RU2617060C1 - Method of predicting risk of infectious endocarditis development - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used for prediction of genetic predisposition or increased risk of infectious endocarditis (IE) development in patients of risk group. Essence of the method: genotyping by polymorphic locus I462V of gene CYP1A1 and by polymorphic locus I105V of gene of glutathione-S-transferase Pi 1 GSTP1 is performed in patients of risk group, if carrier state of combination of genotypes CYP1A1 I462I and GSTP1 I105V is identified, 22.6-fold risk if infectious endocarditis development in their carriers is predicted. Group of risk includes people with intravenous drug addiction, congenital and acquired heart defects, prosthetic valves and other operations on heart, coronary arteries, and immunodeficiency states.

EFFECT: method makes it possible to identify genetic predisposition to IE in patients of risk group, assists in determination of tactics of patient management and individualise complex of preventive and therapeutic procedures.

2 cl, 2 dwg, 1 tbl

Description

The invention relates to medicine, namely to therapy and surgery, and can be used to predict a genetic predisposition or an increased risk of developing infectious endocarditis (IE) in patients at risk.

It is known that many widespread diseases - cancer, immunodeficiencies, allergic conditions and others, are associated with exposure to the body of harmful environmental factors - xenobiotics. Their neutralization and removal from the body is carried out by a detoxification system consisting of more than 200 enzymes. Detoxification occurs in three stages -1 phase (activation), phase II (conjugation or neutralization) and phase III (disposal). Phase I enzymes carry out the oxidation of fat-soluble toxins by superoxide radicals, which leads to the formation of reactive sites on them for the addition of water-soluble molecules by phase II enzymes and subsequent excretion from the body. The risk of pathology is maximized with high activity of phase I enzymes, since reactive sites formed on their participation on xenobiotic molecules provide easy attachment of the latter to DNA and proteins, which leads to cell damage. At the same time, with inertness of phase II enzymes, reactants toxic to cells accumulate in the body.

IE, which is a serious nosology, often with a fatal outcome, often develops with drugs, glucocorticosteroids, cytostatics, AIDS, in people with congenital or acquired heart defects, prosthetic valves and with immunodeficiency states. However, the relationship of IE with the enzymes of the detoxification system that metabolize drugs and drugs has not been studied. The activity of detoxification enzymes depends on the characteristics of allelic variants (polymorphisms) of the genes encoding them. The association of IE with polymorphisms of detoxification enzyme genes has not been investigated. The search for the connection of IE with allelic variants of other genes is still ineffective. Thus, it was shown that polymorphisms in the platelet receptor gene do not affect the course of IE (Daga S., Shepherd JG, Callaghan JG, Hung RK, Dawson DK, Padfield GJ, Hey SY, Cartwright RA, Newby DE, Fitzgerald JR Platelet receptor polymorphisms do not influence Staphylococcus aureus-platelet interactions or infective endocarditis. Microbes Infect. 2011; 13 (3): 216-225). No apparent associations with IE of gene variants of some toll-like receptors and interleukins - TLR4, IL 10, IL IB, IL6 (M. Weinstock, I. Grimm, J. Dreier, C. Knabbe, T. Vollmer. Genetic Variants in Genes of the Inflammatory Response in Association with Infective Endocarditis. PLoS One. 2014; 9 (10): el 10151. Published online Oct 9, 2014. doi: 10.1371 / journal.pone.0110151 PMCID: PMC4192365).

Closest to the claimed method is a method for determining the high risk of IE in carriers of variant Q polymorphism R753Q (rs 5743708) in the toll-like receptor 2 (TLR2) gene (Bustamante J., Tamayo Ε., Florez S., Telleria JJ, Bustamante Ε ., Lopez J., San Roman JA, Alvarez FJ Toll-Like Receptor 2 R753Q Polymorphisms Are Associated With an Increased Risk of Infective Endocarditis. Rev. Esp. Cardiol. 2011; 64 (11): 1056-1059). The method consists in isolating genomic DNA from blood samples taken in test tubes with an anticoagulant - ethylenediaminetetraacetic acid (EDTA), determining the R753Q polymorphism in the TLR2 gene using a polymerase chain reaction and restriction analysis of the obtained PCR products. The authors examined 65 patients with infectious endocarditis in comparison with 66 practically healthy individuals. In a statistical analysis of the results of genotyping, the authors found a significant association with IE R753Q polymorphism of the TLR2 gene and found an increased risk (3-13 times) of IE development in carriers of the Q allele. However, the indicated pathological variant (Q allele) was found by the authors only in 33% of patients infectious endocarditis and in 12.9% of practically healthy people. The revealed relatively low frequency of the Q allele in patients with IE encourages the search for other more informative genetic risk markers. In addition, other researchers did not confirm the claimed association of R753Q polymorphism in the TLR2 gene with IE (Golovkin AS, Ponasenko AV, Yuzhalin A.E., Salakhov RR, Khutornaya MV, Kutikhin AG, Rutkovskaya NV, Savostyanova YY, Barbarash LS An association between single nucleotide polymorphisms within TLR and TREM-1 genes and infective endocarditis. Cytokine. 2014, 8; 71 (1): 16-21).

