RU2598878C1 - Method for prediction of risk of poag with ineffective local hypotensive therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention aims at predicting risk of developing primary open-angle glaucoma (POAG) with ineffective local antihypertensive therapy. DNA is extracted from peripheral venous blood of Russian natives of Central Black Earth Region and polymorphisms are analysed VEGF-A c.-958 C>T (rs833061) and IGFR-1 g.99181663 C>T (rs4965425). If allele T IGFR-1 or a combination of allele C VEGF-A with alleles T IGFR-1 is observed, a high risk of POAG with ineffective local antihypertensive therapy is predicted.

EFFECT: invention provides new criteria for assessing risk of POAG with ineffective local antihypertensive therapy in Russian natives of Central Black Earth Region.

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Description

The invention relates to the field of medical diagnosis, can be used to predict the risk of developing primary open-angle glaucoma with ineffective local antihypertensive treatment.

Glaucoma is one of the most severe forms of ophthalmopathology, which has great medical and social significance due to the constant increase in the incidence, high prevalence and severity of disease outcomes leading to blindness and disability. Among the causes of permanent blindness in Russia, glaucoma occupies one of the leading places, exceeding the average European indicator by 1.5-2 times [Libman E.S. Epidemiological characteristic of glaucoma / Libman E.S. //Glaucoma. - 2009. - No. 1 (App.). - C.2-3]. Primary open-angle glaucoma (hereinafter referred to as POAG) is the most common form of the glaucomatous process [E. Egorov. Glaucoma. National Leadership / Ed. E.A. Egorova. - M.: GEOTAR-Media. - 2013. - 824 p.]. POAG is a multifactorial disease, in the etiopathogenesis of which an important role is played by multifunctional cytokines - growth factors that regulate the production of extracellular matrix, affecting angiogenesis, proliferation and apoptosis of cells [Banerjee D. Comprehensive analysis of myocilin variants in east Indian POAG patients / Banerjee D.ee, Bhatt A., Ponda A., Ray K. // Molecular Vision. - 2012. Vol. 18. - P.1548-1557].

One of the key mechanisms for the development of glaucoma optical neuropathy (hereinafter referred to as GON) is the dysregulation of the optic nerve blood flow. In response to tissue hypoxia, endothelial cells secrete pro-angiogenic factors, such as vascular endothelial growth factor VEGF-A and insulin-like growth factor receptor IGFR-1. This process stimulates a cascade leading to the activation of apoptosis of retinal ganglion cells [Resch H. Endothelial dysfunction in glaucoma. / Resch H., Garhofer G., Fuchsjager-Mayrl G. // Acta Opthalmologica. - 2009. - vol. 87. - P.4-12].

Elevated intraocular pressure (IOP) is one of the leading risk factors for the progression of glaucoma optic neuropathy [Kuroedov A.V. Risk factors for the progression of primary open-angle glaucoma. / Kuroedov A.V., Gorodnichy V.V. // Glaucoma: theory, trend, technology: Sat. scientific Articles VI Int. conf. - M., 2008. - S. 370-385]. POAG refers to irreversible pathology, stabilization of the process depends on timely treatment. In cases where local antihypertensive treatment of POAG is ineffective, IOP is higher than tolerant, it is necessary to proceed to surgical treatment. Thus, the earlier normalization of IOP will be made, incl. surgically, the greater the likelihood of maintaining visual function [Egorov E.A. Glaucoma. National Leadership / Ed. E.A. Egorova. - M.: GEOTAR-Media. - 2013. - 824 p.].

The receptor for insulin-like growth factor-1 IGFR-1 is a gene expression product located in humans on the distal portion of the long arm of chromosome 15, structurally similar to the insulin receptor gene and consists of 22 exons. The IGF-1 receptor gene is expressed in almost all tissues [Baserga R. The IGF-1 receptor in cancer biology. / Baserga R., Peruzzi F., Reiss K. // Int. J. Cancer. - 2003. - Vol. 107. - P. 873-877].

