RU2753270C1 - Method for predicting risk of developing primary open-angle glaucoma in women according to genetic data - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and is intended to predict the risk of developing primary open-angle glaucoma (POAG) in unrelated Russian patients, a native of the Central Black Earth region of the Russian Federation. DNA is isolated from the peripheral venous blood. Analysis of genetic markers of matrix metalloproteinases is carried out. A high risk of developing POAG in women is predicted if the GG haplotype is detected at the rs2250889 and rs17577 loci of the MMP9 gene.

EFFECT: invention provides criteria for assessing the risk of POAG development in unrelated women of Russian nationality, natives of the Central Black Earth region of the Russian Federation.

1 cl, 2 dwg, 2 ex

Description

Glaucoma is one of the most severe forms of ophthalmopathology, which is of great medical and social importance due to the high prevalence, constant increase in the incidence and severity of disease outcomes leading to blindness and disability [A metabolomics profiling of glaucoma points to mitochondrial dysfunction, enescence, and polyamines deficiency [Text ] / S. Leruez, A. Marill, T. Bresson [et al.] Invest. Ophthalmol. Vis. Sci. - 2018. - Vol. 59, No. 11. - P. 4355-4361]. Among the clinical forms of the disease, the most common is primary open-angle glaucoma (POAG), which accounts for 72.3 to 96.1% of all forms of glaucoma [MicroRNA-related polymorphisms in pseudoexfoliation syndrome, pseudoexfoliative glaucoma, and primary open-angle glaucoma [Text] / A. Chatzikyriakidou, P. Founti, A. Melidou [et al.] // Ophthalmic. Genet. - 2018. - Vol. 39, No. 5. - P. 603-609]. In the world, more than 90 million people suffer from glaucoma, and by 2030 the number of such patients is expected to double [Biochemical and structural-biomechanical features of the human scleral matrix in primary open-angle glaucoma [Text] / EH Iomdina, N. Yu. Ignatieva, HA Danilov [et al.] // Bulletin of Ophthalmology. - 2011. - T. 127, No. 6. - S. 10-14]. In Russia, according to some sources, there are 750 thousand patients with glaucoma [Violation of vascular-platelet hemostasis as a risk factor for the progression of primary open-angle glaucoma [Text] / NI Kurysheva, VN Trubilin, E. Yu. Irtegova [and others] // Ophthalmology. - 2015. - T. 12, No. 3. - S. 54-62].

POAG is a chronic disease characterized by optic neuropathy, progressive degeneration of ganglion cells and a layer of retinal nerve fibers [Biochemical and structural-biomechanical features of the human scleral matrix in primary open-angle glaucoma [Text] / EH Iomdina, N. Yu. Ignatieva, HA Danilov [and etc.] // Bulletin of ophthalmology. - 2011. - T. 127, No. 6. - S. 10-14].

A feature of POAG is an asymptomatic course and a rather complex and laborious diagnosis at the initial stages, therefore, the detection of this disease in most cases occurs at stages accompanied by already irreversible changes in the optic nerve [Krzyїanowska-Berkowska, P. Relationship between the rate of change in lamina cribrosa depth and the rate of retinal nerve fiber layer thinning following glaucoma surgery [Electronic resource] / P. Krzyїanowska-Berkowska, K. Czajor, I. Helemejko [et al.] // PLoS One. - 2018. - Vol. 13, no. 11. - Art. ID 0206040. - Mode of access: https://journals.plos.org/plosone/article/file?id=10.1371/journal. pone.0206040 & type = printable].

Vision lost as a result of illness is not restored. Despite the achieved success in treatment, more than half of patients continue to lose visual function even after surgical and laser treatment. This disease is one of the main causes of visual impairment and blindness among people of working age in developed countries [Blood pressure, ocular perfusion pressure and open-angle glaucoma in patients with systemic hypertension [Text] / E. Cantor, F. Méndez, C. Rivera [et al.] // Clin. Ophthalmol. - 2018. - Vol. 12. - P. 1511-1517]. To date, there is no unambiguous theory of the etiopathogenesis of the glaucomatous process in POAG with an understanding of the causes and sequence of the development of the glaucomatous process as a whole, while matrix metalloproteinases (hereinafter MMP) are of great importance in the development of POAG.

The family of matrix metalloproteinases is a family of zinc and calcium dependent endopeptidases and consists of more than 20 enzymes that are capable of cleaving almost all components of the extracellular matrix of connective tissue. MMPs serve as the main regulators of the composition of the extracellular matrix.

