Ignite Creation Date:
2024-05-06 @ 7:51 AM
Last Modification Date:
2024-10-26 @ 11:53 AM
Study NCT ID:
NCT02615015
Status:
UNKNOWN
Last Update Posted:
2017-03-23
First Post:
2015-11-12
Brief Title:
SNPs in the DNase 1 Gene Impair Its Activity and Are Increased in a STE-ACS Patient Cohort Compared to Healthy Controls
Sponsor:
Medical University of Vienna
Organization:
Medical University of Vienna
Study Overview
Official Title:
Single Nucleotide Polymorphisms in the Deoxyribonuclease 1 Gene Impair Its Activity and Are Increased in a ST-elevation Acute Coronary Syndrome Patient Cohort Compared to Healthy Controls
Status:
UNKNOWN
Status Verified Date:
2017-03
Last Known Status:
ENROLLING_BY_INVITATION
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:
Neutrophil extracellular traps NETs and deoxyribonuclease DNase activity determine outcome in ST elevation acute coronary syndrome STE-ACS DNase single nucleotide polymorphisms SNPs were increased in a japanese cohort
In the present study the investigators seek to measure DNase SNPs frequency in a caucasian STE-ACS cohort compared to healthy controls each n400 The investigators will compute polymorphisms DNase activity NET surrogate markers and clinical variables in regression models
In the present study the investigators seek to measure DNase SNPs frequency in a caucasian STE-ACS cohort compared to healthy controls each n400 The investigators will compute polymorphisms DNase activity NET surrogate markers and clinical variables in regression models
Detailed Description:
Background Acute coronary syndrome ACS is among the leading causes of death1 Atherosclerosis and coronary thrombotic occlusion are driven by inflammatory pathomechanisms2 The investigators have shown neutrophilic activation and neutrophil extracellular traps NETs at the culprit lesion site of ST-elevation ACS STE-ACS patients3 NETs are well described components of thrombi building a condensation nucleus for platelets erythrocytes and fibrinogen NET-containing thrombi can be effectively lysed by deoxyribonuclease DNase in vitro4 By adding DNase the investigators showed potent acceleration of tissue plasminogen activator-mediated lysis of coronary thrombi ex vivo Surrogate markers of NETs are strong predictors of acute coronary events5 In STE-ACS patients NETs directly correlated with cardiac magnet resonance-measured infarct size Moreover increased coronary DNase activity correlated with smaller infarct size3
Two major endogenous plasmatic DNases have been described namely DNase 1 and DNase gamma6 Several polymorphisms of the gene encoding for DNase 1 affecting its activity are known78 The single nucleotide polymorphism SNP Q222R in the DNase 1 gene leading to impaired extracellular DNase activity was correlated with increased incidence of myocardial infarction in Japanese patients9 SNPs associated with reduced DNAse activity are also known for DNase gamma but no associations with ACS have been investigated Present data implicate a critical balance between formation and degradation of NETs and outcome of ACS
Rationale The investigators hypothesize that coronary endoluminal NET formation is a major component of acute macro- and microvessel atherothrombosis Extracellular DNase degrades NETs thus down-regulating overwhelming NET formation Impaired DNase activity increases the risk for ACS Thus the investigators seek to test the presence of polymorphisms in the DNase gene impairing DNase activity in a STE-ACS population
Aims
1 To compare single nucleotide polymorphisms in the DNase 1 and gamma gene in STE-ACS patients coronary artery disease patients and healthy controls using polymerase chain reaction
2 To test DNase activity in plasma of these patients and healthy controls and to correlate that with the respective gene expression type
3 To test NET formation and NET surrogate markers and to correlate those with DNase activity and DNase SNPs
4 To correlate these data with surrogate markers of infarct size reperfusion long term outcome parameters and risk factors for coronary artery disease
Methods
Patients
