RU2592200C2 - Method for prediction of risk of developing primary open-angle glaucoma with ineffective conservative therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and concerns a method for predicting risk of primary open angle glaucoma (POAG) with ineffective conservative therapy in individuals of Russian nationality, who are natives of Central Black Soil Region of Russia. Method includes carrying out analysis of polymorphisms of TNFα (-308G/A TNFα) and predicting risk of POAG with ineffective conservative treatment, depending on options identified by locus of -308G/A TNFα and other predictors of development of disease according to equation: P=e^y/(1+e^y), (1) where "e" is a mathematical constant equal to 2.72; and "y" is calculated by logistic regression equation: y=15-0.099x₁+1.232x₂-0.099x₃+ 0.091x₄-1.292x₅-1.825x₆-0.911x₇, (2) where x₁ is age in years; x₂ is presence of cardiovascular disease: no - x₂ = 0, yes - x₂ = 1; x₃ is systolic blood pressure in mmHg; x₄ is diastolic blood pressure in mmHg; x₅ is presence of POAG with relatives: no - x₅ = 0, yes - x₅= 1; x₆ is presence of comorbidity eye: no - x₆ = 0, yes - x₆ = 1; x₇ is genetic variant locus -308G/A TNFα: for option AA- x₇ = 1 for variant AG- x₇ = 2, for option GG- x₇ = 3. If P is greater than 60 - predict risk of developing POAG with ineffective conservative therapy, if P is less than 40 - predict effectiveness of conservative treatment of POAG, if P is greater than 40 but less than 60 - more thorough dynamic follow up is necessary.

EFFECT: essence of method lies in that it includes isolation of DNA from peripheral venous blood.

1 cl, 2 dwg, 3 ex

Description

The invention relates to the field of medical diagnosis, can be used to predict the risk of developing primary open-angle glaucoma, then POAG, with ineffective conservative therapy.

It is known that with POAG, increased intraocular pressure leads to an accelerated development of glaucoma optical neuropathy and a decrease in visual functions. The use of effective antihypertensive therapy reduces the level of IOP, and therefore, reduces the risk of developing glaucomatous atrophy of the optic nerve and stabilizes visual function. Against the background of drug antihypertensive treatment, target intraocular pressure is achieved in 33.7% of cases [The effectiveness of drug and laser-surgical treatment of primary open-angle glaucoma with long-term follow-up / C.V. Balalin, V.P. Fokin, E.V. Abrosimova // Materials of the X International Congress “Glaucoma: Theories, Trends, Technologies. HRT Club Russia - 2012. S. 54-58]. Therefore, the ability to predict the risk of developing POAG with ineffective conservative treatment will promptly offer the patient laser or surgical treatment.

POAG has a multifactorial nature [Nesterov A.P. Glaucoma [Text] / A.P. Nesterov. - M .: Medical Information Agency LLC, 2008. - 360 pp.], In the development of which an etiopathogenetic significance is currently attached to disorders in the apoptosis system [The role of apoptosis and Müller cell metabolism in experimental glaucoma [Text] / B.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]. Possessing 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 [Implications of dynamic changes among tumor necrosis factor - alpha (TNF-alpha), membrane TNF receptor, and soluble TNF receptor levels in regard to the severity of dengue infection / L. Wang, R.F. Chen, J.W. Liu et al. // Am. J. Trop. Med. Hyg. - 2007 .-- Vol.77. - No. 2. - P. 297-302]. Starting various intracellular processes, TNFα controls the vital activity of various cells by initiating apoptosis. It was found that TNFα produced by the cells of the central nervous system (microglial, astrocytes, oligodrodrocytes) is able to significantly affect the release of neurotransmitters and the activity of ion channels [Neurotropic effects of cytokines and growth factors / P. C. Vastyanov, A.A. Oleinik // Successes in physiological sciences. - 2007. - T. 38, No. 1. - S. 39-54]. 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 was established [TNFA gene polymorphism and disequilibrium linkage of TNFA and class II HLA genes (DRB1, DQA1 and DQB1) in the Siberian C / Caucasian population. AND. Konenkov, O.V. Golovanova, V.V. Dortman et al. // Immunology. - 2008. - No. 1. - S. 6-10].

In the studied medical and patent literature, the authors did not find a method for predicting the risk of developing POAG with ineffective conservative therapy based on data on the genetic polymorphism -308G / A TNFα and other predictors of the development of the disease.