The main biological function of MMP is to remove components of the extracellular matrix. Metalloproteases regulate the action of growth factors: vascular endothelial factor, fibroblast growth factor receptor, epithelial and insulin-like growth factor (Sawicki G., 2005). MMP-2, -3, -7, -9 promote the activation of transforming growth factor β, which is a chemoatractant for monocytes, releasing it from the matrix [Poteryaeva, ON Matrix metalloproteinases: structure, regulation, role in the development of pathological conditions [Electronic resource ]: literature review / ON Poteryaeva // Medicine and education in Siberia. - 2010. - No. 5. - Art. 2. - Access mode: http://ngmu.ru/cozo/mos/article/pdf.php?id=449]. MMPs play a central role in the metabolism of connective tissue proteins, the processes of normal development of the extracellular matrix, in oncogenesis and angiogenesis [Ganusevich, II The role of matrix metalloproteinases (MMP) in malignant neoplasms. I. Characteristics of MMPs, regulation of their activity, prognostic value [Text]: a review / II Ganusevich // Oncology: adj. to the journal. "Experimental Oncology". - 2010. - T. 12, No. 1. - S. 10-16].

Matrix metalloproteinase-9 (MMP-9, gelatinase B) is involved in inflammatory processes, plays a significant role in tissue remodeling and repair, and determines the mobilization of various growth factors and cytokine factors. Various cytokines and growth factors are involved in the regulation of the biological activity of MMP-9, including fibroblast growth factor, epidermal growth factor, platelet growth factor, transforming growth factor β, vascular endothelial growth factor, nerve growth factor, tumor necrosis factor α, interleukins, interferons, etc. According to the literature, MMP-9 takes part in the destruction of extracellular matrix proteins, activates growth factors (TGF-β and TNF-α) [Oliveras, A. Resistant hypertension: patient characteristics, risk factors, comorbidities and outcomes [Text] / A. Oliveras, A. de la Sierra // J. Hum. Hypertens. - 2014. - Vol. 28, No. 4. - P. 213-217].

In the Russian Federation, studies of the involvement of matrix metalloproteinase genes in the formation of a predisposition to POAG and its complications in women are sporadic and fragmentary, and there are no data on the role of genetic variants rs2250889 and rs17577 MMP-9 in the development of POAG in women.

To assess the current patent situation, a search was carried out on titles of protection for the period from 1990 to 2020. The analysis of the documents was carried out in the direction: a method for predicting the risk of developing primary open-angle glaucoma in women on the basis of molecular genetic data depending on polymorphic markers of matrix metalloproteinases genes. Sources of information: sites of the Federal Institute of Industrial Property http://fips.ru.

In the studied medical scientific and available patent literature, the authors did not find a way to predict the risk of POAG in women based on data on the GG haplotype of the rs2250889 and rs17577 genetic polymorphisms of the MMP-9 gene.

There is a known method for predicting the risk of development and progression of glaucoma according to RF patent No. 2354287 (published on 05/10/2009), in which corneal hysteresis and central corneal thickness are determined and then the biomechanical coefficient of the cornea is calculated using the formula: K = KG / CTR · 50, where K is biomechanical corneal coefficient, CG - corneal hysteresis, CTR - central corneal thickness, and if the value is less than 0.82, the risk of development and progression of glaucoma is predicted. The method provides adequate prediction of the risk of development and progression of glaucoma, taking into account the elastic properties of the cornea and its central thickness, and appropriate treatment. The disadvantages of this method include the fact that it does not take into account the role of genetic polymorphisms and, in addition, provides for the need for expensive ophthalmic equipment: an analyzer of the biomechanical properties of the eye and a pachymeter.

RF patent No. 2483306 (published on 05/27/2013), which describes a method for predicting the disease of primary open-angle glaucoma by taking lacrimal fluid and blood, studying lacrimal fluid and blood serum by enzyme-linked immunosorbent assay using specific test systems. Elevated levels of metalloproteinase-9 (MMP-9), which exceed 52.5 ng / ml in the lacrimal fluid and 274.49 ng / ml in the serum; elevated levels of the complex of metalloproteinase-9 with its tissue inhibitor (MMP-9 / TIMP-1), whose values exceed 0.19 ng / ml in the lacrimal fluid and 4.93 ng / ml in the serum, and elevated levels of secretory immunoglobulin A ( sIgA), which values exceed 47.38 mg / L in the lacrimal fluid and 2.1 g / L in the blood serum, are the criteria for diagnosing primary open-angle glaucoma. This method can be used for early diagnosis of primary open-angle glaucoma in patients suffering from myopia, hypertension, type 2 diabetes mellitus and those at risk of developing the disease. The disadvantages of this method include the fact that it does not take into account the role of genetic polymorphisms and, in addition, provides for the need to study two biological samples: lacrimal fluid and blood serum.