The vascular endothelial growth factor VEGF-A is a heparin-binding glycoprotein with a molecular weight of 45 kDa - it is a specific mitogen of endothelial cells, which stimulates physiological and pathological angiogenesis, growth and proliferation of endotheliocytes, and inhibits the apoptosis of circulating precursors of the endothelial cells N. and FerraEG itsraraEG [FerraEG receptors / Ferrara N., Gerber HP, Le Couter J. // Nat. Med. - 2003 .-- Vol. 9 (6). - P.669-76].

The VEGF-A gene is located on the short arm of chromosome 6 at the 6p21.3 locus and consists of eight exons separated by seven introns with alternative splicing and controls the synthesis of a whole group of proteins. VEGF-A gene expression is modulated by various factors, in particular cytokines, lipopolysaccharides, hormones and hypoxia. VEGF-A expression is highly variable, with at least 25 different polymorphisms that define it [Heist R. VEGF polymorphisms and survival in early-stage non-small cell lung cancer. / Heist R., Zhai R., Liu G. // Journal of clinical oncology. - 2008 .-- Vol.26. - P.856-62].

To assess the current patent situation, a search was performed on the title documents for the period from 1990 to 2014. The analysis of documents was carried out in the direction: A method for predicting the risk of developing POAG with ineffective local antihypertensive treatment.

Sources of information: sites of the Federal Institute of Industrial Property http://fips.ru.

In the studied scientific and medical and accessible patent literature, the authors did not find a way to predict the risk of developing primary open-angle glaucoma with ineffective local antihypertensive treatment.

The method for studying the involvement of genetic variants of tumor necrosis factors and their receptors in the formation of POAG in a group of patients with uncompensated IOP on the background of conservative treatment, outlined in the dissertation by EV Tikunova, was selected for the prototype. "The role of genetic polymorphisms of tumor necrosis factors and their receptors in the development of primary open-angle glaucoma." In the course of this study, the involvement of genetic polymorphisms of tumor necrosis factors and their receptors in the formation of the disease in a group of patients with POAG with uncompensated IOP on the background of conservative therapy in individuals of Russian nationality, natives of the Central Black Earth Region was analyzed. Conservative therapy included the appointment of instillations of various groups of drugs: prostaglandin analogues, β-blockers, carbonic anhydrase inhibitors, α ₂ -adrenoreceptor agonists, in the form of monotherapy or their combinations. Uncompensated IOP was considered the level of ophthalmotonus above tolerant pressure - the upper limit of the individual norm of IOP [Egorov E.A. Glaucoma. National Leadership / Ed. E.A. Egorova. - M.: GEOTAR-Media. - 2013. - 824 p.].

The disadvantages of this method include the fact that it does not take into account the role of genetic polymorphisms of growth factors in forming the risk of developing primary open-angle glaucoma with ineffective local hypotensive treatment.

The objective of this study was to expand the arsenal of methods for diagnosing POAG with ineffective local antihypertensive treatment.

The technical result of using the invention is to obtain criteria for assessing the risk of developing POAG with ineffective local antihypertensive treatment in individuals of Russian nationality, natives of the Central Black Earth Region.

In accordance with the task, a method was developed for predicting the risk of developing primary open-angle glaucoma with ineffective local antihypertensive treatment, including:

• DNA extraction from peripheral venous blood;

• analysis of polymorphisms of the VEGF-A genes S.-958C> T (rs 833061), IGFR-1 g.99181663 C> T (rs 4965425);

• predicting an increased risk of developing primary open-angle glaucoma with ineffective local antihypertensive treatment if a genetic variant T IGFR-1 or a combination of the VEGF-A allele C with the IGFR-1 T allele is detected.