The objective of the present invention was to study the association of IE with polymorphisms of genes of enzymes of the I and II phases of the detoxification system and the development of a new method for predicting the risk of developing IE.

The task was achieved by collecting venous blood from patients, isolating DNA, conducting a polymerase chain reaction, and determining gene polymorphism. Genotyping is carried out at the 1462V polymorphic locus of the CYP1A1 gene and at the I105V polymorphic locus of the glutathione-S-transferase Pi1 GSTP1 gene in risk patients and upon identification of carriage combination of CYP1A1 14621 and GSTP1 I105V genotypes in their carriers predict 22.6-fold endocarditis risk.

The risk group includes people with intravenous drug addiction, congenital and acquired heart defects, prosthetic valves and other heart operations, coronary arteries, immunodeficiency states.

Novelty of invention

1. Genotyping at the 1462V polymorphic loci of the cytochrome P-450 1A1 CYP1A1 gene and the I105V gene of the glutathione-S-transferase Pi1 GSTP1 gene of risk patients allows predicting a 22.6-fold risk of IE in carriers of the combination of the CYP1A114621 and GSTP1 I105V genotypes.

2. The risk group includes persons with intravenous drug addiction, congenital and acquired heart defects, prosthetic valves and other heart operations, coronary arteries, immunodeficiency states.

In the patent and scientific literature there is no information about a similar method for determining the genetic risk of IE.

The combination of essential features of the invention allowed to obtain a new technical result, which consists in establishing a genetic predisposition to IE in patients at risk, which will help determine patient management tactics and individualize the complex of preventive and therapeutic measures.

For the first time, it is proposed to use genotyping by polymorphic loci of the enzyme genes I and II of the detoxification phases CYP1A1 I462V and GSTP1 I105V to identify the maximum genetic risk of IE. In patients with a combination of the CYP1A1 I462I and GSTP1 I105V genotypes, among patients with intravenous drug addiction, congenital and acquired heart defects, prosthetic valves and other heart operations, coronary arteries, and immunodeficiency states, a 22.6-fold risk of developing IE was revealed. The tendency to IE, the main etiological factor of which are pathogenic bacteria, mainly Staphylococcus and Streptococcus, in patients at risk seems to be associated with a weakening of innate and adaptive immunity due to the accumulation of toxic reactants in the body due to the genetically dependent high activity of the P-450 enzyme CYP1A1 and reduced - the enzyme GSTP1.

The method is as follows.

Genomic DNA is isolated from peripheral blood cells using commercial DNA-express-blood-plus or DNA-express-blood reagents (NPF Litekh, Moscow). For genotyping, commercial reagent kits are used to detect mutations (polymorphisms) in the human genome - “SNP-express” (NPF Litekh, Moscow). Genotyping is carried out at the I462V loci (nucleotide substitution 2455A / G, amino acid substitution Ile462Val) of the cytochrome P-450 1A1 CYP1A1 gene [-4 cytochrome P450 mutation] and I105V (313A / G nucleotide substitution, Ile 105 Val amino acid substitution) GSTP1 mutation (mutation Pi-glutathione-S-transferase) [NPF Litekh, Moscow] using the allele-specific polymerase reaction method. According to the instructions for the kits, two amplification reactions are carried out simultaneously with a sample of isolated DNA, with two pairs of allele-specific primers, for the parallel detection of mutants of the normal and normal types. The reaction mixture for PCR consists of 5 μl of the studied DNA sample and 20 μl of the working amplification mixture containing 0.2 μl of Taq polymerase working solution. Amplification is carried out in the Tertsik automatic thermal cycler (DNA-Technology, Moscow). The amplification program, according to the instructions, includes the following temperature conditions - 1 cycle at 93 ° C for 1 min, 35 cycles with DNA denaturation steps for 10 sec at 93 ° C, primer annealing for 10 sec at 64 ° C and chain synthesis for 20 seconds at 72 ° C, and 1 cycle at 72 ° C for 1 min. The analysis of PCR products is carried out after their electrophoretic separation in 50 ml of 3% agarose gel in 50 × TAE buffer, into which 5 μl of a 1% solution of ethidium bromide are added before solidification. Two rows of holes are cut out in each gel — to detect the normal type allele and mutant allele. Fragments of the analyzed DNA appear in the form of luminous bands. Gels are analyzed in an ECX-15M UV-transilluminator (Vilber Lourmat, France) using a GL-2 gel-documenting video system (Russia) and / or photographed using a Canon PowerShot A590 IS digital camera in transmitted ultraviolet light with a wavelength of 310 nm.

