Ignite Creation Date:
2024-05-06 @ 2:44 PM
Last Modification Date:
2024-10-26 @ 1:36 PM
Study NCT ID:
NCT04411563
Status:
UNKNOWN
Last Update Posted:
2020-06-02
First Post:
2020-05-25
Brief Title:
Epigenetic Tools as Prognostic Predictors in Covid19
Sponsor:
Bakirkoy Dr Sadi Konuk Research and Training Hospital
Organization:
Bakirkoy Dr Sadi Konuk Research and Training Hospital
Study Overview
Official Title:
The Effect of miRNA and Epigenetic Modifications on Prognosis in Covid-19 Infection
Status:
UNKNOWN
Status Verified Date:
2020-05
Last Known Status:
RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The necessity for early detection and hence improving the outcome of treatment of pneumonia is critical especially SARS-CoV-2 induced cases This work was designed to evaluate the potential application of measuring circulating epigenetic markers namely miR-744 miR-24 miR-124 miR-155 miR-19a miR-122 miR-21 miR-223 let-7f miR-146 miR-196 miR-136 P13-K miR-9 expression and DNA methylation profiling of the ACE2 TMPRSS2 PARP HOX1 genes in Coronavirus disease 2019 COVID-19 due to SARS-CoV-2 withwithout pneumonia and severe acute respiratory syndrome SARS patients as an attempt to evaluate the potential benefits of these new circulating prognostic epigenetic markers for Turkish patients
Detailed Description:
The objectives of this observational study are 1 To investigate why COVID-19 infection causes different biological behavioral patterns in individuals 2 To determine the role of epigenetic mechanisms here to detect the difference between individuals 3To determine the effectiveness of this behavior model and mechanisms on clinical treatment and to ensure that the results obtained are a step for further treatment studies 4 To evaluate whether the data obtained in MicroRNA analysis is a predictive marker on prognosis Material and methods Study design This is an observational prospective cohort study In the power analysis performed with the G power 31 programs related to our study the effect size for the lymphocyte percentage and C-reactive protein was found to be 057 Effective Treatment of Severe COVID-19 Patients with Tocilizumab alpha probability of error 005 in the sample width analysis taking the power value 080 the total number of patients to be collected was 105 1053 35 for each group
Patients criteria This study was approved by the Clinical Research Ethics Committee 2011- CREC-40 at Bakırköy DrSadi Konuk Training and Research Hospital and informed consent will be obtained from all patients according to the rules of the Declaration of Helsinki Approval of the local ethical committee number is 2020143 2011-CREC-40 Patients enrolled in this study will be admitted to the Infectious Diseases and Clinical Microbiology Department and the Intensive Care Department from May 2020 to September 2020 Diagnosis of COVID 19 will be depending upon the presence of specific lesions in lungs confirmed by computed tomography CT andor laboratory findings according to the Republic of Turkey Ministry of Health diagnostic criteria
Inclusion criteria All patients between the ages of 18 and 65 included will suffer from COVID 19 and specific treatment should not yet be initiated COVID 19 diagnosis of patients will be based on full medical history thorough clinical examination radiological imaging laboratory assessment and SARS-CoV-2 analysis by RT-PCR
Exclusion criteria
1 People with chronic lung disease or previous AC surgery
2 People with immune deficiency or cancer patients
3 Patients receiving immunosuppressive therapy for autoimmune disease
4 People with neurological diseases affecting the respiratory muscles such as hypotonia
On the basis of the inclusion and exclusion criteria 105 patients subjects will be included in this study divided into three groups group I n 35 patients are mild group II n 35 patients are moderate and group III n 35 patients are severe and critical Mild and moderate cases are usually defined as those without severe pneumonia or severe acute respiratory syndrome Mild symptoms are low-grade fever not more than 38 degrees celsius dry cough fatigue sore throat headache the new loss of taste and smell Patients with normal or mild pneumonia findings of radiological imaging and blood lymphocyte count 800 µl and serum CRP40 mg l ferritin 500ngml D-Dimer 1000 ngml will be included in group I Moderate symptoms are fever of about 385-39 degrees celsius chills deep cough fatigue and body aches muscle pain the general feeling of being unwell Patients with bilateral diffuse pneumonia findings of radiological imaging or blood lymphocyte count 800 µl or serum CRP 40 mg l or ferritin 500ng ml or D-Dimer 1000 ng ml will be included in group II Severe symptoms are all the common symptoms mentioned above along with shortness of breath chest discomfort confusionunresponsiveness bluish facelips possible gastrointestinal issues like diarrhea or nausea Patients with ICU intensive care unit admission criteria such as confusion or tachycardia 125 min or respiratory distress or tachypnea 22 min or hypotension 9060 mmHg or SPO2 93will be included in group III Also patients with the central nervous system and heart involvement will be directly included in the severe case group
