WES ON GASTRO-ESOPHAGEAL TUMORS TO IDENTIFY BIOMARKERS OF RESPONSE TO EGFR INHIBITION
DAC to regulate access to Olink targeted proteomics data in Chronic rhinosinusitis (CRS) patient cohort.
This study includes 1,220 cases with young onset stroke (stroke before age 60 years) who are participants of the larger RACE study. Risk Assessment of Cerebrovascular Events (RACE) is an on-going existing case-control study of stroke now involving over 5000 imaging confirmed cases of stroke and 5000 controls, recruited from seven centers in Pakistan. The study is aimed to investigate the genetic, biomarker and lifestyle determinants of stroke and its subtypes. Cases are eligible for inclusion in the study if they: (i) are aged at least 18 years; (ii) present with a sudden onset of neurological deficit respecting a vascular territory with sustained deficit at 24 hours verified by medical attention within 72 hours after onset (onset is defined by when the patient was last seen normal and not when found with deficit); and (iii) the diagnosis is supported by CT/MRI; and (iv) present with a Modified Rankin Score < 2 prior to the stroke. Findings from patient's history, 12-lead ECG and CT or MRI of the brain. The mandatory procedures for inclusion in this investigation are: (i) clinical verification of cerebrovascular event within 72 hours of onset; (ii) neuroimaging CT (non-contrast) or MRI (MRI is not a mandatory investigation but recorded whenever ordered by the attending physician); and (iii) 12-lead ECG. All other ancillary investigations ordered by the attending physician are recorded as well. The TOAST classification method is used to classify ischemic stroke based on aetiology whereas the Oxfordshire classification is used to classify stroke neuro-anatomically. Control participants for this subset of young onset stroke were individuals enrolled in the Pakistan Risk of Myocardial Infarction Study (PROMIS), a case-control study of acute MI based in Pakistan. RACE capitalizes on the genetic data (including information on GWAS) that has already been collected from the healthy participants enrolled in PROMIS. RACE and PROMIS share similar methodology of recruitment. Participants from both these investigations are derived from similar catchment areas, hence providing an attractive opportunity for RACE to utilize PROMIS controls as common controls for genetic investigations. Controls in PROMIS were recruited following procedures and inclusion criteria as adopted for RACE cases. In order to minimize any potential selection biases, PROMIS controls selected for this stroke substudy were frequency matched to RACE cases based on age and gender and were recruited in the following order of priority: (1) non-blood related or blood related visitors of patients of the out-patient department; (2) non-blood related visitors of stroke patients; (3) patients of the out-patient department presenting with minor complaints (e.g. back pain, minor gastric complaints). Control subjects from the PROMIS study were genotyped at the Wellcome Trust Sanger Institute on the Illumina 660W Quad array. The Center for Non-Communicable Diseases, Pakistan, serves as the coordinating center for both RACE and PROMIS. More information on these research investigations can be found at www.cncdpk.com. This young onset stroke component to the RACE study was funded through the Gene Environment Association Studies initiative (GENEVA, www.genevastudy.org as one of three studies designed to assess the genetics of young onset stroke and modification of genetic effects by smoking. GENEVA is part of the trans-NIH Genes, Environment, and Health Initiative (GEI). Genotyping of 1,220 young onset stroke cases was performed at the Johns Hopkins University Center for Inherited Disease Research (CIDR). Data cleaning and harmonization were done at the GEI-funded GENEVA Coordinating Center at the University of Washington. This study is part of the Gene Environment Association Studies initiative (GENEVA, http://www.genevastudy.org) funded by the trans-NIH Genes, Environment, and Health Initiative (GEI). The overarching goal is to identify novel genetic factors that contribute to stroke through large-scale genome-wide association studies of cases and controls recruited within Pakistan. Genotyping was performed at the Johns Hopkins University Center for Inherited Disease Research (CIDR). Data cleaning and harmonization were done at the GEI-funded GENEVA Coordinating Center at the University of Washington.
