RU2812643C2 - Method of early prediction of risk of development of diabetic nephropathy in patients with type 2 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 2 diabetes mellitus. DNA is isolated from venous blood lymphocytes. The GSTP-1 gene is genotyped by polymerase chain reaction followed by restriction analysis. When a homozygous genotype for the G allele of the I105V polymorphism of the GSTP-1 gene is detected and the level of malondialdehyde, superoxide dismutase, catalase and glutathione transferase activity is exceeded, a high degree of risk of developing diabetic nephropathy in the patient is determined.

EFFECT: method provides the ability to early predict the risk of developing diabetic nephropathy in patients with type 2 diabetes mellitus by identifying reliable and informative molecular genetic and biochemical markers.

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Description

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

Type 2 diabetes mellitus (T2DM) is a polygenic, multifactorial disease characterized by chronic hyperglycemia, which results from impaired insulin secretion, insulin action, or both. Multifactorial diseases are based on genetic predisposition and environmental factors. Multifactorial diseases (MFDs) 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 ulcers, liver cirrhosis, diabetes mellitus, etc., which are accompanied by various complications. A special role in the prognosis of the course of the disease and the patient’s quality of life is played by such a formidable complication of type 1 diabetes as diabetic nephropathy (DNP), which ultimately leads 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 I.I., Shestakova M.B., 2016). Moreover, the mortality rate 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 influence the severity and rate of progression of nephropathy, primarily due to the 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 DNP. In connection with the active development of genetic technologies and increased 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 amount of available data indicates the involvement of various polymorphic genes in the formation of predisposition to multifactorial pathology, both across the entire spectrum and to individual nosological forms [Risch N.J., 2000; Hirschhorn J.N. et al., 2002; Lohmueller K.E. et al., 2003; Baranov B.S., 2009]. In the conditions of modern technogenic civilization, it is believed that the most significant contribution to the formation of a predisposition to socially significant human diseases can be made by genes of enzymes of the body’s protective and adaptive systems - these are, first of all, genes of components of the immune surveillance system, antioxidant defense (AOD) and enzymes of xenobiotic biotransformation ( 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 during operation (ROS), which are highly reactive and are potential factors in the intensification of free radical oxidation processes (SRO) and LPO will make it possible to predict and proactively include drugs with antioxidant and nephroprotective effects in the complex therapy of diabetes. All of the above determines the relevance of studying the role of polymorphic variants of genes of the xenobiotic biotransformation system in the development of pathobiochemical disorders in patients with DNP and will allow us to determine additional effective methods for monitoring the therapy at the molecular genetic level.

There is a known method for predicting the development of chronic kidney disease (CKD) in patients with diabetes mellitus (DM) (2021115852, 06/02/2021 IPC A61B 5/00 (2006.01) G01N 33/50 (2006.01)), including determining the type of diabetes mellitus and a set of diagnostically significant indicators: gender (x gender), age at diagnosis (x age), history of myocardial infarction (x heart attack), history of diabetic coma (x coma), presence of diabetic retinopathy (x retinopathy), body mass index value (BMI) (x BMI), based on the data obtained and in the presence of type 1 diabetes, calculate the probability of developing CKD after 5 years in patients with type 1 diabetes (p) using the formula:

,

There is a known method for diagnosing the risk of developing atherosclerosis or diabetic retinopathy in a subject (application 2001130755/14, C12Q 1/68, BIPM No. 24, 08/27/2003), including determining in a patient with type 2 diabetes the presence of polymorphism in the signal peptide region of the prepro- Human NPY. The polymorphism consists of the replacement of leucine with proline at position 7 in the human prepro-NPY signal peptide region and is an indicator of an increased risk of developing diabetic retinopathy and atherosclerosis. But, unfortunately, this study did not include patients with type 1 diabetes, and glycemic control was different for all subjects. This does not allow us to judge the absence of 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 using the polymerase chain reaction method followed by restriction analysis, characterized in that genotyping of interleukin-6 is carried out and when the *G allele of the polymorphic variant -572 G/C of the interleukin-6 gene - IL-6 is identified, the risk of developing diabetic retinopathy is predicted.

