RU2507520C1 - Method for prediction risk of development of diabetic angiopathy of lower extremities in patients suffering type 2 diabetes mellitus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: DNA is recovered from peripheral venous blood; polymorphism of the tumour necrosis factor α gene -308G/A is analysed, and if observing the allele -308A TNFα, a high risk of the development of angiopathy of the lower extremities in the patients suffering type 2 diabetes mellitus is predicted.

EFFECT: obtaining the new criteria for assessing the risk of the development of diabetic angiopathy of the lower extremities in the patients suffering type 2 diabetes mellitus.

1 dwg, 1 tbl, 1 ex

Description

The invention relates to the field of medical diagnosis, can be used to predict the risk of developing lower limb angiopathy in patients with type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is an endocrine disease that occurs as a result of pancreatic b-cell damage or the development of insulin resistance [Joseph NA., Greenberg AS. Adipocytocines and insulin resistance. // J Clin Endocrinol. Metab. 2004. - No. 89 (2). - P. 447-460]. The disease is manifested by hyperglycemia, damage to target organs [Dedov II, Melnichenko GA, Fadeev VV Endocrinology: a textbook for medical universities / Ed. Acad. RAMS I.I.Dedova. - M.: GEOTAR-Media, 2008. - 430 p.].

The course of type 2 diabetes is characterized by the development of various complications. Its most common complications are diabetic retinopathy, angiopathy of the lower extremities, diabetic nephropathy, polyneuropathy of the lower extremities. The relationship between the life expectancy of patients and the complications of diabetes is obvious. One of the terrible complications of type 2 diabetes is angiopathy of the lower extremities, which is manifested by pain in the legs, the development of "intermittent claudication", a decrease in pulsation of the arteries of the feet and the development of cider of diabetic foot. In the future, if untreated, gangrene develops with subsequent amputation of the limb [Williams D.T., Price P, Harding K.G. "An Evaluation of Screening Methods for Arterial Component of Diabetic Foot Disease in the Community." Program and abstractbook of the 4 th international Symposium on the Diabetic foot, 22-24 May 2003, Noordwijekerhout, The Netherlands: 62].

As evidenced by the results of a number of studies, a significant role in the development of complications of type 2 diabetes is played by pro-inflammatory cytokines, such as TNF and its receptors [Rydlovskaya A.V., Simbirtsev A.S. "Functional TNFα gene polymorphism and pathology." Cytokines and inflammation. 2005.Vol. 4, No. 3. C.4-10]. Tumor necrosis factor - extracellular protein, a multifunctional pro-inflammatory cytokine produced mainly by monocytes and macrophages, affects lipid metabolism, coagulation, insulin resistance and endothelial function. This cytokine acts on target cells of almost all organs and tissues. The human TNFα gene (TNFα) was cloned in 1985. The gene is mapped on chromosome 6p21.3, at the locus encoding the molecules of the main histocompatibility complex of the first (HLA-A, B, C) and second classes (HLA-DP, DQ, DR), between the Ltα and Ltβ genes [Glotov OS Baranov BC Genetic polymorphism, multifactorial diseases and longevity // Medical Genetics-2007. - t.6, No. 4 (58) -C.17-29], has a size of 2762 bp and contains 4 exons. The last exon encodes protein by more than 80%. More than 30 polymorphic variants of the gene are known (microsatellites, SNP polymorphisms), but only about half of them affect the expression of TNF-α in vitro. According to the literature, a highly productive allele in the -308G / A TNFα genetic polymorphism is the -308A TNFα allele [Fernandez N., Kopesh B, Fernandes N, Hameed M, Cohen MC, Raveche E, Cohen S. Investigation of promoter polymorphisms in the tumor necrosis factor-alpha and interleukin-10 genes in liver transplant patients. Transplantation 2002; 73: 1886-1891].

There are three main directions of TNFα action: cytotoxic, directed to tumor cells; immunomodulatory and pro-inflammatory, caused by the activation of macrophages, neutrophils, eosinophils and endothelial cells; effect on metabolism. TNFα is actively involved in the process of rapidly increasing cell size and initiating apoptosis. In addition to the direct pro-inflammatory effect, TNFα has a wide range of immunoregulatory effects and is involved in the regulation of metabolic processes [Baranov B.C. Human genome and “predisposition” genes. (Introduction to predictive medicine) / B.C. Baranov, E.V. Baranova, T.E. Ivashchenko // St. Petersburg: "Intermedica", 2000-272 p.].

In the studied medical and accessible patent literature, the authors did not find a method for predicting the risk of developing lower limb angiopathy in patients with type 2 diabetes mellitus based on data on tumor necrosis factor gene polymorphism.

To assess the current patent situation, a search was performed on the title documents for the period from 1990 to 2012. The analysis of the documents was carried out in the direction: a method for predicting the risk of developing lower limb angiopathy in patients with type 2 diabetes mellitus based on molecular genetic data depending on polymorphic markers of tumor necrosis factor genes.