The invention is illustrated in FIG. 1 and FIG. 2.

In FIG. 1 shows the results of genotyping of 12 people according to I462V polymorphism of the CYP1A1 gene in the form of an electrophoregram, which shows the amplification products of the studied locus in the form of luminous bands. To - negative control; lanes 2-8, 10-12 — homozygous normal genotype I462I, lane 1 — homozygous mutant genotype V462V, lane 9 — heterozygous genotype I462V.

In FIG. 2 shows the results of genotyping of 8 patients with infectious endocarditis by I105V polymorphism of the GSTP1 gene in the form of an electrophoregram, which shows the amplification products of the studied locus in the form of luminous bands. To - negative control; lanes 1-4, 6-8 - heterozygous genotype I105V, lane 5 - homozygous genotype I105I.

A total of 32 patients (14 women and 18 men) aged 23 to 75 years who were hospitalized in therapeutic clinics of Novokuznetsk with a diagnosis of “infectious endocarditis” were examined. Most patients suffered from intravenous drug addiction - 13 people, 5 people found rheumatic defect of the mitral and aortic valves (MK, AK), prosthetics were performed, in 2 - prolapse of MK 2-3 degrees, in 4 people - atherosclerotic changes in MK and AK, in 2 - a severe viral infection without a history that was previously weighed down; in 5 people, chronic kidney disease with the need for hemodialysis and a pronounced immunodeficiency state, the 1st coronary artery bypass grafting was performed.

The control group consisted of 109 healthy individuals. Blood sampling and molecular genetic studies were carried out on the basis of the informed consent of the individuals examined. Mathematical processing of the research results was carried out using the statistical software packages InStatII, Microsoft Excel. The critical level of significance in testing statistical hypotheses was assumed to be 0.05. The significance of differences in the frequency distribution of alleles and genotypes between groups of patients and healthy individuals was assessed using Fisher's two-sided exact criterion. The frequency of the allele / genotype was determined by the ratio of the number of carriers to the total number of carriers of the tested alleles / genotypes in the study sample. The relative risk of disease for a particular symptom was calculated as the odds ratio (OR-odds ratio): OR = (a × d) / (b × c), where a and b are the number of patients with and without a mutant allele (genotype), respectively; c and d are the number of people in the control group with and without a mutant allele (genotype), respectively.

Genotyping results for the I462V polymorphic loci of the CYP1A1 gene and I105V gene of the GSTP1 gene in patients with IE and practically healthy individuals are presented in Table 1. They show that 94% of IE patients were carriers of the homozygous variant I462I of the CYP1A1 I462V polymorphism, and 2% of IE patients were carriers of the heterozygous CY variant I462V, homozygous for the mutant CYP1A1 462V allele among sick individuals was not found. Homozygous carriers of CYP1A1 I105I in the control were 18% less. The risk of disease among carriers of this genotype in the risk group was high. When comparing the frequencies of alleles of this polymorphism, a similar risk of disease was revealed in carriers of the CYP1A1 462I allele.

Among patients, the largest number of people were carriers of the heterozygous genotype GSTP1 I105V, and these individuals showed a high predisposition to IE with a 15-fold risk of its development in comparison with the control. An analysis of the carriage of a combination of the studied genotypes revealed the maximum genetic predisposition to the disease in carriers of the homozygous CYP1A1 I462I variant and the heterozygous GSTP1 I105V variant with a 22.6-fold risk of IE. An analysis of the correlation between the studied genotypes and IE confirmed the positive association of genotype I462I and allele 462I of the CYP1A1 gene and genotype I105 V of the GSTP1 gene with IE. The strongest association with IE was found in carriers of a combination of the CYP1A1 I462I and GSTP1 I105V genotypes.

