RU2580308C1 - Method for prediction of risk of developing primary open-angle glaucoma stage iii-iv - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medical diagnostics and provides a method of predicting risk of primary open angle glaucoma stage III-IV, comprising isolating DNA from peripheral venous blood of individuals of Russian nationality who are natives of Central Black Soil Region of Russian Federation, analysis of polymorphisms of genes -308G / A TNFα,+ 250A/G Ltα, +36A/G TNFR1, thus making a forecast of increased risk of primary open angle glaucoma stage III-IV in case of these genetic variants allele -308G TNFα or genotype -308GG TNFα, or +250A allele Ltα or genotype +250A Ltα, or a combination of alleles -308G TNFα genotype +250AA Ltα, or combination of genotypes -308GG TNFα allele +36A TNFR1; and low risk of developing primary open angle glaucoma stage III-IV predict detection of a combination of allele -308A TNFα allele +36A TNFR1 and allele +250G Ltα or a combination of allele -308A TNFα c allele +36A TNFR1.

EFFECT: invention provides higher prediction accuracy.

1 cl, 3 dwg

Description

The invention relates to the field of medical diagnosis, can be used to predict the risk of developing primary open-angle glaucoma of stages III-IV.

Glaucoma is one of the most severe forms of ophthalmopathology, which has great medical and social significance due to the high prevalence, constant increase in the incidence and severity of disease outcomes leading to blindness and disability. Among the clinical forms of the disease, the most common is primary open-angle glaucoma (hereinafter referred to as POAG), which accounts for 72.3 to 96.1% of all forms of glaucoma [Glaucoma: nat. Guide / Ed. E.A. Egorova. - M .: GEOTAR-Media, 2013. - 824 p.]. POAG is a multifactorial disease of the eyes, characterized by an increase in intraocular pressure beyond the tolerance level for the optic nerve, glaucoma optic neuropathy and a typical decrease in visual functions [Glaucoma / A.P. Nesterov. - M .: Medical Information Agency LLC, 2008. - 360 p.]. It has been established that in the age group up to 59 years, the prevalence of POAG is 0.88 cases per 1000 people, from 60 to 70 - 6.44 per 1000 people, among people over 75 years old glaucoma occurs with a frequency of 17.3 per 1000 population [Glaucoma: nat. Guide / Ed. E.A. Egorova. - M .: GEOTAR-Media, 2013. - 824 p.].

According to the literature, in 90% of cases, glaucoma is detected in the later stages [http://ria.ru/moscow/20100426/226916133.html]. Given the fact that in the initial stages of glaucoma is almost asymptomatic, the detection of this disease in most cases occurs at stages accompanied by already irreversible changes in the optic nerve, therefore, predicting the development of POAG III-IV stages will form risk groups among individuals and timely implement these groups necessary treatment and preventive measures to prevent the progression of the disease.

Currently, important etiopathogenetic significance in POAG is given to disorders in the apoptosis system [The role of apoptosis and Muller cell metabolism in experimental glaucoma / V.N. Alekseev, E.B. Martynova, I.A. Samusenko. - Wedge. Ophthalmology, 2005. - No. 2. - S. 52-55]. One of the central links in this process is occupied by tumor necrosis factors and their receptors [Glaucomatous neurodegeneration: An eye on tumor necrosis factor-alpha / R. Agarwal, P. Agarwal // Indian. J. Ophthalmol. - 2012. - Vol.60. - P. 255]. With many biomedical effects, these cytokines can influence the development and progression of POAG [TNF-alpha signaling in glaucomatous neurodegeneration / G. Tezel // Prog Brain Res. - 2008 .-- Vol. 173. - P. 409-421].

TNFα is a multifunctional cytokine that provides a wide range of biological signals [Kashkin, K.P. Immune system cytokines: basic properties and immunobiological activity, Wedge. laboratory diagnostics. - 1998. - No. 11. - S. 21]. Starting various intracellular processes, TNFα controls the vital activity of various cells by initiating apoptosis. In experimental in vitro models, it has been shown that TNFα initiates astrogliosis - the process of enhancing the proliferation of astrocytes [Human astrocytes proliferate in response to tumor necrosis factor alpha /B.P. Barna, M.L. Estes, B.S. Jacobs et al. // Exp. Neurol. - 1990 .-- Vol.30. - P. 239-243], and therefore, even greater production of TNFα, which, by binding to its receptors, initiates the process of cell death. Moreover, the nature and intensity of the process of cell death is determined by the amount of TNFα acting on them, which suggests a dose-dependent effect of the cytokine.