Specimen collection and handling About 10 ml of peripheral venous blood will be collected under strict aseptic conditions by clean venipuncture using vacuum blood collection tubes and will be distributed as 3 ml in EDTA tubes for complete blood count and DNA extraction 3 ml in another sterile special RNA protective tube stored at -80 C for miRNA extraction In addition 4 ml in a plane tube will be allowed to clot at the room temperature and then will be centrifuged at 3000 rpm for 10 minutes and the collected serum will be stored at -80C to be used for analyzing cytokine and other specific testsOn the 7th day of treatment second samples will be taken under the same conditions and finally on the 21st day third samples will be taken
Laboratory investigations RNA Extraction was done according to the miRNeasy SerumPlasma Advanced Kit cat no 217204 https wwwqiagencom
The expressions of selected miRNAs and genes were analyzed using Real-Time PCR The list of required kits chemicals and consumables is as follows
miRCURY LNA RT Kit Cat No 339340 miRCURY LNA miRNA PCR Assay 200 Cat No 339306 miRCURY LNA SYBR Green PCR Kit 600 Cat No 339346 miRCURY LNA SYBR Green PCR Kit 200 Cat No 339345 PAXgene Blood miRNA Kit 50 Cat No 763134 miRNeasy SerumPlasma Kit 50 Cat No 217184 PCR Tubes 02 ml 1000 Cat No 981005 TF10UL FILTER TIPS FOR P-10RAC Cat No TF-10-R-S TF100UL FILTER TIPS FOR Cat No TF-100-R-S TF 1000UL UNIVERSAL FILTER TIPS Cat No TF-1000-R-S
Kit and consumables required for DNA methylation analysis
ZymoResearch EZ DNA Methylation-Gold Kit 200 Preps Cat no D5006 ZymoResearch Human Methylated Non-methylated DNA Set DNA w primers 1 set Cat no D5014 ZymoResearch ZymoTaq qPCR Premix 200 Reactions Cat no E2055 Primary synthesis consumable for 1 gene region AXYGEN 05ml Thin Wall PCR TubesFlat Cap Clear1000 Cat no PCR-05-C Capp 20ml Boil-Proof Microtubes Clear 1000 Cat no MCT-200-C Capp 10 ul Dnase Rnase Free Bulk Pipet tips 10 rack Cat no 5030010 Capp 200 ul Dnase Rnase Free Bulk Pipet ucu Yellow Tips 10 rack Cat no 4130075 Capp 1000 ul Dnase Rnase Free Bulk Pipet ucu Blue Tips 10 rack Cat no 4130135 Bio Molecular Systems Mic Tubes and Caps 1 Box 20 racks of 48 tubesrack 960 Tubes in Strips of 4 with matching caps Cat no MIC-TUBES960
The motivation element behind the choice of miRNAs in the current work is a product of the following integrated online databases for miRNA target prediction and functional analysishttpmicrornaosumcedu httpswwwgenecardsorg httpwwwmirdborgmiRDB Gene association networks with ACE2 TMPRSS2 PARP HOX1 genes in COVID 19 cases were analyzed using GeneMANIA Multiple Association Network Integration Algorithm for prediction of possible functional interactions among them httpwww genemaniaorg
Patients criteria This study was approved by the Clinical Research Ethics Committee 2011- CREC-40 at Bakırköy DrSadi Konuk Training and Research Hospital and informed consent will be obtained from all patients according to the rules of the Declaration of Helsinki Approval of the local ethical committee number is 2020143 2011-CREC-40 Patients enrolled in this study will be admitted to the Infectious Diseases and Clinical Microbiology Department and the Intensive Care Department from May 2020 to September 2020 Diagnosis of COVID 19 will be depending upon the presence of specific lesions in lungs confirmed by computed tomography CT andor laboratory findings according to the Republic of Turkey Ministry of Health diagnostic criteria
Inclusion criteria All patients between the ages of 18 and 65 included will suffer from COVID 19 and specific treatment should not yet be initiated COVID 19 diagnosis of patients will be based on full medical history thorough clinical examination radiological imaging laboratory assessment and SARS-CoV-2 analysis by RT-PCR
Exclusion criteria
1 People with chronic lung disease or previous AC surgery
