLBI) commissioned a working group to identify current challenges in the field. These included a need to define and standardize normal RV structure and function in populations; access to RV tissue for research purposes and the development of complex experimental platforms that recapitulate the in vivo environment; and the advancement of imaging and invasive methodologies to study the RV within basic, translational, and clinical research programs. Specific recommendations were provided, including a call to incorporate precision medicine and innovations in prognosis, diagnosis, and novel RV therapeutics for patients with pulmonary vascular disease.Item DNA Damage and Repair in Pulmonary Arterial Hypertension(MDPI, 2020-10-19) Sharma, Samantha; Aldred, Micheala A.; Medicine, School of MedicinePulmonary arterial hypertension (PAH) is a complex multifactorial disease with both genetic and environmental dynamics contributing to disease progression. Over the last decade, several studies have demonstrated the presence of genomic instability and increased levels of DNA damage in PAH lung vascular cells, which contribute to their pathogenic apoptosis-resistant and proliferating characteristics. In addition, the dysregulated DNA damage response pathways have been indicated as causal factors for the presence of persistent DNA damage. To understand the significant implications of DNA damage and repair in PAH pathogenesis, the current review summarizes the recent advances made in this field. This includes an overview of the observed DNA damage in the nuclear and mitochondrial genome of PAH patients. Next, the irregularities observed in various DNA damage response pathways and their role in accumulating DNA damage, escaping apoptosis, and proliferation under a DNA damaging environment are discussed. Although the current literature establishes the pertinence of DNA damage in PAH, additional studies are required to understand the temporal sequence of the above-mentioned events. Further, an exploration of different types of DNA damage in conjunction with associated impaired DNA damage response in PAH will potentially stimulate early diagnosis of the disease and development of novel therapeutic strategies.Item Food for Thought: The Emerging Role of Intestinal Microbiota in Pulmonary Arterial Hypertension(American Thoracic Society, 2022) Aldred, Micheala A.; Medicine, School of MedicineItem Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA(Elsevier, 2023) Xiao, Sihao; Kai, Zhentian; Murphy, Daniel; Li, Dongyang; Patel, Dilip; Bielowka, Adrianna M.; Bernabeu-Herrero, Maria E.; Abdulmogith, Awatif; Mumford, Andrew D.; Westbury, Sarah K.; Aldred, Micheala A.; Vargesson, Neil; Caulfield, Mark J.; Genomics England Research Consortium; Shovlin, Claire L.; Medicine, School of MedicineDespite whole-genome sequencing (WGS), many cases of single-gene disorders remain unsolved, impeding diagnosis and preventative care for people whose disease-causing variants escape detection. Since early WGS data analytic steps prioritize protein-coding sequences, to simultaneously prioritize variants in non-coding regions rich in transcribed and critical regulatory sequences, we developed GROFFFY, an analytic tool that integrates coordinates for regions with experimental evidence of functionality. Applied to WGS data from solved and unsolved hereditary hemorrhagic telangiectasia (HHT) recruits to the 100,000 Genomes Project, GROFFFY-based filtration reduced the mean number of variants/DNA from 4,867,167 to 21,486, without deleting disease-causal variants. In three unsolved cases (two related), GROFFFY identified ultra-rare deletions within the 3' untranslated region (UTR) of the tumor suppressor SMAD4, where germline loss-of-function alleles cause combined HHT and colonic polyposis (MIM: 175050). Sited >5.4 kb distal to coding DNA, the deletions did not modify or generate microRNA binding sites, but instead disrupted the sequence context of the final cleavage and polyadenylation site necessary for protein production: By iFoldRNA, an AAUAAA-adjacent 16-nucleotide deletion brought the cleavage site into inaccessible neighboring secondary structures, while a 4-nucleotide deletion unfolded the downstream RNA polymerase II roadblock. SMAD4 RNA expression differed to control-derived RNA from resting and cycloheximide-stressed peripheral blood mononuclear cells. Patterns predicted the mutational site for an unrelated HHT/polyposis-affected individual, where a complex insertion was subsequently identified. In conclusion, we describe a functional rare variant type that impacts regulatory systems based on RNA polyadenylation. Extension of coding sequence-focused gene panels is required to capture these variants.Item Identification and validation of a novel pathogenic variant in GDF2 (BMP9) responsible for hereditary hemorrhagic telangiectasia and pulmonary arteriovenous malformations(Wiley, 2022) Balachandar, Srimmitha; Graves, Tamara J.; Shimonty, Anika; Kerr, Katie; Kilner, Jill; Xiao, Sihao; Slade, Richard; Sroya, Manveer; Alikian, Mary; Curetean, Emanuel; Thomas, Ellen; McConnell, Vivienne P. M.; McKee, Shane; Boardman-Pretty, Freya; Devereau, Andrew; Fowler, Tom A.; Caulfield, Mark J.; Alton, Eric W.; Ferguson, Teena; Redhead, Julian; McKnight, Amy J.; Thomas, Geraldine A.; Genomics England Research Consortium; Aldred, Micheala A.; Shovlin, Claire L.; Medicine, School of MedicineHereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant multisystemic vascular dysplasia, characterized by arteriovenous malformations (AVMs), mucocutaneous telangiectasia and nosebleeds. HHT is caused by a heterozygous null allele in ACVRL1, ENG, or SMAD4, which