RU2793531C1 - Method for early prediction of the risk of development of diabetic nephropathy in patients with type 1 diabetes mellitus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to endocrinology, genetics, nephrology, genetic engineering, biochemistry, and can be used for early prediction of the risk of developing diabetic nephropathy (DNP) in patients with type 1 diabetes mellitus. DNA is isolated from venous blood lymphocytes. The CYP2C19 (G681A) gene is genotyped by polymerase chain reaction followed by restriction analysis. If a patient has a heterozygous polymorphism G681A of the CYP2C19 gene and an excess of the activity of malondialdehyde (MDA), catalase (CAT), glutathione-S-transferase (G-ST) and superoxide dismutase (SOD) over the reference values — indicators of the control group of healthy donors, the high risk of developing NPD is predicted.

EFFECT: method allows at an early stage to reliably and informatively predict the risk of developing rapidly progressive nephropathy, to take timely measures to correct this condition and to prevent the early development of such severe complications of DNP as anemia, arterial hypertension, pathology of phosphorus-calcium metabolism, dyslipidemia, proteinuria, etc. due to determination of heterozygous polymorphism G681A of the CYP2C19 gene and excess activity of MDA, CAT, G-ST and SOD in a patient.

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Description

The present invention relates to medicine, namely to endocrinology, genetics, nephrology, genetic engineering, biochemistry, and can be used to predict the severe course of diabetic nephropathy in people with type 1 diabetes mellitus.

Type 1 diabetes mellitus (DM 1) is a polygenic multifactorial disease, which is based on immune-mediated or idiopathic destruction of pancreatic β-cells, leading to absolute insulin deficiency (Clinical guidelines for the treatment of type 1 diabetes mellitus, 2021). Multifactorial diseases are based on genetic predisposition and environmental factors. Multifactorial diseases (MFD) are the most numerous and diverse group of diseases, accounting for more than 90% of all human somatopathology and characterized by the highest growth rates of morbidity, mortality and disability of the working population in modern populations [Bochkov N.P. et al., 2011]. The most common MDs include: rheumatoid arthritis, coronary heart disease, hypertension and peptic ulcer disease, cirrhosis of the liver, diabetes mellitus, etc., which are accompanied by various complications. A special role in the prognosis of the course of the disease and the quality of life of the patient is played by such a formidable complication of type 1 diabetes as diabetic nephropathy (DNP), leading in its outcome to Chronic kidney disease. DNP is formed as a result of hemodynamic and metabolic factors and occurs in approximately 20-40% of patients with type 1 diabetes (Dedov II, Shestakova MB., 2016). At the same time, mortality in people with type 1 diabetes and DNP is 2 times higher than in patients without DNP (Dreval A.V., Misnikova I.V., 2010). Identification of predictors that, in addition to already known damaging factors (hyperglycemia, arterial hypertension, dyslipidemia, immune inflammation, oxidative stress), can affect the severity and speed of progression of nephropathy, primarily due to excessive formation of membrane and cytotoxic products of lipid peroxidation (LPO), is an important problem for deepening knowledge about the mechanisms of development and course of NPD. In connection with the active development of genetic technologies and the increase in knowledge in the field of molecular genetic research, the study of the relationship between individual allelic genes and pathological processes is becoming widespread (Kuzmina L.P., Bezrugavnigova L.M., 2006). A significant number of available data indicates the involvement of various polymorphic genes in the formation of predisposition to multifactorial pathology, both across the spectrum and to individual nosological forms [Risch N.J., 2000; Hirschhorn J.N. et al., 2002; Lohmueller K.E. et al., 2003; Baranov B.C., 2009]. In the conditions of modern technogenic civilization, it is believed that the most significant contribution to the formation of predisposition to socially significant human diseases can be made by the genes of the enzymes of the protective and adaptive systems of the body - these are, first of all, the genes of the components of the immune surveillance system, antioxidant defense (AOD) and xenobiotic biotransformation enzymes ( FBK) [Polonikov A.V. et al., 2008]. Detection of polymorphic alleles of xenobiotic biotransformation genes, which, on the one hand, neutralize substances dangerous to the body, and, on the other hand, produce reactive oxygen species in the process of functioning (ROS), which are highly reactive and are potential factors intensifying free-radical oxidation processes (SRO) and lipid peroxidation, will allow predicting and in advance including antioxidant and nephroprotective drugs in the complex therapy of DM. All of the above determines the relevance of studying the role of polymorphic variants of the genes of the xenobiotic biotransformation system in the development of pathobiochemical disorders in patients with DNP and will make it possible to determine additional effective methods for monitoring ongoing therapy at the molecular genetic level.