The gene encoding the TNFα protein, includes 4 exons and 3 introns, has a size of 2762 bp and maps to the human genome on the short arm of the sixth chromosome (6p21.3), located next to the genes of the main histocompatibility complex [Tumor Necrosis Factor and Lymphotoxin Alfa Genetic Polymorphisms and Outcome in Pediatric Patients With Non-Hodgkin's Lymphoma: Results From Berlin-Frankfurt-Münster Trial NHL-BFM 95 / K. Seidemann [et al.] // Journal of Clinical Oncology. - 2005 .-- Vol.23. - No. 33. - P. 8414-8421]. The most common type of TNFα gene mutations are single nucleotide substitutions in the promoter region. More than 30 polymorphic variants of this gene are known, but only about half of them affect the expression of TNFα in vivo. For most of them, the effect on the level of transcriptional activity of the TNFα gene promoter, and, consequently, on the production of the cytokine itself [Cytokine gene polymorphism in human disease / M.V. Hollegaard, J.L. Bidwell // Genes Immun. - 2006. - Vol. 7. - Suppl. 3. - P. 269-276].

TNFβ (lymphotoxin-α, Ltα) is a glycoprotein containing 171 amino acid residues and having a molecular weight of about 33 kDa. The Ltα gene is located on the sixth chromosome (6p21.3), located at 1,100 polynucleotide bases from the TNFα gene. The lymphotoxin gene has an exon-intron structure similar to the tumor necrosis factor gene. The amino acid homology of TNFβ and TNFα is about 30% [Tandem arrangement of the genes coding for tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) in the human genome / S.A. Nedospasov, A.N. Shakhov, R.L. Turetskaya et al. // Cold Sping Harbor Symp. Quant. Biol. - 1986. - Vol. 51. - P. 611-625]. Thus, Ltα has a number of TNFα-like biological activities [Cytokine gene polymorphism in human disease / M.V. Hollegaard, J.L. Bidwell // Genes Immun. - 2006. - Vol. 7. - Suppl. 3. - P. 269-276], binding to the same receptors as TNFα.

Currently, two types of TNFα receptors have been identified: TNFR1 and TNFR2.

TNFR1 is a glycoprotein with a molecular weight of 55 kDa and consisting of 435 amino acids. The human TNFR1 gene is located on chromosome 12p13 [TNF alpha and the TNF receptor superfamily: structure-function relationship / H.T. Idriss, J.H. Naismith // Microsc. Res. Tech. - 2000. - Vol.50. - Number 3. - P. 184-195]. The tumor necrosis factor receptor of the first type mediates all types of action of tumor necrosis factors, participating in the inflammatory response and apoptosis due to the content of the death domain DD (death domain), which is an 80-amino acid sequence located at the C-terminus of the intracellular part of the receptor. This structure plays the role of a kind of “bridge” connecting the membrane receptor with adapter molecules, the formation of a complex with which initiates the intracellular chains of biochemical transformations and the implementation of the apoptosis program through activation of caspase-8 [Ligand passing the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor / LA Tartaglia, D. Pennica, D.V. Goeddel // J. Biol. Chem. - 1993 .-- Vol.268. - P. 18542-18548].

In the studied scientific and medical and accessible patent literature, the authors did not find a method for predicting the risk of developing primary open-angle glaucoma of stages III-IV based on data on genetic polymorphisms -308G / A TNFα, + 250A / G Ltα, + 36A / G TNFR1 and their combinations .

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 primary open-angle glaucoma of stages III-IV based on molecular genetic data depending on polymorphic markers of the genes of tumor necrosis factors and their receptors.

The objective of this study is to expand the arsenal of methods for diagnosing individuals of Russian nationality who are natives of the Central Black Earth Region of the Russian Federation, namely, creating a method for predicting the development of primary open-angle glaucoma of the III-IV stages.