2 People with immune deficiency or cancer patients
3 Patients receiving immunosuppressive therapy for autoimmune disease
4 People with neurological diseases affecting the respiratory muscles such as hypotonia
On the basis of the inclusion and exclusion criteria 105 patients subjects will be included in this study divided into three groups group I n 35 patients are mild group II n 35 patients are moderate and group III n 35 patients are severe and critical Mild and moderate cases are usually defined as those without severe pneumonia or severe acute respiratory syndrome Mild symptoms are low-grade fever not more than 38 degrees celsius dry cough fatigue sore throat headache the new loss of taste and smell Patients with normal or mild pneumonia findings of radiological imaging and blood lymphocyte count 800 µl and serum CRP40 mg l ferritin 500ngml D-Dimer 1000 ngml will be included in group I Moderate symptoms are fever of about 385-39 degrees celsius chills deep cough fatigue and body aches muscle pain the general feeling of being unwell Patients with bilateral diffuse pneumonia findings of radiological imaging or blood lymphocyte count 800 µl or serum CRP 40 mg l or ferritin 500ng ml or D-Dimer 1000 ng ml will be included in group II Severe symptoms are all the common symptoms mentioned above along with shortness of breath chest discomfort confusionunresponsiveness bluish facelips possible gastrointestinal issues like diarrhea or nausea Patients with ICU intensive care unit admission criteria such as confusion or tachycardia 125 min or respiratory distress or tachypnea 22 min or hypotension 9060 mmHg or SPO2 93will be included in group III Also patients with the central nervous system and heart involvement will be directly included in the severe case group
Specimen collection and handling About 10 ml of peripheral venous blood will be collected under strict aseptic conditions by clean venipuncture using vacuum blood collection tubes and will be distributed as 3 ml in EDTA tubes for complete blood count and DNA extraction 3 ml in another sterile special RNA protective tube stored at -80 C for miRNA extraction In addition 4 ml in a plane tube will be allowed to clot at the room temperature and then will be centrifuged at 3000 rpm for 10 minutes and the collected serum will be stored at -80C to be used for analyzing cytokine and other specific testsOn the 7th day of treatment second samples will be taken under the same conditions and finally on the 21st day third samples will be taken
Laboratory investigations RNA Extraction was done according to the miRNeasy SerumPlasma Advanced Kit cat no 217204 https wwwqiagencom
The expressions of selected miRNAs and genes were analyzed using Real-Time PCR The list of required kits chemicals and consumables is as follows
miRCURY LNA RT Kit Cat No 339340 miRCURY LNA miRNA PCR Assay 200 Cat No 339306 miRCURY LNA SYBR Green PCR Kit 600 Cat No 339346 miRCURY LNA SYBR Green PCR Kit 200 Cat No 339345 PAXgene Blood miRNA Kit 50 Cat No 763134 miRNeasy SerumPlasma Kit 50 Cat No 217184 PCR Tubes 02 ml 1000 Cat No 981005 TF10UL FILTER TIPS FOR P-10RAC Cat No TF-10-R-S TF100UL FILTER TIPS FOR Cat No TF-100-R-S TF 1000UL UNIVERSAL FILTER TIPS Cat No TF-1000-R-S
Kit and consumables required for DNA methylation analysis
ZymoResearch EZ DNA Methylation-Gold Kit 200 Preps Cat no D5006 ZymoResearch Human Methylated Non-methylated DNA Set DNA w primers 1 set Cat no D5014 ZymoResearch ZymoTaq qPCR Premix 200 Reactions Cat no E2055 Primary synthesis consumable for 1 gene region AXYGEN 05ml Thin Wall PCR TubesFlat Cap Clear1000 Cat no PCR-05-C Capp 20ml Boil-Proof Microtubes Clear 1000 Cat no MCT-200-C Capp 10 ul Dnase Rnase Free Bulk Pipet tips 10 rack Cat no 5030010 Capp 200 ul Dnase Rnase Free Bulk Pipet ucu Yellow Tips 10 rack Cat no 4130075 Capp 1000 ul Dnase Rnase Free Bulk Pipet ucu Blue Tips 10 rack Cat no 4130135 Bio Molecular Systems Mic Tubes and Caps 1 Box 20 racks of 48 tubesrack 960 Tubes in Strips of 4 with matching caps Cat no MIC-TUBES960
The motivation element behind the choice of miRNAs in the current work is a product of the following integrated online databases for miRNA target prediction and functional analysishttpmicrornaosumcedu httpswwwgenecardsorg httpwwwmirdborgmiRDB Gene association networks with ACE2 TMPRSS2 PARP HOX1 genes in COVID 19 cases were analyzed using GeneMANIA Multiple Association Network Integration Algorithm for prediction of possible functional interactions among them httpwww genemaniaorg
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None