Myocardial ischemia occurs when there is a mismatch between coronary oxygen delivery and metabolic requirements of the myocardium, which may be clinically manifested during angina, coronary angioplasty or cardiopulmonary bypass (CPB). Myocardial ischemia may lead to a spectrum of myocardial stunning, hibernating myocardium, and ultimately cell death if the ischemic insult is severe. In the human heart, irreversible damage begins after approximately 20 to 40 minutes of oxygen deprivation. Observed molecular and cellular changes of myocardial ischemia are characteristic of an inflammatory response, but the exact mechanisms that underlie this pathological process are unclear and may not be full emulated by animal models of ischemia or infarction. Thus, we felt it valuable to investigate a human ischemia model. During cardiac surgery, CPB with aortic cross-clamping (AoXC) and cardioplegic arrest is associated with excellent clinical outcomes and suitable operative conditions. However, despite the use of cardioprotective strategies, AoXc during CPB is accompanied by a variable, yet obligate ischemic period lasting 1 to 3 hours, resulting in hypoxia, metabolic substrate depletion, reperfusion injury, apoptosis, and necrosis. Cardiac specific biomarkers of ischemia and infarction, including troponin, are elevated even after routine coronary artery bypass graft surgery and correlate with the duration of ischemia from AoXc.This process of CPB provides us with the ability to examine the transcriptional profile before and after an expected, consistent, and reproducible human ischemic event, albeit induced by cold cardioplegic arrest and not coronary occlusion. In addition, the absence of reperfusion in this time period allows us to examine the transcriptomic response to intermittent ischemia, without having to account for the perturbations of reperfusion injury. Although various animal models have been used to examine the effects of ischemia on cardiac function, no human data exist which examine the early transcriptomic response to a left ventricular (LV) ischemic insult. We therefore characterized the effect of cold cardioplegia induced acute ischemia on the transcriptional profile of the LV by performing whole transcriptome next-generation RNA-sequencing (RNA-seq) in patients undergoing cardiac surgery by sampling human LV tissue prior to, and after, the obligate ischemia during AoXC. We hypothesized that the cold cardioplegia induced ischemic injury will dramatically alter transcription in the human myocardium, and that we would identify genes and pathways, which will identify interventional targets for pharmacological therapy. Methods:We have collected left ventricle tissue samples and blood sample from patients undergoing heart surgery. We obtained punch biopsies (~3-5μg total RNA content) from the site of a routinely placed surgical vent in the anterolateral apical left ventricular wall of patients undergoing elective aortic valve replacement surgery with cardiopulmonary bypass. After an average of 79 minutes of aortic cross-clamping with intermittent cold blood cardioplegia for myocardial protection every 20 minutes, a second biopsy was obtained in the same manner. Tissue samples were immediately placed in RNAlater® (Ambion, ThermoFisher Scientific, Waltham, MA), and after 48 hours at +4°C were stored at -80°C until RNA extraction. Total RNA was isolated with Trizol and RNA quality was assessed using the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). Libraries were prepared by poly(A) mRNA isolation and reverse transcription Polymerase Chain Reaction (RT-PCR), then sequenced on the Illumina HiSeq2000 or HiSeq2500 (Illumina, San Diego, CA). As samples were analyzed at different times, different read lengths were employed, initially using single-end reads (n=20) and then transitioning to paired end reads (n=216), ranging from 36 - 100 base pairs. Raw sequencing files were processed using Sickle, Skewer, and STAR software, and aligned to GrCh37 or UCSC Hg19. DNA was isolated from whole blood using standard methods. SNP genotyping was performed using the Illumina Omni2.5Exome-8 BeadChip array with additional exome content (Illumina, San Diego, CA) chip, version 1.1. We first phased and imputed 93 subjects using a phasing tool called SHAPEIT and an imputation tool called MINIMAC, with 1000 Genomes phase 1 version 3 for the reference panel. We then phased and imputed 26 more subjects using SHAPEIT, an imputation tool called IMPUTE2, and 1000 Genomes phase 3 version 5.