RF patent No. 2517233, published on May 27, 2014, describes a method for predicting the progression of primary open-angle glaucoma, which consists in taking samples of tear fluid and blood, then determining the content of anti-apoptotic protein Bcl-2 in the tear fluid and blood serum. In the absence of it in the lacrimal fluid and / or serum, the progression of the glaucomatous process is predicted. The method makes it possible to predict the progression of the glaucomatous process with the further implementation of appropriate adequate therapeutic measures. The disadvantage is the complexity of the study, because it is necessary to study two biological materials at once, and also that it does not take into account the role of genetic polymorphisms.

For the prototype, the patent of the Russian Federation No. 2558861 was selected according to the application No. 2014132597/15 dated 08/07/2014 "A method for predicting the risk of developing primary open-angle glaucoma." In the course of this study, the method of discriminant analysis (hereinafter LDF) was used to study patients with POAG and the control group according to ten predictors: age, the presence of cardiovascular diseases, the level of systolic blood pressure, the level of diastolic blood pressure, the presence of POAG among relatives, the presence of concomitant eye pathology, microvascular disorders in the anterior segment of the eyes, the state of the pigment border of the pupillary edge of the iris, the degree of pigmentation of the anterior chamber angle, genetic variant at locus + 1663A / G TNFR2. The material for the study was venous blood, and genomic DNA was isolated by phenol-chloroform extraction. The typing of the + 1663A / G TNFR2 locus was carried out using the polymerase chain reaction of DNA synthesis on an IQ5 amplifier (Bio-Rad) in real time. The disadvantage of the prototype is that it does not take into account the influence on the development of POAG of other genetic polymorphisms and their combinations, as well as gender.

The objective of this study is to expand the arsenal of diagnostic methods, namely, to create a method for predicting the risk of POAG in women based on data on the GG haplotype of the rs2250889-rs17577 polymorphic loci of the MMP 9 gene.

The technical result of using the invention is to obtain criteria for assessing the risk of developing POAG in unrelated women of Russian nationality, a native of the Central Black Earth region of the Russian Federation (from the Belgorod, Kursk, Voronezh, Tambov and Lipetsk regions), based on data on the polymorphic loci rs2250889 and rs17577 of the MMP 9 gene, including:

- isolation of DNA from peripheral venous blood;

- analysis of polymorphisms rs2250889 and rs17577 of the MMP 9 gene;

- Predicting a high risk of POAG development in women when detecting the GG haplotype of the rs2250889 and rs17577 genetic polymorphisms of the MMP-9 gene.

The novelty and inventive step lie in the fact that the prior art does not know the possibility of predicting the development of POAG in women based on data on the GG haplotype of the rs2250889-rs17577 polymorphic loci of the MMP 9 gene.

The method is carried out as follows:

Isolation of genomic DNA from peripheral blood is carried out by phenol-chloroform extraction (Mathew, 1984) in two 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) are added to 4 ml of blood with EDTA. The resulting mixture is 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 sediment and resuspended. Then add 0.4 ml of 10% SDS, 35 µl of proteinase K (10 mg / ml) and incubate the sample at 37 ° C for 16 hours.

At the second stage, DNA extraction is carried out sequentially from the obtained lysate with 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 sampled. DNA is precipitated from solution with two volumes of chilled 96% ethanol. After lyophilization, the resulting DNA is dissolved in bidistilled, deionized water and stored at -20 ⁰ С.

Analysis of polymorphic markers rs2250889 and rs17577 of the MMP-9 gene was carried out by the method of polymerase chain reaction (PCR) on a CFX-96 Real-Time System (Bio-Rad) thermal cycler using standard oligonucleotide primers and probes with the risk fоr аthеrоsсlеrоsis disеаse аnd аbdоminаl аоrtiс аnеurysm [Techt] / R. Pradhan-Palikhe, R. Vikatmaa, T. Lajunen [et al.] // Sсand. J. Immunol. - 2010. - Vol. 72, No. 2. - P. 150-157.] (Synthesized at Test-Gen LLC (Ulyanovsk)).

Amplification of genomic DNA was carried out in a reaction mixture with a total volume of 10 μL, including a mixture for MMP PCR - 4 μL, Taq polymerase - 2 μL, a test sample (~ 30 ng DNA / μL) - 1 μL, deionized water - 3 μL.

