Personalized Risk of Rapidly Progressive Atherosclerosis
Condition: Atherosclerosis, Coronary
MeSH terms: Atherosclerosis; Coronary Artery Disease; Myocardial Ischemia
Study type: Observational

Summary: 
      Вackground. Progressive atherosclerosis is accompanied by unfavorable clinical outcomes,
      study and understanding of this process, creation of risk assessment method is necessary for
      individualization of approaches to treatment and prevention of this condition.

      Purpose of the study. Creation of a mathematical model to assess the risk of accelerated
      atherosclerosis development, using methods of factor and correlation analysis.

      Patient Characteristics and Study Methods. A retrospective cohort study included 202 patients
      with coronary heart disease. Group 1 included patients who had had myocardial infarction or
      unstable angina, emergency arterial stenting, stroke, peripheral artery thrombosis, critical
      ischemia, and lower extremity amputation within 2 years before study inclusion. Patients in
      the comparison group did not have these events. The influence of each of the studied
      parameters on the probability of fast progressing atherosclerosis was determined by factor
      and correlation analysis. The prospective part of the study will include follow-up of
      patients from both groups for 12 months. Annual "endpoints": fatal outcome, unscheduled
      coronary revascularization, nonfatal myocardial infarction and stroke, hospitalization due to
      unstable angina pectoris, stent thrombosis, stenting/plasty of lower limb arteries.
    

Detailed description: 
      Study design: open continuous retrospective and prospective study by parallel group method.

      Three control points were defined in the dissertation work: the first point was
      retrospective, 2 years before the study; the second was the day of inclusion in the study;
      and the third was 12 months after inclusion.

      The retrospective point covered 2 years from the date of patient inclusion in the study
      (period from January 2019 to January 2021) with determination of a large spectrum of
      parameters of clinical, instrumental, laboratory nature potentially influencing the
      appearance and progression of atherosclerosis (according to the literature analysis). The
      mentioned examinations were performed and evaluated at two points: the first one - at the
      time of the first preceding coronary event during the two-year retrospective period, the
      second one - at the time of the patient's inclusion in the present study. 202 patients were
      divided into two groups.

      Study Methods. At the moment of inclusion (1 point) in the study all patients underwent
      general clinical examination with assessment of complaints and clinical status, laboratory
      examination with general and biochemical blood tests, including special biochemical
      examination of blood serum to determine the level of high-sensitivity C-reactive protein
      (CRP, mg/L) and lipid spectrum, including total cholesterol (GC, mmol/L), low-density
      lipoproteins (LDL, mmol/L), high-density lipoproteins (HDL, mmol/L), triglycerides (TG,
      mmol/L); urinalysis and instrumental examinations: electrocardiography (ECG),
      echocardiography (Echo-CG), R-graphy of chest organs, selective coronary angiography In
      addition to standard research methods, all patients will be determined molecular genetic
      parameters. Standard methods of investigation will be repeated after one year (2 points).

      Preparation of DNA preparations. DNA extraction from blood was performed by phenol-chloroform
      extraction. Five to six volumes of buffer A (10 mM Tris-HCl, pH=7.5; 10 mM NaCl; 3 mM MgCl2)
      were added to 1 volume of blood sample and clots were rubbed in a homogenizer. After
      centrifugation at 2500g for 15 min, the precipitates were washed three times with buffer A
      and resuspended in 1 ml of buffer B (10 mM EDTA; 100 mM NaCl; 50 mM Tris-HCl, pH=8.5). After
      adding SDS to 0.5% and proteinase E to 200 μg/mL, the mixture was incubated for 12 hours at
      56°C. Deproteinization was performed sequentially with phenol-chloroform mixture (1:1),
      water-saturated phenol, phenol-chloroform mixture (1:1), and chloroform. DNA was precipitated
      by adding NaCl solution to 1 M and 1 V isopropyl alcohol. After that, the solution was cooled
      for 1 h at -20 °C. The precipitate obtained by centrifugation on an Eppendorf microcentrifuge
      at 12000g for 15 min was washed three times with 75% ethanol followed by centrifugation for 5
      min. 12000g and, after drying at 56 °C, dissolved in deinanilized water to a DNA
      concentration of 0.5 µg/μl.Genotyping of polymorphisms was performed using real-time PCR
      according to the manufacturer's protocol (TaqMan probes, Thermo Fisher Scientific, USA) on a
      StepOnePlus instrument. They were selected according to the results of international full
      genome-wide association studies (GWAS), which confirmed the association of these SNPs with
      coronary heart disease (CHD), an inflammatory process that plays a significant role in the
      progression of atherosclerosis.

      Methods of statistical analysis. SPSS 22.0 software package will be used for statistical
      analysis of molecular-genetic data. The first stage will be to determine the frequencies of
      genotypes and alleles of the studied SNPs in the group of myocardial infarction patients with
      elevated values of cardiospecific markers and the comparison group, where cardiospecific
      markers will be within normal limits; then we will assess compliance of genotype frequencies
      with Hardy-Weinberg equilibrium in the control group (using the chi-square criterion). There
      will be a comparison of the levels of indicators such as age; cre-atinine level; diagnostic
      level of biomarkers of myocardial necrosis: MB creatine kinase and troponin, fatty acid
      binding protein (FABP); body mass index; waist circumference; serum C-peptide concentration;
      glycemia levels at admission and discharge; blood hemoglobin levels at admission and
      discharge; Systolic and diastolic blood pressure at admission; heart rate at admission; left
      ventricular ejection fraction value by echocardiography; serum HCMP concentration;
      lipidogram: serum concentration of low-density lipoproteins (LDL), high-density lipoproteins
      (HDL), total cholesterol, triglycerides; height, weight, body mass index, in carriers of
      different genotypes were performed after checking normality of distribution of these features
      by Kolmogorov-Smirnov test. If the traits would meet the criteria of normal distribution, a
      single-factor analysis of variance would be used. Significance of differences between two
      genotypic classes will be additionally checked using t-test for two independent samples. If a
      trait under study does not meet the criteria of normal distribution, comparison of the level
      of this trait in carriers of different genotypes will be performed using the Kruskal-Wallis
      test; the reliability of differences between the two genotypic classes will be additionally
      checked using the Mann-Whitney test for two independent samples. The association of SNPs with
      risk factors related to categorical variables will be tested with conjugation tables using
      Pearson chi-square test. In the case of four-field tables, comparison of samples by genotype
      and allele frequencies will use Fisher's exact two-sided criterion. The relative risk (OR -
      odds ratio) of disease outcome/risk factor presence for a particular allele or genotype will
      be calculated as odds ratio.

      Influence of clinical, demographic, functional, metabolic, inflammatory parameters and
      myocardial necrosis markers on long-term prognosis will be assessed by odds ratio (0.95
      confidence interval).
    

Intervention name(s): without Intervention
Intervention type(s): Other
Intervention description(s): without Intervention
Observational model: Cohort
Time perspective: Other
Sampling method: Probability Sample
Study population: 
        A retrospective cohort study included 202 patients with coronary heart disease. Group 1
        included patients who had had myocardial infarction or unstable angina, emergency arterial
        stenting, stroke, peripheral artery thrombosis, critical ischemia, and lower extremity
        amputation within 2 years before study inclusion. Patients in the comparison group did not
        have these events. The influence of each of the studied parameters on the probability of
        fast progressing atherosclerosis was determined by factor and correlation analysis.
      
Biospecimen description: 
      frozen blood clot
    
Keywords: rapidly progressive atherosclerosis; personalized risk