The novelty and inventive step lies in the fact that the possibility of predicting the risk of developing primary open-angle glaucoma with ineffective local antihypertensive treatment according to the genetic variants of the VEGF-A loci S.-958C> T (rs 833061), IGFR-1 g .99181663 C> T (rs 4965425) and combinations thereof.

The invention is characterized in the following figures.

- CС VEGF-A,

- ТТ VEGF-A,

- СТ VEGF-A,

- отрицательный контроль.In FIG. 1 shows discrimination of alleles at the VEGF-A locus c. -958C> T (rs 833061), where

- CC VEGF-A,

- TT VEGF-A,

- ST VEGF-A,

- negative control.

- CС IGFR-1,

- ТT IGFR-1,

- СТ IGFR-1,

- отрицательный контроль.In FIG. Figure 2 shows the allele discrimination at the IGFR-1 locus g.99181663C> T (rs 4965425), where

- CC IGFR-1,

- TT IGFR-1,

- ST IGFR-1,

- negative control.

In FIG. 3 in table 1 presents a comparative assessment of the frequencies of alleles and genotypes of polymorphisms of growth factors in patients with POAG with ineffective local antihypertensive treatment and in the control group.

In FIG. 4 in table 2 presents the prevalence of combinations of alleles / genotypes of growth factor genes in patients with POAG with ineffective local antihypertensive treatment and in the control group.

The method is as follows.

DNA is isolated from samples of peripheral venous blood of individuals in 2 stages. At the first stage, 4 ml of blood is mixed with lysis buffer, which contains 320 g of sucrose, 1% Triton X - 100, 5 ml MgCl ₂ , 10 ml Tris - HCl (pH = 7.6), centrifuged for 20 minutes at 4 ° C on 4000 rpm Then add 4 ml of EDTA - Soline, 400 μl of 10% SDS, 35 μl of proteinase K (10 mg / ml) and incubate for 16 hours at 40 ° C.

At the second stage, DNA is extracted from equal lysate in equal proportions of phenol, phenol-chloroform, chloroform and centrifuged for 10 min at 4000 rpm. After centrifugation, the aqueous layer was removed and two volumes of chilled ethanol 96% were added. The resulting DNA is dissolved in deionized, double-distilled water, stored at a temperature of -20 ° C and subsequently used for polymerase chain reaction.

Analysis of genetic polymorphisms of VEGF-A S.-958C> T, IGFR-1 g.99181663C> T is carried out by PCR DNA synthesis, which is carried out on a CFX96 amplifier (Bio-Rad Laboratories, USA) using oligonucleotide probes and primers for polymerase chain reaction Synthol company. Genotyping is carried out using CFX Manager ™ software.

The possibility of using the proposed method for assessing the risk of developing primary open-angle glaucoma with ineffective local antihypertensive treatment is confirmed by an analysis of the observation results of POAG patients with ineffective local antihypertensive treatment - 162 people (227 eyes) and 191 people (382 eyes) in the control group. The sampling of patients and control was carried out on the basis of the ophthalmology department of the Belgorod Regional Clinical Hospital of St. Joasaph and the medical center of eye microsurgery LLC Ark.

The studied groups were formed from individuals of Russian nationality, who are natives of the Central Black Earth Region of the Russian Federation and have no kinship between themselves. The group of patients included patients with POAG with ineffective local antihypertensive treatment. Uncompensated IOP was considered the level of ophthalmotonus above tolerant pressure when using the maximum possible combinations of antihypertensive drugs [Egorov E.A. Glaucoma. National Leadership / Ed. E.A. Egorova. - M.: GEOTAR-Media. - 2013. - 824 p.]. The unit of study in this analysis was the eye [Slepova O.S. Markers of Fas-mediated apoptosis in primary open-angle glaucoma and the possibility of their pharmacological correction / O.C. Slepova, M.A. Frolov, N.S. Morozova, J.N. Lovpache // Bulletin of Ophthalmology. - 2012. - No. 4. - S. 27-31].