Thus, we have identified the association of IE with xenobiotic detoxification system gene variants encoding the antioxidant enzyme of the detoxification phase I, cytochrome P-450 1A1, and the antioxidant enzyme of the detoxification phase II, glutathione S-transferase P1. A high risk of IE was identified in carriers of the homozygous variant I462I of the CYP1A1 gene, in which the presence of the normal CYP1A1 462I allele in both chromosomes determines the maximum catalytic activity of the enzyme, as well as in carriers of the heterozygous variant I105V of the GSTP1 gene, in which the presence of mutant S mutant form molecules P1 transferase leads to a decrease in the catalytic ability of the enzyme in relation to a number of substrates. The combination of these options in the genome leads to a 22.6-fold increase in the risk of developing IE compared with the control.

Thus, the proposed method for genotyping I462V polymorphic loci of the CYP1Al gene and I105V of the GSTP1 gene allows one to detect a very high risk of developing infectious endocarditis in carriers of the I462I homozygous CYP1A1 genotype when it is combined with the carriage of the heterozygous variant I105V of the inherited GSTP1 gene among individuals heart defects, prosthetic valves and other heart operations, coronary arteries, immunodeficiency states.

Clinical example No. 1. Patient R., 36 years old, was admitted to the treatment department on 12.12.2014 with complaints of weakness, sweating, shortness of breath during normal physical exertion, pain in the chest, aggravated by deep inhalation, coughing, difficult to separate purulent sputum, fever up to 39 -40 ° C, chills, pain in muscles, joints. Sick for 2 weeks, was treated independently to no avail with antipyretic drugs, ceftriaxone. Delivered by an ambulance crew. In the anamnesis: hepatitis, tuberculosis, HIV - denies. I / O drugs for 15 years (hunk, salt, heroin). Smokes for 15 years at 1 pack per day. Colds 3-4 times a year. There is no permanent job. Upon examination, the state of moderate severity, body temperature 38 ° C, the skin is dry, pale, no swelling, low nutrition. In the lungs, vesicular breathing, scattered dry rales over the entire pulmonary surface, BH 22 per min. Heart sounds are muffled, rhythmic, heart rate 110 per minute, systolic murmur at all points, blood pressure 120/80 mm RT. Art. The abdomen is soft, painless. Liver +1.5 cm from under the edge of the costal arch. Stool, diuresis is normal. During the examination: AT for HIV, HBV, treponema - not detected. HCV AT detected. General blood test - HB 112 g / l, er. 3.5, lake. 8.4, p / a 20, lymph.-13, ESR 50 mm / h. Urinalysis - beats. weight 1012, protein 0.3 g / l, lake. in large numbers, er. -1-2 in s / sp, cylinders hyaline 1-2 in s / sp. Urine analysis according to Nechiporenko: lake. - 201500, er. 2500, cylinders 3000. B / x an. blood: sugar 5.7, creatinine 155 (No. 40-140) μmol / L, urea 8.0, ALT 132, ACT-66 (elevated), fibrinogen 5.25, protein 78.1, bilirubin 11.7. Blood for blood culture No. 3 - negative. Ultrasound - splenomegaly, liver +1.5 cm from the edge of the costal arch, dimensions are normal. Diffuse changes in the renal parenchyma. ECHO KG - the right cavities are moderately dilated, the hypoechoic vegetation center 13 × 5 mm on the TC, regurgitation 2-3 tbsp, slight pericardial effusion, no signs of pulmonary hypertension, PV 67%. ECG - sinus tachycardia 117 per min. Chest x-ray - bilateral septic pneumonia with disintegration. Genotyping results for the I462V polymorphic loci of the CYP1A1 gene and I105V of the GSTP1 gene: a combination of the CYP1A1 I462I and GSTP1 I105V genotypes was revealed, indicating a 22.6-fold risk of IE.

Dz: Acute infective endocarditis with damage to the tricuspid valve, primary. TK insufficiency. CHF 1-2a Art. Bilateral septic pneumonia, with decay, severe, prolonged course. Addiction. Chr. viral hepatitis C.

The treatment was carried out: cefuroxime, vancomycin, fraksiparin, gentamicin. Discharged with improvement on further outpatient treatment.

