RU2808824C1 - Method of predicting the risk of developing normotensive glaucoma using data on adrb1 gene polymorphism - Google Patents

Abstract

FIELD: medicine; ophthalmology.

SUBSTANCE: invention can be used to predict the risk of developing normotensive glaucoma. A method of predicting the risk of developing normotensive glaucoma (NGL) is described. The age, weight, height, gender of the individual, and the presence of a burdened family history are determined. The buccal epithelium is taken. DNA is isolated from the buccal epithelium. The rs1801253 polymorphism of the ADRB1 gene is analyzed. Then the probability of developing NGL is determined using the formula: p = 1/(1+e-Z), where e is a mathematical constant approximately equal to 2.71828; Z = (-23.7907) + 2.24 X1 + 0.1376⋅ X2 + 2.2736 X3 + (-0.0441)⋅ X4 + 2.4056 X5 + 0.0859⋅ X6, where X1 is a genetic variant at the rs1801253 locus, where GG — X1 = 1; GC or CC — X1 = 0, X2 — age in years, X3 — gender, where men — 0; women — 1, X4 — weight in kg, X5 — family history, where no — 0, yes — 1, X6 — height in cm. With a p value equal to or greater than 0.26, a high risk of developing normotensive glaucoma is predicted. The advantage provided by the given set of signs is the possibility of early assessment of the risk of developing NGL. This method has high sensitivity and specificity, there is no need for instrumental examination.

EFFECT: ability to predict the risk of developing normotensive glaucoma based on data on the polymorphic locus in combination with data on other risk factors, such as age, height, gender, weight, and a family history.

1 cl, 2 dwg, 2 tbl, 2 ex

Description

The invention relates to the field of medicine, in particular ophthalmology, and can be used to predict the risk of developing normotensive glaucoma.

Glaucoma is an optical neuropathy characterized by a chronic progressive course. Glaucoma is considered one of the leading causes of irreversible blindness in the world, with more than 60 million people suffering from glaucoma in 2010. Currently, more than 105 million people are diagnosed with glaucoma; in Russia, more than 1 million 250 thousand people have this disease. Due to the rapid growth of the elderly population, it is statistically predicted that the number of patients with glaucoma will increase, with the majority being diagnosed with primary open-angle glaucoma (POAG). Normal tension glaucoma (NTG) is a clinical subtype of POAG in which glaucomatous damage to the optic nerve occurs when the maximum intraocular pressure is below 21 mmHg. [Volkov V.V. Glaucoma with pseudonormal pressure.: Moscow: “Medicine”, 2001. 352 p.]. Early suggestions that glaucoma with low intraocular pressure is a rare pathology have not been confirmed in numerous epidemiological studies. Thus, in a study by Beaver Dam [Klein B.E.K. and others. Prevalence of Glaucoma // Ophthalmology. 1992. T. 99. No. 10. pp. 1499-1504.] it was shown that in almost one third of patients with glaucoma, intraocular pressure was not increased. Shiose et al examined 8126 people over 40 years of age and found that two thirds of Japanese patients with glaucoma had GND [Shiose Y. et al. Epidemiology of glaucoma in Japan—a nationwide glaucoma survey // Jpn. J. Ophthalmol. 1991. T. 35. No. 2. pp. 133-155]. In Asian populations, the prevalence of GND ranged from 50 to 92% of the number of people with POAG [Cho H., Kee C. Population-based glaucoma prevalence studies in Asians // Surv. Ophthalmol. 2014. T. 59. No. 4. P. 434-447.], which significantly exceeds the corresponding figures for Europeans: 30-38% [Bonomi L. et al. Prevalence of glaucoma and intraocular pressure distribution in a defined population // Ophthalmology. 1998. T. 105. No. 2. pp. 209-215].

Currently, there are a number of factors influencing the development of GND, such as changes in the immune system, cerebrospinal fluid pressure, and disturbances in vascular tone [Petrov S. Yu. Modern view on normal-tension glaucoma // Vestn. Oftalmol. 2020. T. 136. No. 6. P. 57]. The exact mechanism of influence of the above-mentioned risk factors for the development of glaucoma is still not fully understood, these data suggest that factors not related to intraocular pressure (IOP) are critical for the development of GND. It is known that glaucoma and related endophenotypes (intraocular pressure, central corneal thickness, optic nerve head parameters are largely inherited [Asefa N.G. et al. Heritability of glaucoma and glaucoma-related endophenotypes: Systematic review and meta-analysis // Surv. Ophthalmol. 2019. T. 64. No. 6. P. 835-851.] A significant number of genetic loci identified in numerous molecular genetic studies of glaucoma confirm the polygenic nature of the disease [Aboobakar I.F., Wiggs J.L. The genetics of glaucoma: Disease associations, personalized risk assessment and therapeutic opportunities-A review // Clin. Experiment. Ophthalmol. 2022. T. 50. No. 2. pp. 143-162; Zukerman R. et al. Molecular Genetics of Glaucoma: Subtype and Ethnicity Considerations // Genes. 2020 T. 12. No. 1. P. 55].