encode proteins mediating bone morphogenetic protein (BMP) signaling. Several missense and stop-gain variants identified in GDF2 (encoding BMP9) have been reported to cause a vascular anomaly syndrome similar to HHT, however none of these patients met diagnostic criteria for HHT. HHT families from UK NHS Genomic Medicine Centres were recruited to the Genomics England 100,000 Genomes Project. Whole genome sequencing and tiering protocols identified a novel, heterozygous GDF2 sequence variant in all three affected members of one HHT family who had previously screened negative for ACVRL1, ENG, and SMAD4. All three had nosebleeds and typical HHT telangiectasia, and the proband also had severe pulmonary AVMs from childhood. In vitro studies showed the mutant construct expressed the proprotein but lacked active mature BMP9 dimer, suggesting the mutation disrupts correct cleavage of the protein. Plasma BMP9 levels in the patients were significantly lower than controls. In conclusion, we propose that this heterozygous GDF2 variant is a rare cause of HHT associated with pulmonary AVMs.Item Mutational and phenotypic characterization of hereditary hemorrhagic telangiectasia(American Society of Hematology, 2020-10-22) Shovlin, Claire L.; Simeoni, Ilenia; Downes, Kate; Frazer, Zoe C.; Megy, Karyn; Bernabeu-Herrero, Maria E.; Shurr, Abigail; Brimley, Jennifer; Patel, Dilipkumar; Kell, Loren; Stephens, Jonathan; Turbin, Isobel G.; Aldred, Micheala A.; Penkett, Christopher J.; Ouwehand, Willem H.; Jovine, Luca; Turro, Ernest; Medicine, School of MedicineHereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Care delivery for HHT patients is impeded by the need for laborious, repeated phenotyping and gaps in knowledge regarding the relationships between causal DNA variants in ENG, ACVRL1, SMAD4 and GDF2, and clinical manifestations. To address this, we analyzed DNA samples from 183 previously uncharacterized, unrelated HHT and suspected HHT cases using the ThromboGenomics high-throughput sequencing platform. We identified 127 rare variants across 168 heterozygous genotypes. Applying modified American College of Medical Genetics and Genomics Guidelines, 106 variants were classified as pathogenic/likely pathogenic and 21 as nonpathogenic (variant of uncertain significance/benign). Unlike the protein products of ACVRL1 and SMAD4, the extracellular ENG amino acids are not strongly conserved. Our inferences of the functional consequences of causal variants in ENG were therefore informed by the crystal structure of endoglin. We then compared the accuracy of predictions of the causal gene blinded to the genetic data using 2 approaches: subjective clinical predictions and statistical predictions based on 8 Human Phenotype Ontology terms. Both approaches had some predictive power, but they were insufficiently accurate to be used clinically, without genetic testing. The distributions of red cell indices differed by causal gene but not sufficiently for clinical use in isolation from genetic data. We conclude that parallel sequencing of the 4 known HHT genes, multidisciplinary team review of variant calls in the context of detailed clinical information, and statistical and structural modeling improve the prognostication and treatment of HHT.Item Mutations causing premature termination codons discriminate and generate cellular and clinical variability in HHT(American Society of Hematology, 2024) Bernabéu-Herrero, Maria E.; Patel, Dilipkumar; Bielowka, Adrianna; Zhu, JiaYi; Jain, Kinshuk; Mackay, Ian S.; Chaves Guerrero, Patricia; Emanuelli, Giulia; Jovine, Luca; Noseda, Michela; Marciniak, Stefan J.; Aldred, Micheala A.; Shovlin, Claire L.; Medicine, School of MedicineFor monogenic diseases caused by pathogenic loss-of-function DNA variants, attention focuses on dysregulated gene-specific pathways, usually considering molecular subtypes together within causal genes. To better understand phenotypic variability in hereditary hemorrhagic telangiectasia (HHT), we subcategorized pathogenic DNA variants in ENG/endoglin, ACVRL1/ALK1, and SMAD4 if they generated premature termination codons (PTCs) subject to nonsense-mediated decay. In 3 patient cohorts, a PTC-based classification system explained some previously puzzling hemorrhage variability. In blood outgrowth endothelial cells (BOECs) derived from patients with ACVRL1+/PTC, ENG+/PTC, and SMAD4+/PTC genotypes, PTC-containing RNA transcripts persisted at low levels (8%-23% expected, varying between replicate cultures); genes differentially expressed to Bonferroni P < .05 in HHT+/PTC BOECs clustered significantly only to generic protein terms (isopeptide-bond/ubiquitin-like conjugation) and pulse-chase experiments detected subtle protein maturation differences but no evidence for PTC-truncated protein. BOECs displaying highest PTC persistence were discriminated in unsupervised hierarchical clustering of near-invariant housekeeper genes, with patterns compatible with higher cellular stress in BOECs with >11% PTC persistence. To test directionality, we used a HeLa reporter system to detect induction of activating transcription factor 4 (ATF4), which controls expression of stress-adaptive genes, and showed that ENG Q436X but not ENG R93X directly induced ATF4. AlphaFold accurately modeled relevant ENG domains, with AlphaMissense suggesting that readthrough substitutions would be benign for ENG R93X and other less rare ENG nonsense variants but more damaging for Q436X. We conclude that PTCs should be distinguished from other loss-of-function variants, PTC transcript levels increase in stressed cells, and readthrough proteins and mechanisms provide promising research avenues.