Genetic, Genomic, Proteiomic and Bioinformatic Studies of Acute Lung Injury and Pulmonary Arterial Hypertension

Proteome-Genome (PG) Core for Acute Respiratory Distress Syndrome (ARDS)

The PG Core is one of three research cores within the Garcia Lab at the University of Arizona Health Sciences (UAHS) and it includes several investigators from the Division of Translational and Regenerative Medicine (Drs. Jason Yuan, Ankit Desai, Stephen Black, Xiaoguang Sun, and Saad Samanni). The PG Core provides essential, cutting edge, proteomic, transcriptomic and genetic expertise to facilitate the translational impact of the integrative studies on acute respiratory distress syndrome (ARDS), ventilator-induced lung injury (VILI) and inflammation-induced lung injury. The Core is responsible for providing the research investigators with a well characterized ARDS biobank (i.e. DNA, serum, plasma) for three ethnic groups (European, African, and Hispanic descent), a complete list of tagging single nucleotide polymorphisms (tSNPs) for each various candidate genes, mid-throughput genotyping services, and data analysis tools to test for association between target SNPs and susceptibility and severity of ARDS. The functional non-synonymous coding SNPs will be screened in silico to identify their impact on protein structure and function (protein-protein interaction, catalytic activity, post-translational modification) by computational protein structure molecular modeling, with protein-protein interaction validated by surface plasmon resonance screening (SPR). Effects of exon-intron boundary SNPs on alternative splicing of these key genes will be analyzed by in silico prediction and validated by SNP-containing minigene constructs. The Core is also responsible for the isolation of good quality and quantity of total RNA from lung tissues or cell cultures of ARDS in vitro/in vivo models, and design a novel exon microarray precisely targeting key disease or function related spliceable exons in the genes studied by investigators in the field of ARDS, in order to characterize the splicing patterns of the signal molecules in the pathway of endothelial cytoskeleton regulation. The Core provides the service to generate the mutated DNA constructs (with disease-associated SNP) to generate recombinant protein used in SPR, or GRF-labeled protein for cellular location tracking in endothelial cells.  In addition, the Core validates the genomic expression data by proteomic approaches (2-D protein gel and mass spectrometry). On top of the protein level characterization, the Core defines the protein translational modification (PTM) status including phosphorylation, nitration, and ubiquitination via traditional mass spectrometry analysis. The Core also utilizes up-to-date technologies to provide a proteomic-genomic study platform for all the Projects on ARDS and generate novel information on the genes targeted for investigation in studying pathogenic and therapeutic mechanisms of ARDS and VILI. The proposed aims of the Proteome-Genome Core are: 1) to provide an efficient/cost-effective mid-throughput genotyping services to project investigators; 2) to provide an integrated laboratory facility to support gene expression and genotyping of samples from well-characterized patients, in vitro/in vivo models of ARDS, experiment and project management, primer and assay design, and computational/analytical activities associated with genomic and genetic data; 3) to provide proteomic analysis of samples (in vitro/in vivo) to characterize protein expression and post-translational modification; and 4) to test and validate the association between patients (or experimental groups) and controls in the prioritized candidate genes from each Project.

NHLBI Pulmonary Vascular Disease Phenomics (PVDOMICS) Program

A new research consortium, entitled “Redefining Pulmonary Hypertension through Pulmonary Vascular Disease Phenomics” (PVDOMICS) Program, was established and funded in 2014 by the National Heart, Lung, and Blood Institute (NHLBI) at the National Institutes of Health (NIH). The purpose of this initiative was to select highly-active and highly-qualified clinical centers and investigators to participate in the PVDOMICS program to perform comprehensive, deep phenotyping across the current WHO-classified pulmonary hypertension (clinical Groups 1 through 5). The PVDOMICS consortium includes six clinical centers and one data coordinating center:

  • Brigham and Women’s Hospital, Boston, MA
  • Columbia University and Cornell University, New York, NY
  • Johns Hopkins University, Baltimore, MD
  • Mayo Clinics, Rochester, MN
  • University of Arizona, Tucson, AZ
  • Vanderbilt University, Nashville, TN
  • Cleveland Clinic, Cleveland, OH (data coordinating center)

The University of Arizona Health Sciences is one of the NHLBI-funded clinical centers (U01 HL125208) designed to integrate physiologic, genomic and epigenetic strategies to sub-phenotype Latino and non-Latino patients with pulmonary hypertension. Our genome wide association studies and whole genome sequencing to identify novel SNPs in Latinos with PAH provides a powerful genomics research hob and training venue for highly translational studies to potentially generate novel category-specific biomarkers, identifying groups at high-risk for PH, and for personalizing PH therapies.