A known method for the diagnosis of metabolic compensation disorders and a method for predicting the risk of complications in patients with type 2 diabetes mellitus on the background of lymphocyte apoptosis according to RF patent No. 2452965, publ. 06/10/2012, "A method for diagnosing metabolic compensation disorders and predicting the risk of complications in patients with type 2 diabetes mellitus by assessing lymphocyte apoptosis." The invention relates to medicine, namely to endocrinology, and can be used to diagnose metabolic compensation disorders and predict the risk of complications in patients with type 2 diabetes mellitus (type 2 diabetes). The method includes collecting peripheral venous blood and determining the percentage of apoptotic lymphocytes An + / PI-, when the content of An + / PI- lymphocytes is lower than 3.5%, a state of compensation of carbohydrate metabolism is diagnosed and a favorable course of the disease is predicted for the coming year, when the content of An + / PI lymphocytes - in the range from 3.5 to 6%, the state of subcompensation is diagnosed and a moderate risk of complications is predicted, the level of An + / PI lymphocytes above 6% characterizes the state of decompensation and a high risk of developing x ronic complications of diabetes.

The disadvantage of this method is that it can only be used to predict the risk of complications, but it is not possible to predict which complication a patient may have.

For the prototype of the selected RF patent No. 2461005, publ. 09/10/2012, "A method for predicting the risk of developing diabetic retinopathy in type 2 diabetes mellitus in Yakut", which proposes a method for predicting the development of diabetic retinopathy by typing DNA from peripheral venous blood lymphocytes and detecting adiponectin gene polymorphism (ADIPOQ) by T45G polymerase chain reaction. When the * T / * T genotype and * T allele are identified, the risk of developing diabetic retinopathy is predicted.

The disadvantage of the prototype is that it considers only one of the complications of type 2 diabetes mellitus - diabetic retinopathy, and is intended only for patients of a certain ethnicity, namely Yakuts.

The objective of this study is to expand the arsenal of diagnostic methods, namely, to create a method for predicting the risk of developing diabetic angiopathy of the lower extremities in individuals of Russian nationality according to the genetic polymorphism of tumor necrosis factor-α (-308G / A TNFα).

The technical result of using the invention is to obtain criteria for assessing the risk of developing diabetic angiopathy in individuals of Russian nationality with type 2 diabetes mellitus.

In accordance with the task, a method was developed for predicting the risk of developing diabetic angiopathy in patients with type 2 diabetes mellitus, including:

- DNA isolation from peripheral venous blood;

- analysis of tumor necrosis factor gene polymorphism (-308G / A TNFα);

- predicting an increased risk of developing diabetic angiopathy of the lower extremities in patients with type 2 diabetes mellitus in the case of the detection of the -308A TNFα allele.

The novelty and inventive step consists in the fact that the possibility of predicting the risk of developing lower limb angiopathy in patients with type 2 diabetes mellitus by the presence of the -308A allele of the tumor necrosis factor α gene (polymorphism -308G / A TNFa) is not known from the prior art.

The method is as follows.

DNA is isolated from samples of peripheral venous blood of patients with type 2 diabetes in 2 stages. At the first stage, 25 ml of lysis buffer containing 320 mM sucrose, 1% Triton X-100, 5 mM MgCl ₂ , 10 mM Tris-HCl (pH 7.6) is added to 4 ml of blood. The resulting mixture was stirred and centrifuged at 4 ° C, 4000 rpm for 20 minutes. After centrifugation, the supernatant is decanted, 4 ml of a solution containing 25 mM EDTA (pH 8.0) and 75 mM NaCl are added to the precipitate and resuspended. Then, 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) are added and the sample is incubated at 37 ° C for 16 hours.

At the second stage, DNA is sequentially extracted from the obtained lysate in equal volumes of phenol, phenol-chloroform (1: 1) and chloroform with centrifugation at 4000 rpm for 10 minutes. After each centrifugation, the aqueous phase is selected. DNA is precipitated from solution in two volumes of chilled 96% ethanol. The formed DNA is dissolved in bidistilled deionized water and stored at -20 ° C.

The isolated DNA is then subjected to polymerase chain reaction using standard oligonucleotide primers (table 1).

Typing of the polymorphic locus of tumor necrosis factor α is carried out by polymerase chain reaction of DNA synthesis on an IQ5 amplifier (Bio-Rad) using standard oligonucleotide primers and probes (Table 1), followed by analysis of polymorphism by allele discrimination method. The reaction mixture with a volume of 25 μl includes: 67 mm Tris-HCl (pH 8.8), 2.5 mm MgCl ₂ , 0.1 μg of genomic DNA, 10 pM of each primer, 5 pmol of each probe, 200 μM dATP, dGTP, dCTP, dTTP and 1 unit of active Taq polymerase. After denaturation (5 min at 95 ° C), 40 amplification cycles were performed according to the scheme: primer annealing - 1 min. at 52 ° C; denaturation - 15 sec at 95 ° C.

