Clinical Retrospective Study And Epigenome-wide DNA Methylation Study Of COVID-19 In Shandong Province

BackgroundSevere acute respiratory syndrome coronavirus type 2(SARS-CoV-2)is a new virus,which caused the outbreak of Corona Virus Disease 2019(COVID-19)in 2019 and is now rapidly spreading around the world.This newly emerged highly pathogenic virus belongs to RNA envelope virus.The receptor binding domain is highly homologous to severe acute respiratory syndrome coronavirus(SARS-CoV)and can lead to death of acute respiratory distress syndrome(ARDS).A few days ago,the cumulative number of people diagnosed with COVID-19 in the world exceeded 170 million,and the death toll exceeded 3.81 million.Several studies in China have systematically expounded the clinical and epidemiological characteristics of COVID-19.At present,the literature shows that the clinical features such as increased lactic acid,lymphocytopenia and elevated D-dimer may indicate a poor prognosis.Other studies have shown that advanced age(? 75 years old),multi-lobe involvement and pleural effusion are positively correlated with the severity of the disease.Identifying the early clinical characteristics of critically ill patients,looking for the best treatment strategies and preventing disease progression are of great significance to reduce mortality and social panic.COVID-19 caused by SARS-CoV-2 has led to a serious global epidemic and brought about worldwide health system challenges.One year after the outbreak of COVID-19,a large number of patients who were clinical cured have been discharged from hospital.As of June 15,2021,China has confirmed a total of 91492 cases of COVID-19,of which 86369 have been cured and discharged from hospital.According to the discharge criteria issued by the National Health Commission,the conditions for discharged patients include no clinical symptoms,improved CT imaging,no fever for more than 3 days and negative reverse transcriptase polymerase chain reaction(RT-PCR)tests at intervals of at least 24 hours.Existing studies have found that after discharge,some recovered patients are returned to hospital again because of a positive SARS-CoV-2 nucleic acid test in nasopharyngeal swabs,feces or sputum samples,that is,the socalled re-detectable positive(RP).It is not known whether RP patients will spread the virus and infect others,which has raised public concern about the existing discharge standards of COVID-19.However,there is a lack of long-term follow-up studies on the infectivity and health status of COVID-19 recovered patients,which has a great impact on the prevention and control of the epidemic.Therefore,there is an urgent need to conduct more investigations on discharged patients.SARS-CoV-2 is highly contagious and may lead to serious clinical outcomes,which requires researchers to explore its biological characteristics and virus-host interaction.Epigenetic modification is involved in a variety of diseases by affecting genomic stability and intracellular homeostasis.In viral infection,epigenetics is also considered to participate in the regulation of pathophysiology and affect the severity of the disease.It has been suggested that RNA viruses,such as coronaviruses,can drive abnormal gene methylation to resist host immune response and contribute to virus replication.Angiotensin converting enzyme 2(ACE2)is the cell receptor of SARS-CoV-2.Recent studies have shown that gene methylation and post-translational histone modification of ACE2 are potential targets for the prevention and treatment of COVID-19.The analysis of the difference of DNA methylation level in COVID-19 patients is helpful to further explore the factors affecting the susceptibility and severity of the disease,and provide new ideas for prevention and accurate treatment for COVID-19.AimIn this study,we review the differences in clinical characteristics between critically ill and critically ill patients,and the long-term follow-up data of 527 patients discharged from COVID-19 in Shandong province for 6 months,and evaluated the clinical characteristics,especially the level of antibody,to find out the risk factors of disease progression,and to further improve the discharge criteria.Genome-wide methylation microarray analysis was carried out in 14 mild and severe COVID-19 patients to find potential biomarkers and therapeutic targets.We aim to help clinicians treat critically ill COVID-19 more effectively and to improve the management strategy.Methods1.Clinical features for severely and critically ill patients with COVID-19 in Shandong:a retrospective cohort studya)The retrospective cohort included 62 critically ill and critically ill COVID-19 patients(35 males and 27 females)who were admitted to Shandong COVID-19 designated Hospital(including