The investigators will contact patients n400 who have been hospitalized in the General Hospital of Vienna in the past 10 years for ST-elevation acute coronary syndrome undergoing primary percutaneous coronary intervention pPCI with TIMI 0-1 These patients will be invited for a consultation The investigators will perform the following investigations with patients willing to join the study
Medical history
Detailed clinical examination
Electrocardiography
Echocardiography
Routine blood draw
Blood draw for research experiments Any patient with relevant clinical findings will be led to further diagnostic evaluation MACE occurring between the acute coronary event until presence will be surveyed at the consultation All patients will be contacted once a year to assess MACE
Healthy donors Blood samples from healthy donors n400 will be obtained by a peripheral blood draw from the cubital vein The probands will be recruited by bulletins in the General Hospital of Vienna
Real time polymerase chain reaction Cells will be lysed and DNAwill be isolated using a DNAeasy kit Applied Biosystems DNA or will be analyzed for DNase SNPs using primerprobe assays Applied Biosystems The ABI PRISM 7000 Sequence Detection System and software Applied Biosystems will be used
NET surrogate marker determination
Nucleosomes For the detection of DNA-histone complexes nucleosomes an ELISA-Cell death detection kit Roche Diagnostics GmbH Germany will be employed Optical density OD values will be normalized to the internal positive control and expressed as arbitrary nucleosome unitsml NUml The intra-assay positive control equals 1000 NUml
MPO-DNA complexes Myeloperoxidase MPO-associated DNA fragments will be identified using a capture ELISA 96-well microplates will be coated with an anti-MPO monoclonal capturing antibody ABD Serotec UK As detection antibody the investigators will use a peroxidase labeled anti-DNA monoclonal antibody Cell Death Detection ELISA Plus Kit Roche Diagnostics GmbH Germany All measurements will be performed in duplicates and values will be specified as mean ODs
Double stranded DNA For the detection of double stranded DNA dsDNA in patient plasma the investigators will employ a Quant-iT PicoGreen dsDNA Assay Invitrogen USA on 96-well microplates PicoGreen is a fluorescent nucleic acid stain for the quantification of dsDNA in solution Fluorescence will be measured by a Varioskan Flash microplate reader Thermo Scientific USA and normalized to the provided standard 1000 ngml
DNase activity assay Endogenous deoxyribonuclease DNase activity will be measured employing a DNase Activity Assay Orgentec Diagnostika GmbH Mainz Germany A DNA-coated microplate will be incubated with plasma samples for 60 minutes Coated DNA will be degraded in proportion to the DNase activity of the respective sample Optical density of residual DNA will be inversely proportional to DNase activity All assays will be performed following the manufacturers instructions All measurements will be performed in duplicate Plates will be read on a VersaMax microplate reader Molecular Devices Sunnyvale CA USA
Polymorphonuclear PMN cell culture and netosis assays PMNs ie mainly neutrophils from healthy donors will be isolated with Polymorphprep Axis-Shield Dundee Scotland Erythrocytes will be lysed using a one-step lysis buffer NH4Cl 154mmolL KHCO3 10mmolL EDTA 01mmolL The cell suspension will then be washed twice with HBSS supplemented with 10 of fetal calf serum FBS Isolated PMNs will be counted and resuspended at 2x106 cellsml in HBSS supplemented with 10 of FBS Cell viability will be determined by trypan blue exclusion
Immunofluorescence staining of NETs Fixed NETs will be stained using a mouse anti-DNA Histone H1 antibody Millipore After incubation a secondary donkey anti-mouse IgG antibody coupled to Alexa Fluor 555 Invitrogen Life Technologies Grand Island NY USA will be added For detection of nuclear DNA 46-diamidino-2-phenylindole DAPI Vectashield mounting medium with DAPI Vector Laboratories Burlingame CA USA will be used For staining of neutrophil elastase NE and myeloperoxidase MPO rabbit anti-human NE and MPO antibodies Abcam are used with a secondary Alexa Fluor 488 coupled donkey anti-rabbit IgG antibody Invitrogen Images will be obtained with an Axio Imager 2 fluorescence microscope using AxioVision Carl Zeiss Microscopy GmbH Göttingen Germany
Statistics Normally distributed data will be expressed as mean standard deviation SD otherwise median and interquartile range IQR will be presented Paired Students t-test will be applied to compare normally distributed variables otherwise Wilcoxon signed rank test will be used Distribution of data will be tested using the Kolmogorov-Smirnov test the Shapiro-Wilk test and histograms data not shown For comparison of multiple groups one-way analysis of variance with post-hoc Scheffé-procedure will be performed Spearmans rank correlation rs will be applied to calculate correlations data Multivariate regression models will be calculated and bootstrap correction techniques will be applied The area under the curve AUC of CK-MB values will be expressed in arbitrary units and be calculated employing the trapezoidal formula 10 if at least 5 consecutive values over a period of 3 days post admission were available Bonferroni-Holm correction will be used for multiple testing A p-value below 005 will be considered significant Statistical analyses were performed using IBM SPSS Statistics 200 for Windows New York NY USA Figures will be generated using GraphPad Prism 5
Two major endogenous plasmatic DNases have been described namely DNase 1 and DNase gamma6 Several polymorphisms of the gene encoding for DNase 1 affecting its activity are known78 The single nucleotide polymorphism SNP Q222R in the DNase 1 gene leading to impaired extracellular DNase activity was correlated with increased incidence of myocardial infarction in Japanese patients9 SNPs associated with reduced DNAse activity are also known for DNase gamma but no associations with ACS have been investigated Present data implicate a critical balance between formation and degradation of NETs and outcome of ACS
Rationale The investigators hypothesize that coronary endoluminal NET formation is a major component of acute macro- and microvessel atherothrombosis Extracellular DNase degrades NETs thus down-regulating overwhelming NET formation Impaired DNase activity increases the risk for ACS Thus the investigators seek to test the presence of polymorphisms in the DNase gene impairing DNase activity in a STE-ACS population
Aims
1 To compare single nucleotide polymorphisms in the DNase 1 and gamma gene in STE-ACS patients coronary artery disease patients and healthy controls using polymerase chain reaction
2 To test DNase activity in plasma of these patients and healthy controls and to correlate that with the respective gene expression type
3 To test NET formation and NET surrogate markers and to correlate those with DNase activity and DNase SNPs
4 To correlate these data with surrogate markers of infarct size reperfusion long term outcome parameters and risk factors for coronary artery disease
Methods
Patients
The investigators will contact patients n400 who have been hospitalized in the General Hospital of Vienna in the past 10 years for ST-elevation acute coronary syndrome undergoing primary percutaneous coronary intervention pPCI with TIMI 0-1 These patients will be invited for a consultation The investigators will perform the following investigations with patients willing to join the study
Medical history
Detailed clinical examination
Electrocardiography
Echocardiography
Routine blood draw
Blood draw for research experiments Any patient with relevant clinical findings will be led to further diagnostic evaluation MACE occurring between the acute coronary event until presence will be surveyed at the consultation All patients will be contacted once a year to assess MACE
Healthy donors Blood samples from healthy donors n400 will be obtained by a peripheral blood draw from the cubital vein The probands will be recruited by bulletins in the General Hospital of Vienna
Real time polymerase chain reaction Cells will be lysed and DNAwill be isolated using a DNAeasy kit Applied Biosystems DNA or will be analyzed for DNase SNPs using primerprobe assays Applied Biosystems The ABI PRISM 7000 Sequence Detection System and software Applied Biosystems will be used
NET surrogate marker determination