The applicants have not found any analogues for methods of complex genetic prediction of the severity of DNP in patients with type 2 diabetes. Namely, nephropathy is one of the main causes of death in patients with diabetes. In addition, in the above-mentioned closest analogue, patients had different levels of glycated hemoglobin, respectively, with different degrees of compensation for carbohydrate metabolism, which does not allow us to reliably judge the contribution of only the studied polymorphism to predicting retinopathy. From a practical point of view, patients should be as similar as possible to each other in age, duration of diabetes, degree of glycemic compensation, and therapy taken (or not taken). This will eliminate to a greater extent the influence of related factors on the development of diabetic complications.

Objective: to determine the most reliable and informative molecular genetic and biochemical markers at earlier stages, leading to the progressive course of DNP in individuals with type 2 diabetes.

The essence of this invention is to isolate a homozygous genotype for the G allele of the I105V polymorphism of the GSTP-1 gene, the excess of the activity of enzymes of the AOD system and the indicators of free radical oxidation processes from the reference values (indicators of the control group of conditionally healthy donors: malondialdehyde - 6.4±0, 5 µmol/l; superoxide dismutase - 75.0±3.07 units/g Hb/min; catalase - 30.5±1.6 nmol H2O2/mg Hb; glutathione transferase - 28.1±1.33 µmol/min/ mg of protein), in the presence of these changes, determine a high risk of developing DNP in the patient.

In connection with the isolation of a mutant homozygous genotype for the G allele of the I105V polymorphism of the GSTP-1 gene and determination of the activity of enzymes of the AOD and detoxification system (catalase, superoxide dismutase, glutathione-S-transferase) and the level of malondialdehyde (MDA), as the main marker of FRO and LPO processes , as well as biochemical markers, such as the level of creatinine in the blood serum, calculating the glomerular filtration rate (GFR) and determining the level of protein in the urine, can allow early prediction of the risk of developing rapidly progressive nephropathy, timely implementation of measures to correct this condition and prevent early development such severe complications of DNP as anemia, arterial hypertension, pathology of phosphorus-calcium metabolism, dyslipidemia, proteinuria, etc.

To prove the solution to the problem, DNA was isolated from venous blood lymphocytes, genotyping of FBK genes using the polymerase chain reaction method, followed by restriction analysis. Identification in a patient of homozygous polymorphism for allele 2 of the GSTP-1 gene (I105V) and associated deviations in the indicators of the pro-/antioxidant system (AOP and MDA enzymes) determine a high risk of progression of DNP to grade 3 severity

To conduct laboratory studies, use blood serum samples obtained by centrifuging tubes with whole venous blood at a speed of 3 thousand rpm for 5 minutes at room temperature. Along with a general clinical examination, patients are assessed for fasting and postprandial glucose levels, as well as a general blood count, biochemical blood test, general urinalysis, and determination of glycated hemoglobin. Microalbuminuria is assessed in a single sample of urine after complete stabilization of the glycemic profile (in the control general urine test before discharge). In order to assess the oxidative status before the patient’s discharge from the hospital, after complete stabilization of glycemia, blood was drawn to determine the state of the pro-/antioxidant system (MDA - an indicator of the processes of FRO and LPO and the enzymes AOP and FBA - Catalase, Superoxide dismutase, Glutathione-S-transferase) .

The method was tested on two groups of patients: patients with type 2 diabetes and patients without type 2 diabetes. The main group included 70 patients with type 2 diabetes aged 45-70 years, without severe concomitant pathologies, with a diabetes duration of up to 5 years, a glycated hemoglobin level of no more than 10%, and a GFR level of at least 60 ml/min/1.73 m2. Exclusion criteria: type 1 diabetes/other types of diabetes mellitus; the presence of severe complications of diabetes (proteinuria, renal failure, macrovascular complications); the presence of severe concomitant somatic pathology; patients with primary kidney damage (infectious, vascular, toxic, immunoinflammatory, tumor); age younger than 45 and older than 70 years; level of glycated hemoglobin more than 10%; the duration of type 2 diabetes is more than 5 years and the GFR level is less than 60 ml/min/1.73 m2.