Table 1 Gene Polymorphism and its localization
in the gene Primer structure Literature TNFα -308G / A F: 5'-GAAATGGAGGCAATAGGTTTTTGAG-3 ' (T.Kim, 2003) R: 5'-GGCCACTGACTGATTTGTGTGTAG-3 ' (promoter) 5'-FAM-CCGTCCTCATGCC- RTQ1-3 '5' ROX-CCGTCCCCATGCC - RTQ1-3 '

- гомозиготы -308АА,

- гомозиготы -308GG,

- гетерозиготы -308GA,

- отрицательный контроль), которая осуществляется методом Tag Man зондов по данным величин RFU, где RFU - это уровень относительной флуоресценции (УОФ) каждого зонда. Зонд с флуоресцентным красителем ROX соответствует аллелю G, зонд с красителем FAM - аллелю А.The invention is characterized in figure 1, which presents the discrimination of alleles at the locus -308G / A TNFa (where

- homozygotes -308AA,

- homozygotes -308GG,

- heterozygotes -308GA,

- negative control), which is carried out by the Tag Man method of probes according to the RFU values, where RFU is the level of relative fluorescence (UOF) of each probe. A probe with a fluorescent dye ROX corresponds to allele G, a probe with a dye FAM corresponds to allele A.

Two bands, vertical and horizontal, divide the graph into four sections: one for each homozygous state, one for the heterozygous state and a section without reaction. Assigning genotypes to unknown samples is determined by plotting the RFU for one fluorophore (on the x axis) relative to the RFU for another fluorophore (on the y axis) in the allele discrimination diagram.

- If the RFU of an unknown sample is above the horizontal strip and to the right of the vertical strip, the genotype is heterozygous (GA).

- If the RFU values of an unknown sample are above the horizontal strip and to the left of the vertical strip, the genotype is homozygous for the G allele (the RFU of the G allele is plotted along the y axis).

- If the RFU of an unknown sample is below the horizontal strip and to the right of the vertical, the genotype is homozygous for the A allele (the RFUs of the A allele are plotted along the x axis).

- If the RFU values of an unknown sample are below the horizontal strip and to the left of the vertical, determination of the genotype is impossible (in this case, an undefined sample is a negative control).

The formation of the database and statistical calculations were carried out using the program "STATISTICA 6.0". Associations of alleles and genotypes of the studied DNA markers with the development of lower limb angiopathy in type 2 diabetes mellitus were evaluated by analyzing 2 × 2 contingency tables with calculation of the χ ² criterion with Yates adjusted for continuity and chance ratios (OR) with 95% confidence intervals (CI) [Rebrova, O.Yu. Statistical analysis of medical data. Application of the STATISTICA application software package [Text] ./ O.Yu. Rebrov. - M .: Media Sphere, 2006 .-- 305 p .; Borovikov, V. Statistical art of data analysis on a computer. / V. Borovikov. - 2nd ed. - SPb .: Peter, 2003 .-- 688 p.: Ill. - (For professionals)].

The possibility of using the proposed method for assessing the risk of developing angiopathy of the lower extremities during type 2 diabetes mellitus is confirmed by the analysis of the observation results of 236 patients with diabetes mellitus and 308 people of population control. Patients were included in the appropriate group of patients only after a diagnosis of the disease was confirmed, using clinical and laboratory-instrumental examination methods.

The study group included individuals of Russian nationality, who are natives of the Central Black Earth Region of Russia and have no kinship with each other.

It was found that patients with advanced lower limb angiopathy have the highest allele frequency of -308A TNFα (17.31%) compared with the control group (11.06%, χ ² = 5.77, p = 0.02, OR = l, 68, 95% CI 1.09-2.59) and type 2 diabetes mellitus without lower limb angiopathy (10.38%, χ ² = 4.49, p = 0.03).

Thus, the obtained data indicate the involvement of the tumor-necrosis factor -308G / A genetic polymorphism α in the formation of lower limb angiopathy. A marker of the development of lower limb angiopathy in patients with type 2 diabetes is the -308A TNFα allele. When a type 2 diabetes mellitus is detected with a genetic risk factor for developing lower limb angiopathy (-308A TNFα), dynamic monitoring of the state of lower limb vessels (determination of peripheral arterial pulsation, ultrasound dopplerography and lower limb vascular dopplerometry) should be recommended for early diagnosis and timely treatment lower limb angiopathies in patients with type 2 diabetes mellitus (the use of vascular drugs, anticoagulants, lipid-lowering therapy).

Claims (1)

A method for predicting the risk of developing lower limb angiopathy in patients with type 2 diabetes mellitus, including the extraction of DNA from peripheral venous blood, characterized in that they analyze the tumor necrosis factor -308G / A TNFα polymorphism and predict an increased risk of developing lower limb angiopathy in patients type 2 diabetes mellitus in case of detection of the -308A TNFα allele.

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