To assess the current patent situation, a search was performed on the security documents of the website of the Federal Institute of Industrial Property http://fips.ru. for the period from 1990 to 2013

A method for predicting primary open-angle glaucoma disease according to RF patent No. 2483306, published on 05.27.2013, is intended for the early diagnosis of primary open-angle glaucoma in patients suffering from myopia, hypertension, type 2 diabetes mellitus and at risk of developing the disease. The method consists in examining lacrimal fluid and blood serum by enzyme-linked immunosorbent assay using specific test systems. Elevated levels of metalloproteinase-9 (MMP-9), the values of which exceed 52.5 ng / ml in the lacrimal fluid and 274.49 ng / ml in the blood serum; elevated levels of the metalloproteinase-9 complex with its tissue inhibitor (MMP-9 / TIMP-1), whose values exceed 0.19 ng / ml in lacrimal fluid and 4.93 ng / ml in blood serum, and increased levels of secretory immunoglobulin A ( sIgA), whose 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.

RF patent No. 2517233, publ. 05/27/2014, describes a method for predicting the progression of primary open-angle glaucoma, which consists in taking samples of lacrimal fluid and blood, then the content of anti-apoptotic protein Bl-2 is determined in tear fluid and serum. In the absence of it in the tear fluid and / or serum, the progression of the glaucomatous process is predicted. The method allows to predict the progression of the glaucomatous process with further appropriate appropriate therapeutic measures. The disadvantage is the complexity of the study, because it is necessary to investigate two biological materials at once.

The objective of this study is to expand the arsenal of methods for predicting the development of primary open-angle glaucoma.

The technical result of using the invention is to obtain criteria for assessing the prognosis of POAG with ineffective conservative therapy based on data on the genetic polymorphism -308 G / A TNFα and other predictors of the development of the disease.

In accordance with the task, a risk method was developed for the development of POAG with ineffective conservative therapy for people of Russian nationality who are natives of the Central Black Earth Region of the Russian Federation, including blood sampling from a patient, which introduced the following new signs:

- DNA is isolated from peripheral venous blood;

- analyze polymorphisms of the -308G / A TNFα gene;

- predict the risk of developing POAG with ineffective conservative therapy, depending on the identified variants at the locus -308G / A TNFα and other predictors of the development of this disease according to the equation:

P = e ^y / (1 + e ^y ), (1)

where "e" is a mathematical constant equal to 2.72,

and “y” is calculated according to the logistic regression equation:

y = 15-0.099x ₁ + 1.232x ₂ -0.099x ₃ + 0.091x ₄ -1.292x ₅ -1.825x ₆ -0.911x ₇ , (2)

where x ₁ - age, years; x ₂ - the presence of cardiovascular disease: no - x ₂ = 0, yes - x ₂ = 1; x ₃ - the level of systolic blood pressure, in mm Hg; x ₄ - the level of diastolic blood pressure, in mm Hg; x ₅ - the presence of POAG among relatives: no - x ₅ = 0, yes - x ₅ = 1; x ₆ - the presence of concomitant eye pathology: no - x ₆ = 0, yes - x ₆ = 1, x ₇ - genetic variant at the locus -308G / A TNFα: for variant AA-x ₇ = 1, for variant AG-x ₇ = 2, for the embodiment GG- x ₇ = 3.

- at the same time, if P is more than 60, the risk of developing POAG with ineffective conservative therapy is predicted, if P is less than 40, the effectiveness of conservative treatment of POAG is predicted, if P is more than 40, but less than 60, they predict the need for combined treatment.

The novelty and inventive step lies in the fact that the possibility of predicting the risk of developing POAG with ineffective conservative therapy by the presence of genetic polymorphisms of the -308G / A TNFα gene in combination with other predictors 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 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 they are resuspended. Then, 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) was added and the sample was 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. Isolated DNA was used to carry out the polymerase chain reaction of DNA synthesis.

The analysis of polymorphism -308G / A TNFα (rs 1800629) is carried out by polymerase chain reaction (PCR) of 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 (Fig. 1 ) 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 the allele discrimination at the locus -308G / A TNFα, where ○ - homozygotes - 308AA, homozygotes □ - 308GG, Δ - heterozygotes - 308GA, ◊ - negative control.

Fig 2, which shows the coefficients of multiple regression and the significance level of the indicators used to predict POAG with ineffective conservative therapy.

Genotyping is carried out using the Tag Man method of probes according to the UOF values (relative fluorescence level) 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 of FIG. 1 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 PF for one fluorophore (on the x axis) relative to PF for another fluorophore (on the y axis) in the allele discrimination diagram.

• 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 G allele (the PF of the G 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 A allele (the PF of the A 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, genotype determination is not possible: in this case, an undefined sample is a negative control.