A known method for predicting the development of chronic kidney disease (CKD) in patients with diabetes mellitus (DM) (2021115852, 06/02/2021 IPC A61 B 5/00 (2006.01) G01N 33/50 (2006.01)), including determining the type of diabetes mellitus and the diagnostic complex significant indicators: gender (x sex), age at diagnosis (x age), history of myocardial infarction (x infarction), history of diabetic coma (x coma), presence of diabetic retinopathy (x retinopathy), mass index value body (BMI) (x BMI), based on the data obtained and in the presence of type 1 diabetes, the probability of developing CKD after 5 years in patients with type 1 diabetes (p) is calculated using the formula:

Where

A method is known for diagnosing the risk of developing atherosclerosis or diabetic retinopathy in a subject (application 2001130755/14, C12Q 1/68, BIPM No. 24, 27.08.2003), which includes determining the presence of polymorphism in a patient with type 2 diabetes mellitus in the region of the signal peptide prepro- human NPY. The polymorphism consists in the substitution of leucine for proline at the 7th position in the human prepro-NPY signal peptide region and is an indicator of an increased risk of developing diabetic retinopathy and atherosclerosis. But, unfortunately, patients with type 1 diabetes did not participate in this study and glycemic control was different in all the subjects. That does not allow us to judge the absence of the influence of other external factors on the formation of retinopathy.

The closest analogue is a method for predicting the risk of developing diabetic retinopathy in type 2 diabetes mellitus in Yakuts (Application: 2011137055/15, 09/07/2011 G01N 33/50 (2006.01) C12Q 1/68 (2006.01)), including DNA extraction from venous blood lymphocytes, genotyping of the interleukin gene by polymerase chain reaction followed by restriction analysis, characterized in that genotyping of interleukin - 6 is carried out and if the *G allele of the polymorphic variant -572 G / C of the interleukin-6 gene - IL-6 is detected, the risk of developing diabetic retinopathy is predicted.

The applicants have not found analogues for the methods of complex genetic prediction of the severity of NPD in patients with type 1 diabetes. Namely, nephropathy is one of the main causes of death in patients with diabetes mellitus. In addition, in the above closest analogue, patients had different levels of glycated hemoglobin, respectively, with different degrees of carbohydrate metabolism compensation, which does not allow us to reliably judge the contribution of only the studied polymorphism to the prediction of retinopathy. From a practical point of view, patients should be as similar as possible to each other in terms of age, duration of diabetes, degree of glycemic control, and accepted (or not accepted) therapy. This will eliminate the influence of related factors on the development of diabetic complications to a greater extent.

Objective: to determine the most reliable and informative molecular genetic and biochemical markers in the early stages, leading to a progressive course of DNP in people with type 1 diabetes.

The essence of this invention is the detection of heterozygous polymorphism G681A of the CYP2C19 gene, excess activity of enzymes of the AOD system and indicators of free radical oxidation processes from reference values - indicators of the control group of healthy donors, in the presence of these changes, determine a high risk of developing NPD.

Technical result: in connection with the isolation of the heterozygous polymorphism G681A of the CYP2C19 gene and the determination of the activity of enzymes of the AOD system and the indicators of free radical oxidation processes (catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (G-S-T)) and the level of malonic dialdehyde (MDA), as the main marker of the processes of FRO and LPO, as well as biochemical markers, such as the level of creatinine in the blood serum, the calculation of the glomerular filtration rate (GFR) and the determination of the level of protein in the urine, can allow at an early stage to reliably and informatively predict the risk development of rapidly progressive nephropathy, timely take measures to correct this condition and prevent the early development of such severe complications of DNP as anemia, arterial hypertension, pathology of phosphorus-calcium metabolism, dyslipidemia, proteinuria, etc.