The technical result of using the invention is to obtain criteria for assessing the risk of developing POAG of the III-IV stages based on data on genetic polymorphisms of -308G / A TNFα, + 250A / G Ltα, + 36A / G TNFR1 and their combinations.

In accordance with the task, a method was developed for predicting the risk of developing primary open-angle glaucoma of stages III-IV, including:

- DNA extraction from peripheral venous blood of individuals of Russian nationality, who are natives of the Central Black Earth Region of the Russian Federation;

- analysis of gene polymorphisms -308G / A TNFα, + 250A / G Ltα, + 36A / G TNFR1;

- predicting an increased risk of developing primary open-angle glaucoma of the III-IV stages in case of revealing the genetic variants of the -308G TNFα allele or the -308GG TNFα genotype, or the + 250A Ltα allele or the + 250AA Ltα genotype, or a combination of the -308G TNFα allele with the + 250AA Ltα genotype or combinations: of the -308GG TNFα genotype with the + 36A TNFR1 allele;

- predicting a low risk of the formation of this disease of the III-IV stages when a combination is detected: the -308A TNFα allele with the + 36A TNFR1 allele and the + 250G LTα allele, or the combination: the -308A TNFα allele with the + 36A TNFR1 allele.

The novelty and inventive step consists in the fact that the possibility of predicting the risk of developing primary open-angle glaucoma of the III-IV stages according to the genetic variants of the loci -308G / A TNFα, + 250A / G Ltα, + 36A / G TNFR1 and their combinations is not known from the prior art .

The method is as follows.

DNA is isolated from samples of peripheral venous blood of individuals in 2 stages. At the first stage, 25 ml of lysis buffer containing 320 mM sucrose, 1% Triton X-100, 5 mM MgCl2, 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 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 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. Isolated DNA is used to carry out the polymerase chain reaction of DNA synthesis.

Analysis of the -308G / A TNFα loci, + 250G / A Ltα, + 36A / G TNFR1 loci is carried out by polymerase chain reaction (hereinafter PCR) DNA synthesis. PCR is performed on an IQ5 apparatus (Bio-Rad) in real time using Thermus aquaticus DNA polymerase manufactured by Sileks-M and oligonucleotide primers and probes synthesized by Syntol followed by analysis of polymorphisms by allele discrimination. To discriminate alleles, the Bio-Rad program “IQ5-Standart Edition” is used.

The invention is characterized in the following figures.

In FIG. Figure 1 shows allele discrimination at the locus -308G / A TNFα, where • - -308AA TNFα, ■ - -308GG TNFα, ▲ - -308GA TNFα, ♦ - negative control.

In FIG. Figure 2 shows the discrimination of alleles at the locus + 250A / G Ltα, where • - + 250GG Ltα, ■ - + 250AA Ltα, ▲ - + 250AG Ltα, ♦ - negative control.

In FIG. Figure 3 shows the allele discrimination at the locus + 36A / G TNFR1, where • - + 36AA TNFR1, ■ - + 36GG TNFR1, ▲ - + 36AG TNFR1, ♦ - negative control.

In figures 1-3, two bands, vertical and horizontal, divide the graph into four sections: one for each homozygous state, one for the heterozygous state and the section without reaction. Assigning genotypes to unknown samples is determined by plotting the level of relative fluorescence (hereinafter referred to as UOF) for one fluorophore on the x axis, relative to the UOF for another fluorophore on the y axis on the allele discrimination diagram. A probe with a fluorescent dye ROX corresponds to allele A, a probe with a FAM dye corresponds to allele G.

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

- If the PFV of an unknown sample is above the horizontal strip and to the left of the vertical strip, the genotype is homozygous for the A allele (the PF of the A allele is plotted along the y axis).

- If the PFV of an unknown sample is below the horizontal strip and to the right of the vertical one, the genotype is homozygous for the G allele (the PF of the G allele is plotted along the x axis).

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

The possibility of using the proposed method to assess the risk of developing primary open-angle glaucoma of the III-IV stages in individuals is confirmed by the analysis of the observation results of 126 people (148 eyes) with POAG III-IV stages and 191 people (382 eyes) in the control group. Sampling of patients and control was carried out on the basis of the eye microsurgery department of Belgorod Regional Clinical Hospital of St. Joasaph.