Shandong Thoracic Hospital,etc.)from January 20 to March 12.The severity ofthe disease was classified according to guideline for diagnosis and treatment of COVID-19(version 8.0)issued by the National Health Commission.b)Through the electronic medical system,the routine medical records,nursing records,laboratory examination,imaging examination and microbiological examination of all patients were collected.c)Sequential organ failure assessment score(SOFA)and acute physiology and chronic health score system(APACHEII)were used to evaluate the patient's condition.d)The differences of clinical parameters were analyzed by Mann Whitney U test,chi-square test or Fisher's exact test.Univariate and multivariate logistic regression analysis of age,underlying diseases,lymphocyte count,Apache II score and SOFA score was conducted to explore the risk factors related to the progression of severe COVID-19 patients.2.Characteristics of 527 discharged COVID-19 patients undergoing long-term follow-upa)This study included 527 discharged COVID-19 patients(male 294,female 233)treated in COVID-19 designated Hospital of Shandong province from January 21 to February 16,2020.A re-positive patient is defined as a patient who returns to hospital after being re-detectable positive for nucleic acid in pharyngeal swabs,anal swabs,feces,or sputum samples.The severity of the disease was graded according to COVID-19 's guidelines for diagnosis and treatment(version 5.0-8.0).b)Review the medical records of all patients,including baseline data,medical records and CT imaging results in the electronic medical system.c)The samples for SARS-CoV-2 PCR test were taken from pharynx swab,anal swab,sputum and fecal samples.According to the the 2nd edition of COVID19 Laboratory testing Technology Guide.ORF1ab/N gene nucleic acid detection kit was used for PCR detection.d)The differences of clinical parameters were calculated by Mann-Whitney U test,chi-square test or Fisher exact test.SPSS version 23.0 was used for statistical analysis.3.An epigenome-wide DN A methylation study of patients with COVID-19a)This study included 14 inpatients diagnosed with COVID-19 in designated hospital in Shandong Province from January 27,2020 to May 18,2020,including 7 mild patients,7 severe patients and 7 healthy controls.Blood samples were collected before discharge.All the subjects were not related by blood.The severity of the disease was graded according to COVID-19 Management Guide(version 5.0-8.0).b)Genomic DNA was extracted from peripheral blood using QIAamp blood DNA extraction kit.gDNA was obtained after bisulfite transformation.Illumina Infinium Methylation EPIC BeadChip(850K chip)was used to scan the global genome to screen differential methylation loci.c)The signal value was extracted from the original data using a genome studio(genomic software 2011.1,Illumina),and the resulting data were normalized by performing background correction using the methylation module.d)Differential methylation sites were analyzed.Diffscore<-13 or>13 and ??>0.17 or<-0.17 suggesting that it was a differential methylation site.e)Unsupervised cluster analysis of differential methylation sites was carried out by using Rstudio software.f)VENN analysis further screened the marker differential methylation gene(DMGs).g)Functional enrichment(GO)and pathway enrichment(KEGG)analysis of differentially methylated genes were performed to clarify the functional significance of differentially methylated genes in biological processes,signal transduction and immune system.P<0.05 is considered to be significant.h)The interaction network based on differentially methylated genes was constructed by Cytoscape,and the key genes were screened by Cytohubba plugin.i)The data expression profiles of COVID-19 patients were searched in the GEO database for methylation and transcriptome analysis.Adj.P.Value<0.05 was considered to be statistically significant.Results1.Clinical features for severely and critically ill patients with COVID-19 in Shandong:a retrospective cohort studya)Of all 62 critically ill patients,56%were male,with a median age of 56.5 years;30 developed critically ill,with a median age of 63.0 years;and 7 died.73%of the diagnosed patients had a clear epidemiological contact history.b)The most common clinical symptoms were fever(97%)and cough(71%).The initial symptoms of 15 patients(24%)were not fever,and the median time from onset to fever was 3 days.c)30 patients(48%)had underlying diseases,of which cardiovascular disease was the most common(34%of hypertension and 5%of coronary heart disease).d)There were significant differences in white blood cell count,hemoglobin,lymphocyte count,D-dimer and procalcitonin levels between severe patients and critically ill patients.e)Chest