RU2569458C1 - Method of diagnostics of chronic heart failure by using circulating levels of microrna - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of diagnostics of chronic heart failure (CHF). The method comprises the study of human blood by polymerase chain reaction with hybridization-fluorescence detection of amplification products. The circulating levels of miRNA are determined, and in the miRNA-concentration-423-5r of over 35500 copies/ml the chronic heart failure is detected.

EFFECT: invention enables to carry out diagnostics with increased reliability of CHF at early stages of the disease.

2 ex

Description

The invention relates to medicine, namely to cardiology, and can be used in the diagnosis of chronic heart failure (CHF).

Despite the successes achieved in the study of the pathogenesis and treatment of chronic heart failure (CHF), the prognosis for this condition remains unfavorable and comparable with that for various malignant neoplasms. This is due to the fact that in recent years no new pathogenetic patterns of the disease have been identified, which are potential therapeutic targets.

A known method for the diagnosis of chronic heart failure, including the study of serum or human plasma and bringing into contact of a biological sample with an antibody to proBNP (1-108), detecting proBNP (1-108) in the sample and the correlation between the number of detected antigen-antibody complexes with the clinical the state of the subject.

(RF patent No. 2315773, IPC С07K 16/26, published on January 27, 2008)

The disadvantages of this method are due to the fact that there are a number of other conditions in which the content of a given substance increases. This includes pregnancy, chronic renal failure, thromboembolic complications. Accordingly, it is difficult to make an accurate differential diagnosis. In addition, in a number of categories of patients, in particular in the elderly, patients with overweight, the pro-BNP content does not increase even in the case of clinically significant chronic heart failure.

The objective of the invention is to provide a reliable method for the diagnosis of chronic heart failure by using circulating levels of miRNAs, allowing early diagnosis of heart failure and monitoring the effectiveness of the treatment of this disease.

The technical result consists in increasing the reliability of the method and the possibility of diagnosing heart failure in the early stages of the disease.

This is achieved by the fact that in the claimed method for the diagnosis of chronic heart failure, including the study of a biological sample of a person according to the invention, circulating microRNA levels are determined in the blood of a person using the polymerase chain reaction with hybridization-fluorescence detection of amplification products and when the concentration of miRNA-423-5p is more than 35500 copies / μl establish chronic heart failure.

In recent years, the possibility of influencing the main processes of regulation of intracellular transcription, manifested in a change in the processes of metabolism and fibrosis, has been actively studied. These processes naturally change during the formation of chronic heart failure and depend on both the stage and etiology.

Despite the fact that these processes are complex, complex and multimodal, there is the possibility of a multidirectional effect on them, modulating epigenetic activity. This is due to the presence of systems that regulate these intracellular processes at the upper (root) level. These systems include micro-RNA.

MicroRNAs are a large number of different types of single-stranded RNA molecules involved in the processes of epigenetic regulation of the synthesis of a large number of different proteins, enzymes, structural and regulatory molecules, as well as post-transcriptional suppression of gene activity.

The prospects for using miRNAs in the diagnosis of cardiovascular diseases are primarily associated with the recent discovery of a new class of RNA - circulating miRNAs. Although until recently it was believed that the lifetime of RNA molecules outside the cells was very short, the introduction of new, more sensitive methods made it possible to detect a class of miRNAs constantly present in blood and physiological fluids. The resistance of miRNAs to endonucleases is explained by the formation of supramolecular complexes with protein molecules, as well as the encapsulation of miRNAs in the lipid layer with the formation of nanostructure complexes called exosomes.

The implementation of the method

Whole blood samples are collected in tubes containing EDTA and centrifuged at 3000 g for 5 minutes. Isolation of total RNA (chain length less than 1000 bp) is carried out by adsorption column chromatography on a resin from 200 μl of blood plasma. Then carry out the reaction of reverse transcription and synthesis of the first strand of cDNA. To determine the levels of 5 of the studied individual microRNAs 423-5p, chemical modifications of LNA specific primers are used and analyzed by PCR with hybridization-fluorescence detection of amplification products “in real time”. Data on miRNA expression normalize to the original volume of whole blood.

When miRNA-423-5p concentration is more than 35500 copies / μl, chronic heart failure is established.