Criteria for inclusion in the studied samples:

1. Individuals of Russian nationality, who are natives of the Central Black Soil of the Russian Federation and do not have kinship with each other.

2. Voluntary patient consent for the study.

3. The group of patients included individuals with a newly established or confirmed diagnosis of glaucoma. Diagnosis was carried out on the basis of the results of standards generally accepted in the world of ophthalmological research on the optic nerve head and the state of the visual fields, taking into account ophthalmotonometry data [National Guide to Glaucoma, 2011].

4. The control group included individuals who did not have acute eye diseases at the time of the examination, as well as somatic pathology, leading to secondary eye damage.

Exclusion criteria from the studied samples:

1. Patients with secondary glaucoma of any etiology, as well as patients with a closed iridocorneal angle.

2. Patients in whom glaucoma was combined with another eye pathology or systemic disease affecting the state of the visual field.

3. The presence of severe somatic pathology (cardiac, respiratory, renal failure).

4. Individuals who abandoned the study.

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". Associations of alleles and genotypes of the studied DNA markers with the development of stage III-IV POAG were evaluated by analyzing 2 × 2 contingency tables with calculation of the χ ² criterion adjusted for Yates continuity and odds ratios (OR) with 95% confidence intervals (CI). The role of combinations of genetic variants –308G / A TNFα, + 250G / A Ltα, + 36A / G TNFR1 in the formation of POAG III – IV stages was studied using the APSampler software [http://sources.redhat.com/cygwin/], using the Monte Carlo method by Markov chains and Bayesian nonparametric statistics [FavorovA.V. et al., 2005]. In order to minimize errors of the first kind associated with obtaining false-positive results when conducting multiple comparisons, the Bonferroni correction was introduced - the significance level p was recalculated for multiple pairwise comparisons according to the formula: p _cor = p × n, where p is the obtained level of statistical significance, n is the number of paired comparisons. For a statistically significant level, p _cor ≤ 0.05 [Statistical analysis of medical data. Application of STATISTICA application package / O. Yu. Rebrova. - [3rd ed.]. - M .: Media Sphere, 2006. - 305 p.: Ill].

Among patients with POAG with stages III-IV, the maximum frequency of the following genetic variants is observed: the -308G TNFα allele (94.1%), either the -308GG TNFα genotype or the + 250A Ltα allele (78.13%) or genotype + 250AA Ltα (61.81%) than in the control group (86.74%, χ ² = 10.51, p = 0.002, OR = 2.44, 95% CI 1.39-4.32 ; 76.80%, χ ² = 7.68, p = 0.007, p _cor = 0.02, OR = 2.26, 95% CI 1.25-4.13; 69.34%, χ ² = 7 44, p = 0.007, OR = l, 58, 95% CI 1.13-2.21 and 46.96%, χ ² = 8.50, p = 0.005, p _cor = 0.015, OR = l, 83 95% CI 1.21-2.77, respectively).

Using bioinformatics approaches, the maximum frequency of combinations of genetic variants was identified: the -308G TNFα allele with the + 250AA Ltα genotype (61.81%) or the -308GG TNFα genotype with the + 36A TNFR1 allele (69.01%) in patients with POAG with stages III-IV compared with the control group (46.33% and 53.98%). These combinations are risk factors for the development of stages III-IV of the disease (p = 0.001, p _cor = 0.006, OR = l, 87, 95% CI 1.26-2.79 and p = 0.001, p _cor = 0.006, OR = 1 90, 95% CI 1.26-2.87, respectively).

Conversely, among the patients of the control group, higher frequencies of genetic combinations are observed: the -308A TNFα allele with the + 36A TNFR1 allele with the + 250G Ltα allele (16.86%) or the -308A TNFα allele with the + 36A TNFR1 allele (16.48%) compared with patients with POAG with stages III-IV (6.34%, p = 0.001, p _cor = 0.008 and 6.34%, p = 0.001, p _cor = 0.004). These combinations have a protective value for the development of stages III-IV of the disease (OR = 0.33, 95% CI 0.16-0.69 and OR = 0.34, 95% CI 0.17-0.71, respectively.