CT scan changes in critically ill patients included bilateral lung involvement(98%),patchy shadow(65%),ground glass shadow(63%)and consolidation(47%).f)The median APACHEII score and SOFA score were 8.0 and 3.0,respectively.ARDS is the most common complication,and critically ill patients usually have a poor prognosis.g)Univariate regression analysis showed that aging,underlying diseases,high SOFA and APACHEII scores,and lymphocyte count<109/L at admission were risk factors for disease progression.Multivariate Logistic regression analysis showed that the lymphocyte count<109/L at admission was significantly correlated with the severity of the disease.2.Characteristics of 527 discharged COVID-19 patients undergoing long-term follow-upa)Of the 527 discharged patients,32(6.1%)were re-detectable positive for SARSCoV-2 nucleic acid during follow-up.11 and 4 were detected negative in respiratory samples,but positive results were obtained from fecal samples and anal swab samples,respectively.b)Adolescents(<18 years)were more likely to be re-detectable positive than other age groups,and the time span was shorter(average 8.8 days[6.0-9.0 days]),while the middle-aged group was on the contrary(average 17.5 days[14.0-17.5 days]).c)The improvement of CT imaging features in RP group was similar to that in non-RP groupd)Three months and six months after discharge,the antibody negative rates were 14.2%and 25.0%,respectively.Cases in which the antibody turned negative in 6 months after discharge were more common in the adolescent group(40%?15.6%).3.An epigenome-wide DNA methylation study of patients with COVID-19a)A total of 214,245,228 differential methylation sites were detected(severe ?control group,severe ? mild group,mild ? control group),of which 114,126,113 sites were hypermethylation sites and 100,119,115 hypomethylation sites,respectively.Abnormal hypermethylation sites were significantly distributed on the CpG shores,while abnormally low methylation sites were significantly distributed on CpG shores and shelves.b)The results of cluster analysis showed that there were significant differences in gene methylation between COVID-19 patients with different severity and healthy controls.c)35 potential "marker" genes for SARS-CoV-2 were screened by Venn crossover analysis,including 12 genes located in the promoter region(ATHL1,CHN2,CHST15,CPLX2,CRHR2,DCAKD,GNAI2,HECW1,HYAL1,MIR510,PDE11A and SMG6).d)GO analysis of differentially methylated genes in severe and mild patients showed that the gene functions were mainly concentrated in neuropeptides,IL13,chemokine secretion,T helper cell differentiation and mast cell degranulation.The significant pathways involved in KEGG pathway analysis include sphingomyelin signal pathway,Hippo signal pathway,phospholipase D signal pathway,hypertrophic cardiomyopathy,oxytocin signal pathway,dilated cardiomyopathy,type I diabetes and 5-hydroxytryptamine pathway.e)Differential methylation genes in severe and mild patients were analyzed by PPI network analysis,and four key genes with the highest degree of interaction were identified(GNG7,GNAS,PRKCZ and PRKAG2).Combined methylation and transcriptional analysis showed that the expression of GNG7 and GNAS was down-regulated in severe patients,which emphasized the importance of GNG7 and GNAS2 in the process of SARS-CoV-2 infection.Conclusions1.The decrease of lymphocyte count at admission(<109/L)was the most important independent risk factor,which was closely related to the progression of critical illness.Aging,underlying diseases(especially hypertension and coronary heart disease),increased D-dimer,decreased hemoglobin,and higher SOFA and APACH scores are also considered as risk factors for disease progression.Blood cell count and procalcitonin level have certain predictive value for consequently bacterial infection.2.In adolescent discharged patients,nucleic acid test is more likely to be re-detectable positive,and negative SARS-CoV-2 antibody is more common in adolescent patients,which may increase the risk of reinfection.Stool samples may be more likely to be re-detectable positive than samples collected from other sources.3.There were significant abnormalities in genome-wide methylation in patients with different severity of COVID-19.35 differentially methylated genes were screened that may predict SARS-CoV-2 susceptibility.Differential methylation genes between severe and mild groups were enriched in a variety of pathways,including inflammatory and immune activation pathways.PPI analysis identified GNG7,GNAS,PRKCZ and PRKAG2 as hub genes,and combined transcriptome analysis found that GNG7 and GNAS genes were down-regulated in severe patients,indicating that GNG7 and GNAS played an important role in the development of COVID-19.