To prove the solution of the problem, DNA was isolated from venous blood lymphocytes, genotyping of PBK genes by the polymerase chain reaction method, followed by restriction analysis. Detection in a patient of heterozygous polymorphism G681A of the CYP2C19 gene and, associated with this, an increase in the activity of malondialdehyde (MDA), catalase (CAT), glutathione-S-transferase (G-S-T) and superoxide dismutase (SOD) over the reference values - indicators of the control group of healthy donors predicts a high risk of developing diabetic nephropathy.

For laboratory studies, blood serum samples obtained by centrifuging tubes with whole venous blood at a speed of 3 thousand rpm for 5 minutes at room temperature are used. Along with a general clinical examination, fasting glucose levels and postprandial glycemia are assessed in patients, as well as a complete blood count, a biochemical blood test, a general urinalysis, and the determination of glycated hemoglobin. Microalbuminuria is assessed in a single portion of urine after complete stabilization of the glycemic profile (in the control general urinalysis before discharge). In order to assess the oxidative status before the patient was discharged from the hospital, after complete stabilization of glycemia, blood was taken to determine the state of the pro-/antioxidant system (MDA - an indicator of the FRO and LPO processes and AOD and FBK enzymes - CAT, SOD, G-S-T) .

The method was tested on 50 patients with type 1 diabetes, having a similar level of glycated hemoglobin (the average level in the experimental group is 8.7%), and the duration of type 1 diabetes is 7-10 years. The control group was formed from 20 apparently healthy volunteers matched in age, gender and ethnicity, who were not relatives of patients from the main groups and did not have a history of diabetes mellitus and other chronic diseases.

Whole venous blood, which was taken once when the patient was included in the study, served as the material for the molecular genetic study. To isolate genomic DNA, a sorbent method was used using the DNA-express blood reagent kit (Litech, Russia), then the genotyping of the CYP2C19 gene was performed using the real-time leukocyte fraction polymerase chain reaction (RT-PCR) on a RotorGene analyzer. G681A. For this, the appropriate reagent kits (Litech, Russia) were used. Registration of FAM and HEX made it possible to determine three variants of the genotype:

гомозигота по основному аллелю (аллель 1),

homozygous for the main allele (allele 1),

гетерозигота,

heterozygote,

гомозигота по минорному аллелю (аллель 2) Было проведено сравнение различных вариантов полиморфизма G681A гена CYP2C19 с показателями СКФ, микроальбуминурии, потребностью в инсулине и показателями общего антиоксидантного статуса. Значимые отклонения в вышеуказанных показателях выявлены для гетерозиготного варианта полиморфизма G681A гена CYP2C19.

homozygous for the minor allele (allele 2) Different variants of the G681A polymorphism of the CYP2C19 gene were compared with indicators of GFR, microalbuminuria, insulin requirement, and indicators of the general antioxidant status. Significant deviations in the above indicators were found for the heterozygous variant of the G681A polymorphism of the CYP2C19 gene.

The study of indicators of the pro-/antioxidants system was carried out according to the following methods:

1) MDA: Steel I.D., Garishvili T.G., 1977.

2) Superoxide dismutase (SOD): Sirota T.V., 1999.

3) Glutathione-S-transferase (G-S-T): Karpishchenko A.I., 1999.

4) Catalase (CAT): Korolyuk M.A. et al., 1988.

The indicators of patients in the control group were taken as normal (reference) values of the activity of enzymes of the AOD system.

The significance of differences in the distribution of genotype frequencies between groups of patients and healthy individuals was assessed using the F 2 test. Quantitative indicators in the clinical characteristics of patients were assessed using Student's t-test. The calculations were performed using the BIOSTAT program. Differences were considered statistically significant at p less than 0.05.