Example 1

A 62-year-old man with a history of long-term arterial hypertension. Combined diseases - diabetes mellitus, obesity. Complaints of shortness of breath, decreased exercise tolerance. Echocardiography shows signs of diastolic dysfunction. MNP 90 pg / ml. The determination of possible heart failure, as the cause of this symptomatology, is made according to the claimed method. The concentration of miRNA-423-5p was 62400 copies / μl, which exceeds the threshold value of 35500 copies / μl. In this regard, a diagnosis of chronic heart failure is made. The prescribed therapy allowed to achieve a significant improvement in clinical symptoms.

Example 2

A 69-year-old woman with a long history of hypertension and smoking. Combined disease - chronic obstructive pulmonary disease. Complaints of shortness of breath. To determine the possible heart failure as the cause of this symptomatology and to resolve the issue of treatment tactics, circulating levels are determined according to the claimed method. The concentration of miRNA-423-5p was 2726 copies / μl, which is less than the threshold value of 35500 copies / μl. In this regard, the diagnosis of chronic heart failure is rejected. Prescribed therapy for chronic obstructive pulmonary disease, against which there is an improvement in well-being

The study included 100 people: of which 56 patients with clinically and instrumentally confirmed CHF and 44 made up the control group, among which 15 (34%) had previously established CVD without heart failure and 29 (66%) were practically healthy volunteers. The average age of patients with heart failure was 59.4 ± 11.4 and ranged from 25 to 78 years, 40.5 ± 17.5 years were volunteers. The selection criteria for patients included in the first group were: the presence of systolic heart failure (with a decrease in EF less than 40%), heart failure with a preserved LV ejection fraction, regardless of the fact of compensation / decompensation at the time of inclusion. In the latter case, to confirm the diagnosis, a medical history was required either at the time the decompensation of the heart failure was turned on or the diagnostic level of BNP was exceeded.

The main reasons for the development of CHF were IHD: Post-infarction cardiosclerosis (IHD: PIKS) 45.5%, dilated cardiomyopathy (DCMP) 18.2%, hypertensive heart (HS) 14.5% and inflammatory cardiomyopathy (HCMP) 10.9%. A small percentage were patients with heart failure on the background of valvular disease and thyrotoxic heart. The severity of heart failure was assessed according to the classification of the New York Heart Association (NYHA, 1964) and ranged from I to IV FC. All patients received basic therapy in accordance with treatment standards (ACE inhibitors or ARA, β-blockers, antagonists of mineralocorticoid receptors, diuretics - if indicated). Exclusion criteria were HF, due to ACS, isolated right ventricular failure in the presence of pulmonary embolism. Measurement of the ejection fraction of the left ventricle (LVEF), end-diastolic size of the LV, and BNP concentration were performed only for patients with heart failure.

The expression profile of circulating miRNAs was evaluated. Statistical processing of research data was carried out using software SPSS 17.0, Microsoft Excel 2010.

results

MicroRNA-423-5p was significantly increased in patients with heart failure compared with the control group 2.5 times (p = 0.003).

The discrepancy in the Kapalan-Meyer curves when comparing the cumulative proportion of patients depending on the concentration of miRNA-423-5p and the presence of heart failure was statistically significant both in the interval assessment by confidence intervals, and by the Breslau criterion of early discrepancy (p = 0.002) and by the late discrepancy criterion Log Rank.

The diagnostic significance of miRNA-423-5p was evaluated using ROC analysis. The area under the AUC curve was 0.674 (95% CI 0.566-0.783, p = 0.003).

The threshold concentration of microRNA-423-5p for the presence of heart failure in accordance with the schedule and table of the ROC analysis for the point with the highest sensitivity and specificity was 35500 copies / μl. The sensitivity of this threshold value was 78.6% (95% CI 70.6-86.5%), specificity 63.6% (95% CI 55.4-71.9%), predictive value of a positive result 73.3% (95% CI 64.8-81.9%)), predictive value of negative result 70% (95%) CI 61.1-78.9%), accuracy index 72% (63.3-80.7%) ) The likelihood ratio, which assesses how much the probability of the presence of heart failure exceeds the probability of the absence of heart failure at a concentration of miRNA-423-5p higher or equal to 35500 copies / μl, was 6.417 (95% CI 2,646-15,561).

Thus, the claimed method will allow with high reliability to conduct early diagnosis of heart failure, as well as monitoring the effectiveness of treatment.

Claims (1)

A method for the diagnosis of chronic heart failure, including the study of a biological sample of a person, characterized in that the circulating levels of microRNAs are determined by polymerase chain reaction with hybridization-fluorescence detection of amplification products and at a concentration of miRNA-423-5p more than 35500 copies / μl establish chronic cardiac failure.