The ADRB1 gene encodes the beta-1 adrenergic receptor. The beta-1 adrenergic receptor is a G protein-coupled receptor that is predominantly expressed in cardiac tissue and plays an important role in the regulation of heart rate, myocardial contractility, and relaxation of vascular smooth muscle. The ADRB1 gene is located on chromosome 10q24.3 and consists of two exons. Numerous studies have linked mutations in the ADRB1 gene to various clinical outcomes, including familial naturally occurring short sleep duration, hypertension, heart disease, and metabolic disorders such as diabetes and obesity.

In a foreign study [Inagaki Y. et al. Polymorphism of beta-adrenergic receptors and susceptibility to open-angle glaucoma // Mol. Vis. 2006. T. 12. P. 673-680.] studied the association of ADRB1 polymorphisms with open-angle glaucoma. The study showed that certain polymorphisms of the ADRB1 gene affect the pathophysiology of POAG in Japanese patients. The Arg389Gly polymorphism in the ADRB1 gene showed significantly different allele and genotype frequencies in patients with normal tension glaucoma compared to controls.

Since the onset and progression of GND are asymptomatic, diagnosing the disease in the early stages, before irreversible changes develop, is very important.

There is a known “Method for predicting the risk of developing optic neuropathy in normotensive glaucoma” (Patent RU No. 2593891, IPC A61B 5/00 - 08/10/2016), in which the thickness of the retina is determined in the temporo-outer and infero-outer parts of the macula, and with a value equal to or less than 210 microns predict the risk of developing optic neuropathy in normotensive glaucoma.

The disadvantages of this method are that it does not take into account the role of the ADRB1 gene locus and requires optical coherence tomography.

The “Method for predicting the risk of normotensive glaucoma” is known (Patent RU No. 2573336, IPC A61B 3/00, A61B 3/18, A61B 8/10 - 01/20/2016), in which the central thickness of the cornea and the tonographic indicator - the Becker coefficient and at thickness are determined A cornea equal to or less than 520 μm and a Becker coefficient of more than 110 predict the risk of developing normotensive glaucoma.

The disadvantages of this method are that it does not take into account the role of the ADRB1 gene locus and requires special ophthalmic equipment: a pachymeter and an electronic ophthalmotonograph.

The objective of the claimed invention is to develop a method for diagnosing the risk of developing normotensive glaucoma using genetic data.

The technical result of the claimed invention is the ability to predict the risk of developing normotensive glaucoma based on data on the polymorphic locus in combination with data on other risk factors such as age, height, gender, weight, and the presence of family history.

The technical result of the claimed invention is achieved due to the fact that the values of the following indicators are determined: age, height, gender, the presence of a family history, the genotype is determined at the rs1801253 locus of the ADRB1 gene using the polymerase chain reaction method, the probability of developing NGL is calculated using the formula:

p=1/(1+e ^-Z ),

where e is a mathematical constant approximately equal to 2.71828;

Z=(-23.7907)+2.24⋅Х1+0.1376⋅Х2+2.2736⋅Х3+(-0.0441)⋅Х4+2.4056⋅Х5+0.0859⋅Х6, where

X1 - genetic variant at the rs1801253 locus (GG-X1=1; GC or CC-X1=0),

X2 - age (years),

X3 - gender (men - 0; women - 1),

X4 - weight (kg),

X5 - family history (no - 0, yes - 1),

X6 - height (cm),

with a p value equal to or greater than 0.26, a high risk of developing normotensive glaucoma is predicted.

The advantage provided by the given set of signs is the possibility of early assessment of the risk of developing GND. This method has high sensitivity and specificity, no need for instrumental examination.

Details, features, and advantages of the present invention follow from the following description of the implementation of the claimed technical solution using the drawings, which show:

Fig. 1 - Allelic discrimination by detection of TaqMan probes based on real-time RFU (relative fluorescence units) values of the rs1801253 polymorphism on a CFX96 amplifier, -CC, -CG, - GG.

Fig. 2 - ROC - evaluation curve of the model including genetic data on the rs1801253 locus of the ADRB1 gene.

The method is carried out as follows.

The following medical and biological indicators are determined: age, height, gender, presence of a burdened hereditary history.