The control group was formed from 20 volunteers, comparable in age, gender and ethnicity, who were not relatives of the patients in the main group and had no history of type 2 diabetes, severe kidney damage, or severe concomitant somatic pathology.

Genotyping using PCR made it possible to further divide patients of the main and control groups into subgroups depending on the detected polymorphism of the GSTP-1 gene (I105V): homozygotes with a zero (deletion) genotype (homozygotes for allele 2 - 0/0 - G/G), homozygotes with a normal genotype (homozygotes for allele 1 - +/+ - A/A), heterozygotes with genotype +/0 - A/G for certain genes and gene loci.

The material for the molecular genetic study was whole venous blood, which was taken once when the patient was included in the study. To isolate genomic DNA, the sorbent method was used using a set of DNA-express blood reagents (Litekh, Russia), then the GSTP1 polymorphism I105V (A>G) polymorphism was studied using PCR SNP-express electrophoresis on Tertsik equipment. For all polymorphisms, the corresponding reagent kits were used (Litekh, Russia). Registration of FAM and HEX made it possible to determine three genotype variants:

• homozygous for the main allele (allele 1)

• heterozygote,

• homozygous for the minor allele (allele 2)

A comparison was made of various variants of polymorphisms of the xenobiotic biotransformation gene and the AOD system: GSTP1 polymorphism I105V (A>G), with indicators of GFR, microalbuminuria, insulin requirement and indicators of general antioxidant status. Significant deviations in the above indicators were detected for homozygous polymorphism for allele 2 of the GSTP-1 gene (I105V)

The study of the pro-/antioxidant system indicators was carried out using 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 values of patients in the control group were taken as normal (reference) values for the activity of enzymes of the AOD system.

The reliability of differences in the distribution of genotype frequencies between groups of sick and healthy individuals was assessed using the %2 test, using the method of conjugate tables (four-field table). Numerical distributions of indicators of the antioxidant defense system and lipid peroxidation were checked for compliance with the normal distribution using the Shapiro-Wilk test. During the study, the numerical distributions of indicators corresponded to the normal law. Quantitative indicators in the biochemical characteristics of patients (indicators of PBA and SRO enzyme activity) were assessed using the Student's t test. Differences were considered statistically significant at p less than 0.05. Calculations were performed using the STATISTICA 6.1 program Stat-Soft Inc, USA.

In patients with type 2 diabetes (patients of the main group), the percentage of identified heterozygous carriers (+/0 - A/G) of the GSTP-1 gene (I105V) is 16%, and homozygous for allele 1 (+/+ - A/A) - 76%, homozygous for allele 2 (0/0 - G/G) - 8%, in contrast to the control group, in which the percentage of heterozygous carriers (+/0 - A/G) was 65%, and homozygous carriers for allele 1 (+/+- A/A) - 35%, and carriers of homozygotes for allele 2 (0/0 - G/G) - were not identified. When considering the results of the main group for the components of DNP, no significant differences were found between heterozygous carriers (+/0 - A/G) and homozygous carriers for allele 1 (+/+ - A/A) in the level of GFR or microalbuminuria. Thus, the average level of GFR in the subgroups of homozygous and heterozygous polymorphism of the studied gene in the main group was 64 ml/min/1.73 m2, the average level of microalbuminuria was 0.18 g/l, the daily need for insulin was 26 units/day. But they showed patients who were carriers of mutant homozygotes for allele 2 (0/0 - G/G) were significantly worse. Thus, the level of GFR in this subgroup of patients of the main group was 48 ml/min/1.73 m2, the level of albuminuria in a single portion of urine was 0.9 g/l. The daily need for insulin is 17.6 U/day.

The most pronounced changes in FRO and AOD parameters of the main group (MDA and enzymes CAT and SOD) were in the group of homozygous carriers for allele 1. However, this did not correlate with a decrease in kidney function. The activity indicators of AOP enzymes in the subgroup of mutant homozygous carriers (0/0 - G/G) were significantly higher than in the similar subgroup of the control group, but did not differ significantly from the indicators of the subgroups of heterozygous (+/0 - A/G) and homozygous carriers for allele 1 (+/+ - A/A) of the control group. The level of FRO - MDA in the subgroup of mutant heterozygote carriers (+/0 - A/G) was significantly higher (100.5 μmol/l) than in other subgroups of the main group of patients.