When comparing the experimental and control groups for the CYP2C19 (G681A) gene, there were no significant differences in the percentage between homo- and heterozygous carriers of polymorphic variants of this gene. The number of heterozygous carriers (GA) is 15%, homozygous carriers for allele 1 (GG) - 85%. However, significant differences were found in the level of GFR in the experimental group. Thus, carriers of the heterozygous mutation of the CYP2C19 gene had a lower level of GFR (73.7 ml/min/1.73 m ² ), higher microalbuminuria (0.21 g/l - stage A3) compared with homozygous carriers (GFR - 86, 2 ml / min / 1.73 m ² ; microalbuminuria - 0.18 g / l), while the need for insulin per day was higher in individuals with homozygous carriage - 54.5 U / day. compared to 46.8 U/day in heterozygous carriers. Carriers of the mutant homozygote for allele 2 were not found either in the control or in the experimental group.

Example

Patient A., born in 1994, has been suffering from type 1 diabetes for 8 years. Receives IT (aspart and glargine 300) in an average total daily dose of 48 units. At home, I tried to maintain glycemia in the range of 8-13 mmol / l. Due to strong emotional stress in the last 2-3 weeks, I rarely measured glycemia, skipped insulin injections, did not count bread units, injected insulin “by eye”. The level of glycemia against this background increased to 20-25 mmol / l, there was a pronounced dry mouth, thirst, nausea and single vomiting. I could not cope with the decompensation of diabetes mellitus on my own. Hospitalized in the therapeutic department of the Emergency Hospital. From the anamnesis: DM type 1 for 8 years. Chronic diseases: chronic tonsillitis, compensation stage. Biochemical blood test: Blood potassium - 3.3 (3.4-5.5 mmol / l) Creatinine - 97 g / l, GFR - 70 ml / min / 1.73 m ² . Complete blood count - no features. Urinalysis at admission - glucose +++, ketone bodies +++, protein - 1.0 g/l. Acid-base state: pH - 7.11, sBE, s - (-8) mmol / l. Glycated hemoglobin - 8.4%. Infusion therapy, insulin therapy, therapy with alpha-lipoic acid preparations were prescribed. By the time of discharge, after stabilization of the condition and achievement of normoglycemia, a general urinalysis was repeated. The protein level in the urine was 0.6 g/l (600 mg/l) in a single portion of urine. After stabilization of glycemia, blood was taken to assess the pro-/antioxidant system and FBA. MDA level - 28.9 µM/l, CAT - 55.1 nmol/mg, G-ST - 56.6 µmol/min/mg, SOD - 100.1 arb. units. That significantly exceeds similar indicators in the control group.

The patient was examined by an ophthalmologist. The diagnosis is non-proliferative retinopathy.

The final diagnosis was formulated: Type 1 diabetes mellitus, decompensation with ketoacidosis at admission. Non-proliferative retinopathy, Diabetic nephropathy with slightly reduced GFR (GFR = 70 ml/min/1.73 m2). Diabetic distal polyneuropathy of the lower extremities, sensory type. The target level of glycated hemoglobin is less than 6.5%. Chronic tonsillitis, stage of compensation.

During the study of the CYP2E1 gene, a heterozygous polymorphism was found.

In this connection, the patient was offered antioxidant therapy: Vitamin E 400 mg daily for 1-2 months in courses 2 times a year, alpha-lipoic acid preparations 600 mg 1 time per day for 2-3 months in courses 2 times a year. Yearly follow-up by an endocrinologist with a more thorough assessment of kidney function. And also, in the event of arterial hypertension, the use of angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor antagonists (ARA) for the purpose of nephroprotection.

Claims (1)

A method for early prediction of the risk of developing diabetic nephropathy in patients with type 1 diabetes mellitus, which consists in isolating DNA from venous blood lymphocytes, genotyping the CYP2C19 (G681A) gene by polymerase chain reaction followed by restriction analysis, and if heterozygous polymorphism is detected in a patient G681A of the CYP2C19 gene and excess activity of malondialdehyde (MDA), catalase (CAT), glutathione-S-transferase (G-S-T) and superoxide dismutase (SOD) over the reference values - indicators of the control group of healthy donors, predict a high risk of developing diabetic nephropathy.