Identification of Novel Regulatory Genes in Acetaminophen Induced Hepatocyte Toxicity by a Genome-Wide CRISPR/Cas9 Screen

Title from PDF of title page viewed May 10, 2019Dissertation advisor: Shui Qing YeVitaIncludes bibliographical references (pages 154-165)Thesis (Ph.D.)--School of Biological Sciences and School of Medicine. University of Missouri--Kansas City, 2018Acetaminophen (APAP) is a commonly used analgesic responsible for over 56,000 overdose-related emergency room visits annually. A long asymptomatic period and limited treatment options result in a high rate of liver failure, generally resulting in either organ transplant or mortality. The underlying molecular mechanisms of injury are not well understood and effective therapy is limited. Identification of previously unknown genetic risk factors would provide new mechanistic insights and new therapeutic targets for APAP induced hepatocyte toxicity or liver injury. This study used a genome-wide CRISPR/Cas9 screen to evaluate genes that are protective against or cause susceptibility to APAP-induced liver injury. HuH7 human hepatocellular carcinoma cells containing CRISPR/Cas9 gene knockouts were treated with 15mM APAP for 30 minutes to 4 days. A gene expression profile was developed based on the 1) top screening hits, 2) overlap with gene expression data of APAP overdosed human patients, and 3) biological interpretation including assessment of known and suspected APAP-associated genes and their therapeutic potential, predicted affected biological pathways, and functionally validated candidate genes. This screen is the first genome-wide CRISPR/Cas9 knockout screen of APAP induced hepatocyte toxicity. The top hits from this screen included numerous genes previously not linked to liver injury. We further demonstrated the implementation of intermediate time points for the identification of early and late response genes. A negative selection screen identified genes involved in fundamental processes, including NAAA, ATG2B, and MYOZ3. A positive selection screen identified numerous genes potentially involved in pathogenic processes, including LZTR1, PGM5, and EEF1D. A top essential pathway at 24 hours of APAP treatment was Regulation of Skeletal Muscle Contraction. We additionally identified 6 genes, 3 novel and 3 known, that have drug-gene interactions favorable for re-purposing existing therapies to treat APAP-induced hepatotoxicity. Collectively, this line of research has illustrated the power of a genome-wide CRISPR/Cas9 screen to systematically identify novel genes involved in APAP induced hepatocyte toxicity and to provide potential new targets to develop novel therapeutic modalities.Introduction -- Review of the literature -- Research question -- Methods -- Results and discussion part 1: CRISPR/Cas9 screen -- Results and discussion part 2: our screen in the context of other acetaminophen data sets -- Results and discussion part 3: acetaminophen-associated single nucleotide polymorphisms in the literature -- Results and discussion Part 4: validation of top candidate genes -- Conclusions and future directions -- Appendix A. Supplementary figures -- Appendix B. Supplementary table

Identification of novel coding single nucleotide polymorphisms associated with acute respiratory distress syndrome

Title from PDF of title page, viewed on July 28, 2014Thesis advisor: Shui Qing YeVitaIncludes bibliographical references (pages 59-61)Thesis (M. S.)--School of Medicine. University of Missouri--Kansas City, 2014ARDS is a lung condition characterized by impaired gas exchange with systemic release of inflammatory mediators, causing inflammation, hypoxemia and multiple organ failure. Disease susceptibility and progression are poorly understood and there are few effective therapeutic options. Existing biomarkers have limited effectiveness as diagnostic and therapeutic targets. Whole-exome sequencing is an effective tool in detection of disease-causing genetic variants in complex genetic conditions such as acute respiratory distress syndrome (ARDS). To identify disease-causing variants in ARDS patients, whole-exome sequencing was performed on 96 patient DNA samples from the National Heart, Lung and Blood Institute's ARDS Network (ARDSnet). By comparing these exome data with 625 participants of the 1000 Genomes Project, we have tentatively identified a number of single nucleotide polymorphisms (SNP) which are potentially associated with ARDS. In this study, we validated three SNPs (rs78142040, rs9605146, and rs3848719) in an additional 117 ARDS patients using TaqMan SNP genotyping assays (Life Technologies) to substantiate their associations with the susceptibility, severity and outcome of ARDS. rs78142040 (C>T) occurs within a histone mark in intron 6 of the Arylsulfatase D gene. rs9605146 (G>A)(also known as rs114989947) causes a coding change (proline to leucine) with a deleterious effect in the XK, Kell blood group complex subunit-related family, member 3 gene. rs3848719 (G>A) is a synonymous SNP in exon 5 of gene Zinc-Finger/Leucine-Zipper Co-Transducer NIF1. rs78142040 and rs9605146 are significantly associated with susceptibility to ARDS[minor allele frequency (MAF): 0.219 versus 0.003 (control), p1×10-4) in our cases. These 3 SNPs have not been previously associated with ARDS and represent potential new genetic biomarkers for ARDS. More validations in larger patient populations and further exploration of underlying molecular mechanisms are warrantedAbstract -- List of illustrations -- List of tables -- Glossary -- Acknowledgments -- Introduction - Review of the literature -- Research question -- Methods -- Results -- Discussion -- Conclusions -- Supplementary figures and tables -- Reference lis

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types

A first update on mapping the human genetic architecture of COVID-19

peer reviewe

COVID-19 Host Genetics Initiative. A first update on mapping the human genetic architecture of COVID-19

The COVID-19 pandemic continues to pose a major public health threat, especially in countries with low vaccination rates. To better understand the biological underpinnings of SARS-CoV-2 infection and COVID-19 severity, we formed the COVID-19 Host Genetics Initiative1. Here we present a genome-wide association study meta-analysis of up to 125,584 cases and over 2.5 million control individuals across 60 studies from 25 countries, adding 11 genome-wide significant loci compared with those previously identified2. Genes at new loci, including SFTPD, MUC5B and ACE2, reveal compelling insights regarding disease susceptibility and severity.</p

Whole-genome sequencing reveals host factors underlying critical COVID-19

Altres ajuts: Department of Health and Social Care (DHSC); Illumina; LifeArc; Medical Research Council (MRC); UKRI; Sepsis Research (the Fiona Elizabeth Agnew Trust); the Intensive Care Society, Wellcome Trust Senior Research Fellowship (223164/Z/21/Z); BBSRC Institute Program Support Grant to the Roslin Institute (BBS/E/D/20002172, BBS/E/D/10002070, BBS/E/D/30002275); UKRI grants (MC_PC_20004, MC_PC_19025, MC_PC_1905, MRNO2995X/1); UK Research and Innovation (MC_PC_20029); the Wellcome PhD training fellowship for clinicians (204979/Z/16/Z); the Edinburgh Clinical Academic Track (ECAT) programme; the National Institute for Health Research, the Wellcome Trust; the MRC; Cancer Research UK; the DHSC; NHS England; the Smilow family; the National Center for Advancing Translational Sciences of the National Institutes of Health (CTSA award number UL1TR001878); the Perelman School of Medicine at the University of Pennsylvania; National Institute on Aging (NIA U01AG009740); the National Institute on Aging (RC2 AG036495, RC4 AG039029); the Common Fund of the Office of the Director of the National Institutes of Health; NCI; NHGRI; NHLBI; NIDA; NIMH; NINDS.Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care or hospitalization after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes-including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)-in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease