Identification of Genetic Determinants of the Coronary Microvascular Obstruction Development in Percutaneous Coronary Interventions
Conditions: Myocardial Infarction, No-Reflow Phenomenon
Sponsor: Privolzhsky Research Medical University

STUDY PROTOCOL 
Genetic Determinants of the Coronary Microvascular Obstruction in 
Percutaneous Coronary Interventions 
 
 
 
Study Title: 
Genetic Determinants of the Coronary Microvascular 
Obstruction in Percutaneous Coronary Interventions 
Protocol Number: 
CMVO-SNP-2022 
Study Number: 
NCT05355532 
Document date and 
version: 
Version number 1 from April 24, 2022 
Sponsor: 
Privolzhsky Research Medical University 
Principal 
Investigator: 
Principal Investigator: 
Ilya Grigorievich Pochinka, MD 
Research Center: 
State Budgetary Healthcare Institution of Nizhny 
Novgorod Region "City Clinical Hospital No. 13 of 
Avtozavodsky District of Nizhny Novgorod" 
Research Center Address: 
51 Patriotov Street, Avtozavodsky District, 
Nizhny Novgorod, Russian Federation, 603018 
Research Center Phone: 
+7 (831) 255-28-80 

Study Rationale 
For decades, ischemic heart disease (IHD) has been the leading cause of 
mortality in developed countries. Currently, percutaneous coronary intervention 
(PCI) is the primary method for restoring blood flow in the coronary artery (CA) in 
IHD, significantly reducing mortality in myocardial infarction (MI) and improving 
quality of life in stable angina. However, in 5–10% of cases, PCI leads to coronary 
microvascular obstruction (CMVO, or "no-reflow")—a phenomenon characterized by 
the absence of adequate myocardial perfusion despite successful restoration of 
epicardial coronary artery patency [1]. CMVO substantially worsens prognosis and 
can be a direct cause of death [1]. 
The pathogenesis of CMVO is complex and involves multiple concurrent 
mechanisms with varying degrees of contribution [1]. The most significant clinically 
established predictors of CMVO include: age, time from symptom onset to 
reperfusion, severity of acute heart failure (Killip classification), ineffective 
prehospital thrombolytic therapy, collateral blood flow (Rentrop classification) [6], 
coronary artery thrombus burden (TIMI thrombus grade) [7], baseline coronary flow 
(TIMI flow grade) [8], implantation of ≥3 stents during PCI, direct stenting of the 
target artery, blood neutrophil and glucose levels [2–5]. 
The pathophysiological heterogeneity of CMVO makes its prediction 
challenging. Even the best available models [2–5] demonstrate limited positive 
predictive value (60–70%). This limitation may stem from the omission of genetic 
factors influencing endothelial function, microcirculation, hemostasis, and 
inflammation in current models. 
Identifying genetic determinants of CMVO holds significant scientific novelty 
and could form the basis for a novel diagnostic system to predict this complication. 
Such a system would be clinically valuable, reducing PCI-related risks and improving 
patient outcomes. 
 
Study Objective: to identify genetic determinants of coronary microvascular 
obstruction development during percutaneous coronary interventions. 

Study Design 
A matched case-control study will be conducted to examine the role of specific 
single nucleotide polymorphisms (SNPs) as predictors of coronary microvascular 
obstruction (CMVO) development. The study will enroll 80 patients meeting the 
inclusion/exclusion criteria who have provided written informed consent. Patients 
will be allocated into two groups in a 1:1 ratio: 
• Group 1 (CMVO+): 40 patients with myocardial infarction (MI) who 
developed CMVO following PCI 
• Group 2 (CMVO-, control group): 40 patients with MI who did not develop 
CMVO following PCI 
Group allocation will be performed by the principal investigator. The groups 
will be matched by sex and age using the "matched pairs" method [9]. For each 
patient in the CMVO+ group, a corresponding patient of the same sex and similar age 
(±5 years) will be selected from the pool of MI patients who underwent PCI without 
CMVO development. 
The required sample size (80 patients) was determined based on the following 
parameters: alpha error level – 5 %, study power – 80 %, group ratio – 1:1, minimum 
detectable odds ratio – 4.0, expected prevalence of the studied SNPs in the 
population: 12–69 % (average 50%) [10]. Among the 40 planned matched pairs, 20 
pairs must be discordant (i.e., the paired patients must differ in the presence or 
absence of the predictor). 
The study will be conducted in several stages: 
1. Patient Screening 
o The principal investigator will assess inclusion/exclusion criteria. 
o Determine eligibility for study group allocation. 
o Performed in the catheterization laboratory immediately after PCI 
completion. 
2. Informed Consent Process 
o Eligible patients will provide written informed consent for genetic 
testing. 

o Patients will be assigned to respective study groups. 
o Conducted in the catheterization laboratory. 
3. Biological Sample Collection 
o Blood sampling for genetic analysis. 
o Performed in the catheterization laboratory. 
4. Sample Processing and Transportation 
o Blood sample processing. 
o Transportation to the Central Research Laboratory of the Institute of 
Fundamental Medicine at Privolzhsky Research Medical University 
(Russian Ministry of Health). 
5. Clinical Data Collection 
o The investigator will complete patient registry forms. 
o Includes hospitalization course and outcomes. 
6. Genetic Analysis 
o Performed at the Central Research Laboratory of the Institute of 
Fundamental Medicine at Privolzhsky Research Medical University. 
7. Data Management 
o Collected information will be entered into a unified electronic database. 
8. Statistical Analysis 
o Comprehensive processing of all obtained data. 
 
Ethical Information About the Study 
The study is conducted in accordance with Good Clinical Practice (GCP) 
standards and the principles of the Helsinki Declaration. The study protocol has been 
approved by the local ethics committee. Patient information is processed 
anonymously. Upon inclusion in the study, each patient is assigned a serial number. 
The following data are recorded in the registry of enrolled patients: serial number, 
last name, first name, patronymic, sex, date of birth, study group, date of inclusion in 
the study, medical record number, contact phone number, home address. pair number. 
This registry is stored at the research center. The serial number is indicated on: the 

inclusion/exclusion criteria form; the patient's voluntary informed consent for study 
participation; blood collection tubes with patient samples; the patient registry form 
with hospitalization details and outcomes; the unified electronic database of study 
participants. All work with blood samples outside the research center and statistical 
processing of obtained data is performed in anonymized form. 
 
Inclusion Criteria: 
1. Signed voluntary informed consent for study participation by the patient; 
2. Male or female aged 18 years or older; 
3. ST-segment elevation myocardial infarction (STEMI) type I according to the 
Fourth Universal Definition of Myocardial Infarction criteria [11] and current 
clinical guidelines for this condition [12]; 
4. Enrollment in the study immediately after PCI with possibility of blood 
sampling in the catheterization laboratory after signing informed consent; 
5. For "CMVO+" group: Development of CMVO as a result of PCI (CMVO is 
assessed according to criteria specified in the European Society of Cardiology 
clinical guidelines) [12]; 
6. For "CMVO-" group: Absence of CMVO as a result of PCI (CMVO is 
assessed according to criteria specified in the European Society of Cardiology 
clinical guidelines) [12] and matching with the paired patient from "CMVO+" 
group by sex and age (±5 years). 
 
Exclusion Criteria: 
1. Presentation with subacute-stage MI (>48 hours from symptom onset) or early 
post-infarction angina; 
2. MI occurring during revascularization procedures; 
3. PCI-related complications (coronary artery dissection, perforation, or acute 
intraprocedural thrombosis); 
4. Death during PCI unrelated to CMVO development; 

5. Concurrent terminal illness (unrelated to primary diagnosis) with life 
expectancy <1 month. 
The inclusion/exclusion criteria checklist is completed by the principal 
investigator who signs the voluntary informed consent. The completed checklist is 
then attached to the patient-signed informed consent document. 
 
Diagnostic Criteria for STEMI: 
1. Symptoms of acute coronary syndrome (ACS) within 48 hours prior to 
hospitalization (angina or equivalent symptoms); 
2. ST-segment elevation (>0.1 mV in ≥2 contiguous ECG leads) or newly 
developed left bundle branch block or ST-segment depression (≥0.1 mV in 
precordial leads V1-V4) with findings suggestive of true posterior infarction; 
3. Cardiac troponin levels exceeding the 99th percentile of the upper reference 
limit. 
 
Definition of CMVO and Description of Used Angiographic Scales 
According to the 2017 European Society of Cardiology clinical guidelines for 
the management of STEMI patients [12], CMVO is defined as inadequate myocardial 
perfusion after successful mechanical restoration of the infarct-related artery (IRA) 
lumen. 
Criteria of Inadequate Myocardial Perfusion (at least one required for CMVO 
registration): 
1. IRA blood flow <3 points by TIMI flow grade [8]; 
2. Myocardial perfusion <2 points by Myocardial Blush Grade (MBG) [13]. 
Other 
causes 
of 
coronary 
artery 
obstruction 
(spasm, 
dissection, 
thromboembolism) must be confidently excluded. 
 
Procedure for Signing the Voluntary Informed Consent 
The voluntary informed consent is signed by the patient in the catheterization 
laboratory immediately after completion of PCI. The inclusion/exclusion criteria 

checklist and the signed voluntary informed consent form constitute part of 
the primary study documentation. These documents are placed in the patient file, 
which is stored at the research center. 
 
Protocol for Blood Sample Collection, Storage and Transportation 
The study utilizes human whole peripheral blood. Blood samples are collected 
in the catheterization room immediately after the patient signs the voluntary informed 
consent form. The test tube is labeled with the patient's serial number, initials, and 
blood collection date/time. Sample collection, preprocessing and storage are 
performed according to the manufacturer's protocol for the DNA extraction kit from 
biological material. The following reagent kits are used in this study: 
"CardioGenetics 
Hypertension", 
"CardioGenetics 
Thrombophilia", 
"Folate 
Metabolism Genetics" (manufactured by DNA-Technology, Russia), "SNP-Express-
Cardiogenetics" RV kit for detecting Lys198Asn SNP in the EDN1 gene 
(manufactured by Litech, Russia). 
Whole peripheral blood is collected in 2.0 ml or 4.0 ml Vacuette vacuum 
plastic tubes containing EDTA anticoagulant at a final concentration of 2.0 mg/ml 
(lavender-top tubes). Sodium citrate may also be used as an alternative anticoagulant. 
After collection, the tube should be inverted 2-3 times to mix blood with the 
anticoagulant. Heparin must not be used as an anticoagulant. 
Prior to transportation, samples must be stored in a medical refrigerator at 2-
8°C. Collected samples must be delivered to the genetics laboratory within 24 hours 
of blood collection. Transportation must be performed using a specialized thermal 
container maintaining 2-8°C. 
 
Completion of Patient Registry Card with Hospitalization Course and Outcomes 
At the conclusion of the current hospitalization, the principal investigator 
completes a registry card for each patient containing information about the course 
and outcomes of the hospitalization. The card is anonymous, recording only the 
patient's serial number and initials within the study. This card forms part of the 

primary documentation and is stored in the patient's file at the research center. The 
registry card includes: details of the performed PCI; documented risk factors for 
CMVO development; laboratory test results (complete blood count, biochemical 
blood analysis, brain natriuretic peptide, etc.); instrumental diagnostic findings (ECG, 
ECG monitoring, echocardiography, 6-minute walk test); complications occurring 
during hospitalization (ventricular fibrillation, etc.); hospitalization outcome 
(discharged/deceased). This information is collected solely for registry purposes to 
enable subsequent statistical analysis. All described laboratory and instrumental 
diagnostic methods are performed according to standard clinical protocols for MI 
patient management and their use is unrelated to the study procedures. Copies of all 
test results are stored anonymously in the patient file. Patient angiograms are also 
preserved in anonymized form at the research center. 
 
Purpose and Methodology of Genetic Analysis 
The purpose of genetic analysis is to identify SNPs serving as predictors of 
CMVO development during PCI in patients with MI. For the study, SNPs were 
selected from the "CardioGenetics Hypertension", "CardioGenetics Thrombophilia" 
and "Folate Metabolism Genetics" panels manufactured by "DNA-Technology" 
(Russia), as well as the kit for determining the Lys198Asn SNP in the EDN1 gene 
manufactured by "Litech" (Russia) (Appendix A). This selection is based on literature 
data indicating that the pathogenetic changes associated with these SNPs are similar 
to the mechanisms of CMVO development [1]. 
For the panels from "DNA-Technology", SNP detection will be performed 
using real-time polymerase chain reaction (PCR) with high-resolution melting curve 
analysis using TaqMan fluorescent probes. The method principle involves 
amplification (copying) of DNA fragments containing SNPs through repeated cycles 
of: 1) thermal DNA denaturation, 2) primer annealing to DNA sequences 
complementary to the studied fragment, 3) extension of polynucleotide chains from 
these primers using Taq polymerase.The planned kit includes a "hot start" feature to 
increase reaction sensitivity and specificity, achieved through the use of antibody-

blocked Taq polymerase (which prevents nonspecific primer annealing to DNA 
targets during initial tube heating). 
The amplification mixture contains signal probes with Fam and Hex 
fluorescent labels for each SNP variant being analyzed, which undergo thermal 
melting after PCR completion. The melting temperature of these probes depends on 
their degree of complementarity to the studied DNA targets, which in turn affects 
their fluorescence level. The SNP variant is determined by analyzing the fluorescence 
curve. The mixture also includes a system for amplifying a fragment of human 
genomic DNA, which allows monitoring the DNA quantity in the tube and 
preventing genotyping errors. 
The reagent kit includes amplification mixtures specific for each SNP. Each 
tube uses three fluorescent dyes, enabling simultaneous recording of results from 
multiple amplification reactions occurring in a single tube (determining two alleles 
and assessing genomic DNA quantity). 
For analysis of the Lys198Asn SNP in the EDN1 gene, allele-specific PCR will 
be used (using the "SNP-Express-Cardiogenetics" RV reagent kit, Litech, Russia) 
with real-time reaction product detection (CFX96, BioRad, USA). 
 
 

References 
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10.21688/1681-3472-2020-1-18-27. (In Russ.) 
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10.1161/01.cir.103.21.2550. 
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10. dbSNP 
[Internet]. 
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from: 
https://www.ncbi.nlm.nih.gov/snp. 
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Abbreviations 
ACS – acute coronary syndrome 
CA – coronary artery 
CMVO – coronary microvascular obstruction (no-reflow phenomenon) 
DNA – deoxyribonucleic acid 
ECG – electrocardiogram 
EchoCG – echocardiography 
EDTA – ethylenediaminetetraacetic acid 
IHD – ischemic heart disease 
IRA – infarct-related artery 
MBG – myocardial blush grade 
Me – median 
MI – myocardial infarction 
PCI – percutaneous coronary intervention 
PCR – polymerase chain reaction 
[Q1; Q3] – interquartile range 
SNP – single nucleotide polymorphism 
STEMI – ST-segment elevation myocardial infarction 
TIMI – thrombolysis in myocardial infarction (study group) 
 

Appendix B 
SNPs Planned for Investigation 
Gene 
SNP 
Identifier 
Possible genotypes and 
their proportion in the 
population (reference 
and alternative) 
ADD1 – α-adductin 
1378 G>T (Gly460Trp) 
rs4961 
G=0.806737; T=0.193263 
AGT – angiotensinogen 
704 T>C (Met235Th) 
rs699 
A=0.545132; G=0.454868 
521 C>T (Thr174Met) 
rs4762 
G=0.878133; A=0.121867 
AGTR1 – angiotensin II receptor type 1 
1166 A>C 
rs5186 
A=0.721723; C=0.278277 
AGTR2 – angiotensin II receptor type 2 
1675 G>A 
rs1403543 
G=0.478265; A=0.521735 
CYP11B2 – cytochrome 11b2 – aldosterone 
synthase 
-344 C>T 
rs1799998 
A=0.573264; G=0.426736 
GNB3 – beta 3 subunit of G protein – guanine-
binding protein 
825 C>T 
rs5443 
C=0.668330; T=0.331670 
eNOS – nitric oxide synthase 
-786 Т>C 
rs2070744 
C=0.34927; T=0.65073 
894 G>T (Glu298Asp) 
rs1799983 
T=0.31468; G=0.68532 
EDN1 – endothelin 1 
594 G>T (Lys198Asn) 
rs5370 
G=0.780864; T=0.219136  
F2 – prothrombin 
20210 G>A 
rs1799963 
G=0.98867; A=0.01133 

 
Gene 
SNP 
Identifier 
Possible genotypes and 
their proportion in the 
population (reference 
and alternative) 
F5 – proaccelerin 
1691 G>A (Arg506Gln) 
rs6025 
G=0.951; A=0.049 
F7 – proconvertin 
10976 G>A (Arg353Gln) rs6046 
G=0.882035; A=0.117965 
F13 – fibrinase 
103 G>T (Val34Leu) 
rs5985 
G=0.786; T=0.214 
FGB – fibrinogen 
-455 G>A 
rs1800790 
G=0.81485; A=0.18515 
ITGA2-α2 – integrin 
807 C>T (Phe224Phe) 
rs1126643 
C=0.610691; T=0.389309 
ITGB3-β3 – integrin 
1565 T>C (Leu33Pro) 
rs5918 
T=0.851958; C=0.148042 
SERPINE1 (PAI-1) – serpin 
-675 5G>4G 
rs1799762 
4G=0.48510; 5G=0.51490 
MTHFR – methylenetetrahydrofolate reductase 
677 C>T (Ala222Val) 
rs1801133 
G=0.659842; A=0.340158 
1298 A>C (Glu429Ala) 
rs1801131 
T=0.695830; G=0.304170 
MTR – B12 dependent methionine synthase 
2756 A>G (Asp919Glu) 
rs1805087 
A=0.808600; G=0.191400 
MTRR – methionine synthase reductase 
66 A>G (Ile22Met) 
rs1801394 
A=0.482820; G=0.517180