Genotyping of the studied samples was carried out using the software "CFX-Manager ™" by the method of discrimination of alleles by the values of relative fluorescence units (RFU) (Fig. 1, Fig. 2)

The invention is characterized by the following figures:

- GG,

- СС,

- CG, ♦ - отрицательный контроль.FIG. 1. Discrimination of alleles by the TaqMan probe detection method according to the RFU values (relative fluorescence units) of each probe on the CFX96 amplifier with a real-time detection system of the rs2250889 MMP-9 polymorphism,

- GG,

- SS,

- CG, ♦ - negative control.

- GG,

- AA,

- GA, ♦ - отрицательный контроль.FIG. 2. Discrimination of alleles by the TaqMan probe detection method according to the RFU values (relative fluorescence units) of each probe on the CFX96 amplifier with a real-time detection system of the rs17577 MMR-9 polymorphism,

- GG,

- AA,

- GA, ♦ - negative control.

The calculation of the haplotype frequencies and the analysis of their associations with the formation of POAG in women was carried out using the PLINK v. 2.050 (http://zzz.bwh.harvard.edu/plink/) using the EM algorithm. _{Р рerm} <0.05 was taken as a statistically significant level.

The possibility of using the proposed method for predicting the risk of developing POAG in women is confirmed by the analysis of the observation results of 510 patients, of which 290 patients with primary open-angle glaucoma and 220 women in the control group (POAG was absent). Among the patients, the average age was 70.93 ± 8.70 years, in the control group, the average age was 62.02 ± 11.54 years. The studied groups included unrelated Russian patients, natives of the Central Black Earth region of the Russian Federation (from the Belgorod, Kursk, Voronezh, Tambov and Lipetsk regions). The group of patients included women with a POAG diagnosis confirmed by the necessary research methods in a clinical setting. For the diagnosis of glaucoma, the following criteria were used: high intraocular pressure, the presence of glaucomatous excavation of the optic nerve head and characteristic changes in the peripheral visual field. The patients were examined on the basis of the Ophthalmological Center "Generation" in Stary Oskol, the Department of Ophthalmology of the Regional Clinical Hospital. Saint Joasaph, Belgorod, Medical Center for Eye Microsurgery "Kovcheg", Belgorod. All necessary procedures for examination and examination of patients and individuals of the control group were carried out with their informed consent. The study was carried out under the supervision of the Ethics Committee of the Medical Institute of the National Research University "BelSU".

The typing of molecular genetic markers was carried out at the Department of Biomedical Disciplines, Faculty of General Medicine and Pediatrics, Medical Institute, Belgorod State National Research University.

When calculating the frequencies of haplotypes and analyzing their associations with the formation of POAG in women, a connection was established with the formation of a disease of the GG haplotype of the polymorphic loci rs2250889 and rs17577 of the MMP9 gene. Haplotype GG rs2250889-rs17577 is a risk factor for the development of POAG in women (OR = 1.80; p = 0.009).

As examples of the specific application of the developed method, a survey of Russian patients, natives of the Central Black Earth region of the Russian Federation (from the Belgorod, Kursk, Voronezh, Tambov and Lipetsk regions) and who are not related to each other, is given: a genetic examination was carried out at the rs2250889 and rs17577 loci of the MMP9 gene.

Example 1

Venous blood was taken from patient R., during genotyping of DNA markers, the GG haplotype was identified at the rs2250889 locus and rs17577 of the MMP9 gene, which made it possible to assign the patient to the group of patients with a high risk of POAG development. Further observation confirmed the diagnosis of primary open-angle glaucoma in the patient.

Example 2

Venous blood was taken from patient S., during genotyping of DNA markers, GA haplotype was identified at the rs2250889 locus and rs17577 of the MMP9 gene, which allowed the patient to be assigned to the group of patients with a low risk of POAG development. Further observation did not reveal primary open-angle glaucoma in the patient.

The use of this method will allow at the preclinical stage to form risk groups among women and timely implement in these groups the necessary therapeutic and prophylactic measures to prevent the development of POAG.

Claims (1)

A method for predicting the risk of developing primary open-angle glaucoma in unrelated Russian patients, natives of the Central Black Earth region of the Russian Federation, including DNA extraction from peripheral venous blood, characterized in that analysis of genetic markers of matrix metalloproteinases is carried out, predicting a high risk of developing primary open-angle glaucoma in women if detected haplotype GG at loci rs2250889 and rs17577 of the MMP9 gene.

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