Pulmonary hypertension (PH) is a debilitating and often fatal disease for which there is currently no cure. PH is characterized by sustained pulmonary vasoconstriction and severe vascular remodeling that produce a progressive rise in pulmonary vascular resistance (PVR) and pulmonary arterial pressures (PAP), and results in an abnormal right ventricular (RV) workload, RV failure and death. Current treatment options may limit clinical deterioration but fail to retard or reverse PH vascular remodeling. Less well known are environmental and genetic factors that differentially drive susceptibilities of specific population cohorts to the development of PH and responses to PH therapy. This information gap is exacerbated by the perplexing assortment of clinical characteristics, histo-pathological entities and responses to therapy that constitute the 5 Groups within the current classification of patients with PH: i) pulmonary arterial hypertension (PAH); ii) PH with left heart disease; iii) PH associated with lung diseases and/or hypoxemia; iv) PH attributed to chronic thrombotic and/or embolic disease or chronic thromboembolic pulmonary hypertension (CTEPH); and v) miscellaneous. While providing a framework for treatment, the current classification has proven to have serious limitations in clinical practice. Many patients with PH, in groups other than Group 1 (PAH), succumb to complications of pulmonary vascular disease (such as RV failure) and patients in Groups 2 and 3 may benefit from more rigorous evaluation when PH is "out of proportion" to their underlying cardiac or pulmonary disease. This is true for African American sickle cell disease patients who develop and succumb to severe PH. Information on PH incidence and severity in Latinos is largely unknown; however, at the University of Arizona (UA), a major regional referral center for treatment of patients with all forms of PH, Latinos constitute 25-50% of PH patients depending on the current classification. Clearly highly specific approaches to clinical phenotyping of PH patients are desperately needed. In prior studies, we have employed highly translational approaches to sub-phenotype patients with complex lung disorders such as sarcoidosis, idiopathic pulmonary fibrosis, and lung transplant rejection. In addition, we have conducted genomic and epigenetic sub-phenotyping studies in patients with PAH, in African Americans with sickle cell disease and in CTEPH patients. These studies, utilizing genome-wide gene expression, miRNA arrays, and DNA methylation arrays, allowed the generation of molecular signatures as biomarkers and phenotyping tools in PH patients. Based on these compelling studies, we propose three Specific Aims to employ the-state-of-the-art physiological, genomic and epigenetic strategies to sub-phenotype Latino and non-Latino PH patients across the 5 Groups of PH categories seen at the University of Arizona Medical Center (UAMC): 1) to phenotype UA Latino and non-Latino PH patients using integrated physiological signatures derived from ventriculo-vascular coupling (VVC) and pulmonary vascular impedance measurements; 2) to generate genome-wide, genomic/epigenetic signatures (gene, miRNA, DNA methylation arrays) in peripheral blood mononuclear cells (PBMC) to sub-phenotype UA PH patients across 5 PH Groups; and 3) to utilize whole-genome sequencing to identify novel single nucleotide polymorphisms (SNPs) modulating UA PH sub-phenotypes and SNPs over-represented in Latino PH patients.

The Pulmonary Hypertension Program at UAMC, led by Dr. Franz Rischard, is a leading regional center for patients with pulmonary vascular disease with a large referral base and good relationship with referring physicians. Dr. Rischard and his team have been tracking new visits and performing right heart catheterizations since 2010. Based on the history and data for 2013-2014, Dr. Rischard’s team anticipates an annual growth of 75-100 patients with PH. Dr. Rischard (Co-PI in this study) is a gifted clinician scientist with substantial experience in phenotyping PH patients (145 specialized right heart catheterizations, RHCs) last year including 35 formal exercise catheterizations and assisted in 10 pulmonary angiograms. UAMC enjoys extensive referrals to the PH program including patients with heart failure, adult congenital heart disease, interstitial lung disease, liver transplantation, infectious disease (HIV), sleep apnea, and COPD. In addition, Dr. Rischard is a member of the adult congenital heart disease program with an outreach clinic to the Apache Nation. The UAMC PH program (including dedicated PH nurse coordinator, research nurse), has a thorough and efficient process for phenotyping including specialized echocardiography protocols, cardiopulmonary exercise testing and pulmonary function testing, advanced catheterization techniques (exercise catheterization, pulmonary angiography), cardiac MRI and other standard imaging techniques. UAMC has an existing database with >70 Group I PH (PAH) patients. Dr. Zain Khalpey (Cardiothoracic Surgery and Co-I) receives lung explants from regional transplantation centers for research and analysis available for tissue and physiologic measurements. Finally, the UAMC PH team currently participates in 9 PH clinical trials with ~ 50 patients enrolled annually. The large UA pool of diverse PH patients, large PH referral base, comprehensive UA Bio-Bank initiative, and prior experience with these phenotyping tools, all serve to enhance the feasibility of our novel sub-phenotyping strategy focusing on Latino PH patients. By generating novel Group-specific biomarkers, these studies carry the real potential for increasing personalization of therapy by identifying groups at high-risk for development of PH, for selecting specific therapies for PH patients within a new rational classification paradigm (combination therapy, transplant, surgical referral).