To predict the risk of developing POAG with ineffective conservative therapy, the formula is derived:

P = e ^y / (1 + e ^y ), where

e is a mathematical constant equal to 2.72,

y is a mathematical model using logistic regression analysis, known from the source of Rebrov, O. Yu. [Statistical analysis of medical data. Application of the STATISTICA application software package [3rd ed.]. - Moscow: Media Sphere, 2006. - 305 p.]:

y = b ₀ + b ₁ x ₁ + b ₂ x ₂ + b ₃ x ₃ + ... + b _n x _n ,

where x_onex_n - informative signs, i.e. risk factors b₀-b_n - regression coefficients for these characteristics.

When conducting a logistic regression, the following statistical model was developed for predicting the risk of developing POAG with ineffective conservative therapy, which includes the following informative signs: genetic version at the locus -308G / A TNFα, age, presence of cardiovascular diseases, level of systolic and diastolic blood pressure, presence of POAG in relatives, the presence of concomitant eye pathology. The significance levels of the regression coefficients for all predictors were less than 0.05, i.e. each of them had a statistically significant effect on the risk of POAG with ineffective conservative treatment. The magnitude of the odds ratio for the presented regression model is 22.56, demonstrating that with an increase in the value of the ith sign by unity, the chance of POAG with ineffective conservative therapy increases by more than 22 times. The indicated model correctly predicts the probability of POAG with ineffective conservative treatment in 82% of cases and correctly classifies the control group in 83% of cases.

Using the obtained logistic regression coefficients (Fig. 2), based on data on the genetic variants of the -308G / A TNFα locus, age, the presence of cardiovascular diseases, the level of systolic and diastolic blood pressure, the presence of POAG in relatives, the presence of concomitant eye pathology, it is possible to predict the risk of developing POAG with ineffective conservative treatment according to the equation:

P = e ^y / (1 + e ^y ), (1)

where "e" is a mathematical constant, approximately equal to 2.72,

and “y” is calculated according to the logistic regression equation:

y = 15-0.099x ₁ + 1.232x ₂ -0.099x ₃ + 0.091x ₄ -1.292x ₅ -1.825x ₆ -0.911x ₇ ,

where x ₁ - age, years; x ₂ - the presence of cardiovascular disease: no - x ₂ = 0, yes - x ₂ = 1; x ₃ - the level of systolic blood pressure, in mm Hg; x ₄ - the level of diastolic blood pressure, in mm Hg; x ₅ - the presence of POAG among relatives: no - x ₅ = 0, yes - x ₅ = 1; x ₆ - the presence of concomitant eye pathology: no - x ₆ = 0, yes - x ₆ = 1, x ₇ - genetic variant at the locus -308G / A TNFα: for variant AA-x ₇ = 1, for variant AG-x ₇ = 2, for the variant GG- x ₇ = 3.

Moreover, if P is more than 60, the risk of developing POAG with ineffective conservative therapy is predicted, if P is less than 40, the effectiveness of conservative treatment of POAG is predicted, if P is more than 40, but less than 60, more careful dynamic monitoring is necessary.

The possibility of using the proposed method for predicting the risk of developing POAG with ineffective conservative therapy is confirmed by an analysis of the observation results of 353 individuals: 162 patients with POAG with ineffective conservative therapy and 191 individuals in the control group.

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. Diagnostics was carried out on the basis of the results of standards generally accepted in the world of ophthalmological practice for the study of optic discs and the state of 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.

Clinical and laboratory examination of individuals was carried out on the basis of the Department of Eye Microsurgery of the Belgorod Regional Clinical Hospital of St. Joasaph. 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".

It has been established that if P is greater than 60, the risk of developing POAG with ineffective conservative therapy is likely, if P is less than 40, the effectiveness of conservative treatment of POAG can be expected, if P is greater than 40, but less than 60, more careful dynamic monitoring is necessary.

Examples of using.

Example 1

Patient A., having POAG, has the following indicators: age - 50 years, i.e. x ₁ = 50,

the presence of pathology of the cardiovascular system - no, i.e. x ₂ = 0,

GARDEN - 120 mmHg, i.e. x ₃ = 120,

DBP - 80 mm Hg, i.e. x ₄ = 80,

the presence of POAG in relatives - yes, i.e. x ₅ = 1,

the presence of concomitant eye pathology - no, i.e. x ₆ = 0,

the genetic version of the locus -308G / A TNFα - GG, i.e. x ₇ = 3.

Substituting the values of the corresponding indicators in the logistic regression equation, we obtain for patient A:

y = 15-0.099 * 50 + 1.232 * 0-0.099 * 120 + 0.091 * 80-1.292 * 1-1.825 * 0-0.911 * 3 = 1.425,

respectively, P = 2,721,425 / (1 + 2,721,425) = 0.81.