Nucleosomes For the detection of DNA-histone complexes nucleosomes an ELISA-Cell death detection kit Roche Diagnostics GmbH Germany will be employed Optical density OD values will be normalized to the internal positive control and expressed as arbitrary nucleosome unitsml NUml The intra-assay positive control equals 1000 NUml
MPO-DNA complexes Myeloperoxidase MPO-associated DNA fragments will be identified using a capture ELISA 96-well microplates will be coated with an anti-MPO monoclonal capturing antibody ABD Serotec UK As detection antibody the investigators will use a peroxidase labeled anti-DNA monoclonal antibody Cell Death Detection ELISA Plus Kit Roche Diagnostics GmbH Germany All measurements will be performed in duplicates and values will be specified as mean ODs
Double stranded DNA For the detection of double stranded DNA dsDNA in patient plasma the investigators will employ a Quant-iT PicoGreen dsDNA Assay Invitrogen USA on 96-well microplates PicoGreen is a fluorescent nucleic acid stain for the quantification of dsDNA in solution Fluorescence will be measured by a Varioskan Flash microplate reader Thermo Scientific USA and normalized to the provided standard 1000 ngml
DNase activity assay Endogenous deoxyribonuclease DNase activity will be measured employing a DNase Activity Assay Orgentec Diagnostika GmbH Mainz Germany A DNA-coated microplate will be incubated with plasma samples for 60 minutes Coated DNA will be degraded in proportion to the DNase activity of the respective sample Optical density of residual DNA will be inversely proportional to DNase activity All assays will be performed following the manufacturers instructions All measurements will be performed in duplicate Plates will be read on a VersaMax microplate reader Molecular Devices Sunnyvale CA USA
Polymorphonuclear PMN cell culture and netosis assays PMNs ie mainly neutrophils from healthy donors will be isolated with Polymorphprep Axis-Shield Dundee Scotland Erythrocytes will be lysed using a one-step lysis buffer NH4Cl 154mmolL KHCO3 10mmolL EDTA 01mmolL The cell suspension will then be washed twice with HBSS supplemented with 10 of fetal calf serum FBS Isolated PMNs will be counted and resuspended at 2x106 cellsml in HBSS supplemented with 10 of FBS Cell viability will be determined by trypan blue exclusion
Immunofluorescence staining of NETs Fixed NETs will be stained using a mouse anti-DNA Histone H1 antibody Millipore After incubation a secondary donkey anti-mouse IgG antibody coupled to Alexa Fluor 555 Invitrogen Life Technologies Grand Island NY USA will be added For detection of nuclear DNA 46-diamidino-2-phenylindole DAPI Vectashield mounting medium with DAPI Vector Laboratories Burlingame CA USA will be used For staining of neutrophil elastase NE and myeloperoxidase MPO rabbit anti-human NE and MPO antibodies Abcam are used with a secondary Alexa Fluor 488 coupled donkey anti-rabbit IgG antibody Invitrogen Images will be obtained with an Axio Imager 2 fluorescence microscope using AxioVision Carl Zeiss Microscopy GmbH Göttingen Germany
Statistics Normally distributed data will be expressed as mean standard deviation SD otherwise median and interquartile range IQR will be presented Paired Students t-test will be applied to compare normally distributed variables otherwise Wilcoxon signed rank test will be used Distribution of data will be tested using the Kolmogorov-Smirnov test the Shapiro-Wilk test and histograms data not shown For comparison of multiple groups one-way analysis of variance with post-hoc Scheffé-procedure will be performed Spearmans rank correlation rs will be applied to calculate correlations data Multivariate regression models will be calculated and bootstrap correction techniques will be applied The area under the curve AUC of CK-MB values will be expressed in arbitrary units and be calculated employing the trapezoidal formula 10 if at least 5 consecutive values over a period of 3 days post admission were available Bonferroni-Holm correction will be used for multiple testing A p-value below 005 will be considered significant Statistical analyses were performed using IBM SPSS Statistics 200 for Windows New York NY USA Figures will be generated using GraphPad Prism 5
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None