The table shows the dependence of the level of AOP and FRO enzymes on the genotype of patients in the main and control groups. All results obtained were statistically significant.

Thus, it was revealed that carriers of mutant homozygotes for allele 2 (0/0 - G/G) of the GSTP-1 gene (I105V) have a higher FRO index, as well as a higher level of AOP enzymes: catalase, superoxide dismutase. This also correlates with a more severe course of DNP in this subgroup (the level of GFR is lower and the level of microalbuminuria is higher than in individuals with other genotypes of the main group). It can be assumed that this is one of the reasons for the rapid progression of kidney damage in this cohort of patients.

Example: Patient V., born in 1970, has been suffering from type 2 diabetes for 8 years. Receives IT (glulisine 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 severe emotional stress, in the last 2-3 weeks I rarely measured glycemia, skipped insulin injections, did not count bread units, and injected insulin “by eye.” Against this background, the glycemic level increased to 20-25 mmol/l, severe dry mouth, thirst, nausea and single vomiting appeared. I could not cope with decompensation of diabetes mellitus on my own. Hospitalized in the therapeutic department of the Emergency Hospital. From the anamnesis: type 2 diabetes for 8 years. Chronic diseases: Stage 2 hypertension, risk 4. Biochemical blood test: Blood potassium - 3.3 (3.4-5.5 mmol/l) Creatinine - 97 g/l, GFR - 70 ml/min/1, 73 ^m2 . General blood test - without any features. General urine analysis upon admission - glucose +++, ketone bodies +, protein - 1.6 g/l. Acid-base state: pH - 7.13, sBE, s - (-4) mmol/l. Glycated hemoglobin - 8.9%. Infusion therapy, insulin therapy, and alpha-lipoic acid therapy were prescribed. At the time of discharge, after stabilization of the condition and achievement of normoglycemia, a general urinalysis was repeated. The urine protein level was 0.6 g/L (600 mg/L) in a single urine sample. After stabilization of glycemia, blood was drawn to evaluate the pro-/antioxidant system and FBA. MDA level - 103.9 µmol/l, CAT - 51.1 nmol H2O2/mg Hb, G-ST - 32.6 µmol/min/mg, SOD - 82.1 units/g Hb/min. Which significantly exceeds similar indicators in the control group.

The patient was examined by an ophthalmologist. The diagnosis is preproliferative retinopathy.

The final diagnosis was formulated:

Diabetes mellitus type 2, decompensation with compensated ketoacidosis on admission. Preproliferative 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 7.0%. Concomitant: Hypertension stage 2, risk 4

During the study of the GSTP-1 (I105V) gene, homozygous polymorphism for allele 2 was discovered.

Therefore, the patient was offered therapy with glucagon-like peptide-1 agonists or sodium-glucose cotransporter type 2 inhibitors. But due to financial considerations, the patient decided to refuse this therapy. Antioxidant therapy was also prescribed: 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. Annual observation by an endocrinologist with a more thorough assessment of kidney function. Observation by a neurologist, ophthalmologist, cardiologist. And also - the use of drugs with angiotensin-converting enzyme inhibitors (ACEIs), or angiotensin II receptor antagonists (ARBs) for the purpose of nephroprotection and for antihypertensive purposes.

Claims (1)

A method for early prediction of the risk of developing diabetic nephropathy in patients with type 2 diabetes mellitus, which consists of isolating DNA from venous blood lymphocytes, genotyping the GSTP-1 gene using the polymerase chain reaction method, followed by restriction analysis, and identifying the homozygous genotype for the G allele of the polymorphism in the patient I105V of the GSTP-1 gene and the excess of the activity of enzymes of the antioxidant defense system and indicators of free radical oxidation processes from the reference values - indicators of the control group of conditionally healthy donors: malondialdehyde - 6.4±0.5 µmol/l; superoxide dismutase - 75.0±3.07 units/g Hb/min; catalase - 30.5±1.6 nmol H2O2/mg Hb; glutathione transferase - 28.1±1.33 µmol/min/mg; in the presence of these changes, a high degree of risk of developing diabetic nephropathy in the patient is determined.