Since the P value is greater than 60, the risk of POAG with ineffective conservative treatment in patient A. is high and is about 81%. This patient with POAG was instilled with a combination of groups of prostaglandin analogues and β-blockers, against which IOP remained uncompensated and the patient was offered surgical treatment.

Example 2

For patient B., having POAG, the following indicators were determined:

the presence of pathology of the cardiovascular system - yes, i.e. x ₂ = 1,

GARDEN - 145 mmHg, i.e. x ₃ = 145,

DBP - 80 mm Hg, i.e. x ₄ = 80,

the presence of POAG in relatives - no, i.e. x ₅ = 0,

the presence of concomitant eye pathology - yes, i.e. x ₆ = 1,

the genetic version of the locus -308G / A TNFα - AG, i.e. x ₇ = 2.

Substituting the values of the corresponding indicators into the regression equation, we get for y = 15-0.099 * 73 + 1.232 * 1-0.099 * 145 + 0.091 * 80-1.292 * 0-1.825 * 1-0.911 * 2 = -1.717, respectively P = 2, 72 ^-1.717 / (1 + 2.72 ^-1.717 ) = 0.15, i.e. the risk of POAG with ineffective conservative treatment in patient B. is 15%. Subsequently, patient B. was prescribed an instillation of β-blockers, against which IOP normalized.

Example 3

Patient S., having POAG, has the following indicators:

age - 68 years, i.e. x ₁ = 68,

the presence of pathology of the cardiovascular system - yes, i.e. x ₂ = 1,

GARDEN - 140 mmHg, i.e. x ₃ = 140,

DBP - 90 mm Hg, i.e. x ₄ = 90,

the presence of POAG in relatives - yes, i.e. x ₅ = 1,

the presence of concomitant eye pathology - yes, i.e. x ₆ = 1,

the genetic version of the locus -308G / A TNFα - AA, i.e. x ₇ = 1.

Substituting the values of the corresponding indicators into the regression equation, we obtain for the patient C .:

y = 15-0.099 * 68 + 1.232 * 1-0.099 * 140 + 0.091 * 90-1.292 * 1-1.825 * 1-0.911 * 1 = -0.198, respectively P = 2.72 ^-0.198 / (1 + 2.72 ^-0.198 ) = 0.45, i.e. the risk of POAG with ineffective conservative treatment in patient C. is doubtful and amounts to about 45%, therefore, more careful dynamic monitoring is necessary for the patient.

Thus, we can conclude that the proposed method is operational.

The method allows to predict the risk of developing POAG with ineffective conservative treatment depending on the genetic variants of the locus -308G / A TNFα, in combination with such predictors of disease development: age, the presence of cardiovascular diseases, the level of systolic and diastolic blood pressure, the presence of POAG in relatives and the presence of concomitant eye pathology. That, ultimately, allows you to choose the tactics of managing a patient with POAG.

Claims (1)

A method for predicting POAG with ineffective conservative therapy, including DNA extraction from peripheral venous blood, characterized in that they analyze TNFα gene polymorphisms (-308G / A TNFα) and predict the risk of developing POAG with ineffective conservative therapy for people of Russian nationality who are natives of the Central The chernozem of the Russian Federation, depending on the identified variants at the locus -308G / A TNFα and other predictors of the development of this disease according to the equation:
P = e ^y / (1 + e ^y ), (1)
where "e" is a mathematical constant equal to 2.72,
and “y” is calculated according to the logistic regression equation:
y = 15-0.099x ₁ + 1.232x ₂ -0.099x ₃ + 0.091x ₄ -1.292x ₅ -1.825x ₆ -0.911x ₇ , (2)
where x ₁ - age, years; x ₂ - the presence of cardiovascular disease: no - x ₂ = 0, yes - x ₂ = 1; x ₃ - the level of systolic blood pressure, in mm Hg; x ₄ - the level of diastolic blood pressure, in mm Hg; x ₅ - the presence of POAG among relatives: no - x ₅ = 0, yes - x ₅ = 1; x ₆ - the presence of concomitant eye pathology: no - x ₆ = 0, yes - x ₆ = 1; x ₇ - genetic variant at the locus -308G / A TNFα: for variant AA-x ₇ = 1, for variant AG-x ₇ = 2, for variant GG-x ₇ = 3; at the same time, if P is more than 60, the risk of developing POAG with ineffective conservative therapy is predicted, if P is less than 40, the effectiveness of conservative treatment of POAG is predicted, if P is more than 40, but less than 60, more careful dynamic monitoring is necessary.

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