Thus, the obtained data indicate the involvement of genetic variants of the genes for tumor necrosis factor α (-308G / A TNFα), lymphotoxin α (+ 250A / G Ltα) and tumor necrosis factor 1 type receptor (+ 36A / G TNFR1) and their combinations in the formation of POAG III-IV stages. Genetic variants increase the risk of developing stages III-IV of POAG: allele -308G TNFα (OR = 2.44), genotype -308GG TNFα (OR = 2.26), allele + 250A Ltα (OR = l, 58), genotype + 250AA Ltα (OR = l, 83) and their combinations: -308G TNFα allele with + 250AA Ltα genotype (OR = l, 87); either of the -308GG TNFα genotype with the + 36A TNFR1 allele (OR = 1.90), and the combination of the -308A TNFα allele with the + 36A TNFR1 allele and the + 250G Ltα allele (OR = 0.33) has a protective role in the formation of severe POAG ); or a combination of the -308A TNFα allele with the + 36A TNFR1 allele (OR = 0.34).

Examples confirming the feasibility of the proposed invention.

Patient A. revealed the -308G TNFα allele. With further detailed ophthalmological examination, the patient was diagnosed with stage III POAG.

Patient B., having stage II POAG, revealed the -308GG TNFα genotype. Further dynamic observation revealed the progression of this disease to stage IV.

In patient B., having stage II POAG, the + 250A Ltα allele was detected. Further dynamic observation revealed the progression of this disease to stage III.

Patient G. revealed a genotype of + 250 AA Ltα. With further detailed ophthalmological examination, the patient was diagnosed with stage III POAG.

Patient D. revealed the -308G TNFα allele and the + 250AA Ltα genotype. With further detailed ophthalmological examination, the patient was diagnosed with stage III POAG.

Patient E., having stage II POAG, revealed the -308GG TNFα genotype and the + 36A TNFR1 allele. Further dynamic observation revealed the progression of this disease to stage IV.

In patient I., having stage II POAG, the -308A TNFα allele was detected; allele + 36A TNFR1; allele + 250G Ltα. With further dynamic observation, the progression of this disease was not detected.

Patient K., having stage II POAG, revealed the -308A TNFα allele; allele + 36A TNFR1. With further dynamic observation, the progression of this disease was not detected.

Using this method allows you to predict the risk of POAG III-IV stages in individuals of Russian nationality, who are natives of the Central Black Earth Region of the Russian Federation, and on the basis of this to determine the set of measures to prevent the development of this disease. If genetic risk factors for the development of primary open-angle glaucoma of stages III-IV are identified, dynamic monitoring of the patient's ophthalmological status (regular visit to an ophthalmologist with an intraocular pressure measurement, ophthalmoscopy, perimetry, electronic tonography) for early diagnosis and timely pathogenetic treatment of primary open-angle glaucoma should be recommended . In the presence of combinations: the -308A TNFα allele with the + 36A TNFR1 allele with the + 250G Ltα allele or the -308A TNFα allele with the + 36A TNFR1 allele, the risk of developing primary open-angle glaucoma of stages III-IV should be considered low.

Claims (1)

A method for predicting the risk of developing primary open-angle glaucoma of stages III-IV, including the extraction of DNA from peripheral venous blood of individuals of Russian nationality who are natives of the Central Black Earth Region of the Russian Federation, analysis of gene polymorphisms -308G / A TNFα, + 250A / G Ltα, + 36A / G TNFR1, at the same time, a forecast is made of an increased risk of developing primary open-angle glaucoma of the III-IV stages if the following genetic options are identified: the -308G TNFα allele or the -308GG TNFα genotype, or the + 250A Ltα allele or the + 250AA Ltα genotype, or the combination of the -308G TNFα allele with the + 250AA Ltα genotype, or a combination of the -308GG TNFα genotype with the + 36A TNFR1 allele; and a low risk of the formation of primary open-angle glaucoma of stages III-IV is predicted when a combination of the -308A TNFα allele with the + 36A TNFR1 allele and + 250G Ltα allele or a combination of the -308A TNFα allele with the + 36A TNFR1 allele is detected.

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