The control group was formed from individuals who did not have acute and chronic eye diseases, who did not have any signs of glaucoma, and also did not have severe somatic pathologies, including those leading to eye damage.

Molecular genetic markers were typed in the laboratory of Human Molecular Genetics at the Medical Institute of the Belgorod State National Research University.

The formation of the database and statistical calculations were carried out using the program "STATISTICA 6.0".

The role of combinations of VEGF-A genetic variants S.-958C> T, IGFR-1 g.99181663C> T in the formation of POAG with ineffective local antihypertensive treatment was studied using the APSampler software [http://sources.redhat.com/cygwin/ ] using the Monte Carlo method by Markov chains and Bayesian nonparametric statistics. To validate the associations found, the exact Fisher test (p _f ) and permutation analysis (p _perm ) were used. The p _{perm value} for an allele or allelic combination is defined as the probability that after balanced mixing of the “diseased POAG” labels - “control” (100 permutations) and a new search using APSampler, the significance of the association with the POAG of an arbitrary allele or allelic combination will be no worse than this estimate for the source [Tsareva E.Yu. Pharmacogenomic studies of the effectiveness of treatment of multiple sclerosis with immunomodulating drugs: the dissertation ... candidate of biological sciences: 03.01.03 / Tsareva Ekaterina Yurevna // - Moscow, 2012. - 140 p.]. The differences in the compared frequencies were considered significant if p _f <0.05 and p _perm ≤ 0.005, provided that the 95% CI values for OR do not intersect 1.

A higher concentration of the T IGFR-1 allele (rs 35767) (63.06%) was found in patients with POAG compared with the control group, where the analyzed parameter was 56.02% (χ ² = 4.47, p = 0.03, OR = 1.34; 95% CI 1.02-1.76) (Fig. 3).

When conducting a comprehensive analysis of the carriage of combinations of alleles and genotypes, it was found that the concentration of the combination of the VEGF-A allele C and IGFR-1 T allele among POAG patients with ineffective local antihypertensive treatment (67.82%) is higher than among the control group (52.60% , P _perm = 0.0003) (Fig. 4). This combination of alleles of the studied polymorphisms increases the risk of developing POAG with ineffective local antihypertensive treatment (OR = 1.90, 95% CI 1.29-2.80).

Thus, the results obtained allow us to conclude that the genetic variant of T IGFR-1 and the combination of C VEGF-A alleys with T IGFR-1 are risk factors for the development of POAG with ineffective local antihypertensive treatment.

Examples confirming the feasibility of the proposed invention.

1. Patient A., a native of the Central Black Earth Region of the Russian Federation of Russian nationality, registered with glaucoma and using local medication, found that despite the treatment used, IOP is increased, i.e. local antihypertensive treatment is ineffective. Subsequently, a genetic variant of T IGFR-1 was identified in the patient (rs 4965425). An antiglaucomotamous operation was recommended for this patient.

2. A combination of genetic variants C VEGF-A (rs 833061) and T IGFR-1 (rs 4965425) was revealed in individual V., a native of the Central Black Soil of the Russian Federation of Russian nationality, suffering from POAG. He used topical treatment with combinations of antihypertensive drugs, but IOP was intolerant. Subsequently, he underwent surgical treatment of glaucoma.

Claims (1)

A method for predicting the risk of developing primary open-angle glaucoma (POAG) with ineffective local antihypertensive treatment in individuals of Russian nationality natives of the Central Chernozem region, including blood sampling, characterized in that after DNA extraction from peripheral venous blood, VEGF-A gene polymorphisms s.-958 C are analyzed > T (rs 833061), IGFR-1 g.99181663 C> T (rs 4965425) and they predict an increased risk of developing primary open-angle glaucoma with ineffective local antihypertensive treatment when genetically detected in individuals variant T IGFR-1 or a combination of the C allele VEGF-A with the T allele IGFR-1.

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