Clinical example No. 2. Patient M., 26 years old. She was hospitalized in the department of pulmonology with complaints of dry cough, shortness of breath with slight physical exertion, chills, fever up to 39 ° C, sweating at night, cough with a small amount of purulent sputum. Sick for 2-3 weeks, the day before suffered ARVI, did not seek medical help, symptomatic treatment, fever - in the debut of the disease within 3-4 days. Against the background of treatment - improvement, catarrhal symptoms stopped, the temperature returned to normal. Pronounced weakness continued, shortness of breath with moderate physical exertion. Over the past 2 weeks, chills appeared, the temperature rose again to 39 ° C without the effect of antipyretic drugs, weakness increased, coughing, sweating at night appeared. A history of frequent colds up to 4-6 times a year, frequent use of antibacterial drugs without a doctor's prescription. In 2009, a heart defect was detected, there were no documents with the results of the examination, it was not observed. Drug use denied. The work is related to hypothermia (worked at a cold factory as a packer). Monotonous food, meat, fruits - rarely. Objectively: a state of moderate severity due to intoxication syndrome, body temperature - 38.5 ° C. Nasal breathing is not difficult. The skin is pale, moist, clean, no swelling. In the lungs, breathing is harsh, weakened in the subscapular region on the right, there are no wheezing, heart rate is 18 per minute, heart sounds are deaf, rhythmic heart rate is 110 per minute, auscultation is a gross systolic murmur at all points. During the examination: general blood test -Nv. 91 g / l er 3.4 l 15.0, postbox. 14, lymph. 10.5, young -1, platelets - 415, ESR 52 mm / h. Urinalysis - beats. weight 1005, protein 2.5 g / l, er. 40-50 p / sp., L - 2-6 p / sp. B / x an. blood: ALT 2.26, ACT-3.0, urea 10.2, creatinine 106, protein 59.6, sugar 5.0, procalcitonin 6.1, CRP 460.3 mg / L. D-dimer 2350, AT for HBV, HCV, HIV were not detected. ECG - sinus tachycardia, impaired repolarization processes in the posterior wall of the left ventricle. Chest x-ray - polysigmentary pneumonia on the right with disintegration. ECHO-KG - signs of rheumatic lesions of the mitral valve, stenosis of the valves of the MK. Smo 1.62 cm ² , regurgitation 1-2 tbsp. Aortic valve - stenosis not detected, calcification and fibrosis of the valves, regurgitation 2 degrees. The valves of the tricuspid valve and pulmonary valve are sealed, regurgitation of TK-2 st. In the right atrium along the in / wall there is an additional roundish formation, mobile, of average echogenicity 2.7 × 1.75 cm. Dilation of the cavities of both atria, more than the left, PV 47.8%, paroxysmal movement of the MJP. Ultrasound - hepatosplenomegaly. Diffuse change in renal parenchyma. In dynamics - there was free fluid in the abdominal cavity. According to the results of blood culture, sterility revealed Staphylococcus aureus. In subsequent crops, Pseudo-monas stutseri appeared. Given the severity of the condition, carried out infusion, antibacterial therapy, the patient was placed subclavian catheter. The condition worsened: popliteal artery thrombosis on the right joined, pulmonary embolism recurred, and cardiac and respiratory failure increased. Amputation of the right lower limb to the middle third of the thigh was performed. Genotyping results for the I462V polymorphic loci of the CYP1A1 gene and I105V of the GSTP1 gene: a combination of the CYP1A1 I462I and GSTP1 I105V genotypes was identified, indicating a 22.6-fold risk of IE.

Dz: Secondary infective endocarditis. Chr. rheumatic heart disease: combined mitral disease with a predominance of stenosis. Insufficiency of AK, TC, CHF 2B. FC III. Condition after amputation of the right lower limb to cf. / third of the thigh due to acute arterial thrombosis. Polysegmental pneumonia on the right with decay. Recurrent pulmonary embolism.

Claims (2)

1. A method for predicting the risk of developing infectious endocarditis, including venous blood sampling, DNA extraction, polymerase chain reaction, determination of gene polymorphism, characterized in that genotyping is carried out by the polymorphic locus I462V of the cytochrome P450 1A1 CYP1A1 gene and the polymorphic locus I105V of the glutathione gene S105V -transferases Pi1 GSTP1 of patients at risk and, when a combination of the CYP1A1 I462I and GSTP1 I105V genotypes is detected in their carriers, a 22.6-fold risk of developing infectious endocarditis is predicted. 2. The method according to p. 1, characterized in that the risk group includes persons with intravenous drug addiction, congenital and acquired heart defects, prosthetic valves and other heart operations, coronary arteries, immunodeficiency states.

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