Next, DNA is isolated from the buccal epithelium. DNA extraction is carried out using a set of reagents for DNA extraction using the EDEM express method. To extract DNA, an aliquot of a clinical sample is transferred into a tube with “VKO-diluent”, after which it is subjected to heat treatment, during which the destruction of cell membranes, viral envelopes and other biopolymer complexes occurs and DNA is released. By subsequent centrifugation, the insoluble components are deposited at the bottom of the tube, and the supernatant containing DNA is used for PCR. The internal control sample (ICS) contained in the IKO-diluent reagent undergoes an extraction procedure simultaneously with the DNA contained in the clinical material and, thus, is a marker of the quality of laboratory testing of the clinical sample.

The method of PCR typing of the rs1801253 polymorphism of the ADRB1 gene using oligonucleotide probes assumes the presence in the amplification mixture of probes that strictly correspond to the sequence of allelic variants of the gene and labeled with different fluorophores, as well as a probe with a fluorescence quencher. After amplification of the target gene fragment, the temperature of the reaction mixture is lowered, and probes with a fluorescence source and quencher are hybridized in close proximity to the DNA template. Genotyping is carried out by temperature denaturation of probe duplexes and DNA fragments by measuring fluorescence in “real time”.

Real-time PCR was carried out on a CFX96 amplifier (BioRad Laboratories, USA) using commercial reagents (Synthol, Moscow).

Real-time PCR uses oligonucleotide probes labeled with the fluorescent agents FAM and HEX. Genotyping of the studied samples is carried out using the CFX-Manager™ software using the method of allele discrimination based on the values of relative fluorescence units (RFU) (Fig. 1).

The probability of developing normotensive glaucoma is determined using the formula:

p=1/(1+e ^-Z ),

where e is a mathematical constant approximately equal to 2.71828;

Z=(-23.7907)+2.24⋅Х1+0.1376⋅Х2+2.2736⋅Х3+(-0.0441)⋅Х4+2.4056⋅Х5+0.0859⋅Х6, where

X1 - genetic variant at the rs1801253 locus (GG-X1=1; GC or CC-X1=0),

X2 - age (years),

X3 - gender (men - 0; women - 1),

X4 - weight (kg),

X5 - family history (no - 0, yes - 1),

X6 - height (cm),

with a p value equal to or greater than 0.26, a high risk of developing normotensive glaucoma is predicted.

An example of the method.

137 patients of the Republican Clinical Ophthalmological Hospital of the Ministry of Health of the Republic of Tatarstan named after Prof. E.V. Adamyuk were under observation (37 patients with GND, 100 control group). The groups were formed taking into account the ophthalmological diagnosis: the criterion for inclusion in the GND group was age over 40 years, a diagnosis of normal-tension glaucoma with tonometric intraocular pressure values not exceeding 21 mm Hg.

The diagnosis of normal-tension glaucoma was established based on data from optical coherence tomography of the optic disc and computer perimetry.

DNA extraction was performed from the buccal epithelium of all 137 patients. Typing of single nucleotide polymorphism rs1801253 of the ADRB1 gene was carried out using the polymerase chain reaction method with fluorescent tags and automatic registration of results in real time using kits from Synthol (Sintol, Moscow) on a CFX96 device (BioRad Laboratories, USA).

The normality of the distribution of quantitative characteristics was assessed using the Shapiro-Wilk test. To describe quantitative variables with a normal distribution, the mean value and standard deviation were used. Quantitative data that were not normally distributed were described using the median, 25th, and 75th quartiles. The hypothesis about the equality of the general variances of the two groups was tested using the Bartlett test. Comparison of groups of quantitative indicators with normal distribution was carried out using Student's t-test. The statistical significance of differences in groups of indicators with a distribution that differed from normal was determined using the Mann-Whitney test for indicators. Qualitative data analysis was carried out using the chi-square test (χ ² ). Differences were considered significant when the significance level reached P<0.05. Quantitative indicators such as age, weight and height were significantly different in the groups of patients with GND and the control group (Table 1).

Analysis of the genotype distribution of the rs1801253 polymorphism of the ADRB1 gene revealed an increased incidence of the GG genotype (compared to CG+CC) in the group of patients with GND, the odds ratio was 9.03 (95% CI 1.28 - 64.03, p = 0.03) .

The groups differed significantly in gender (chi-square value (χ ² ) = 8.96; p = 0.003), family history (χ ² value = 14.5; p < 0.001).

Prediction of the risk of developing normotensive glaucoma was carried out using multivariate logistic regression and subsequent ROC analysis [Grigoryev SG, Lobzin Yu. V., Skripchenko NV THE ROLE AND PLACE OF LOGISTIC REGRESSION AND ROC ANALYSIS IN SOLVING MEDICAL DIAGNOSTIC TASK // J. Infectology. 2016. T. 8. No. 4. pp. 36-45]. Logistic model of the form Z=K+β ₁ ⋅Х1+β ₂ ⋅Х2+…+β _n ⋅Xn,

where K is a constant, Xi are predictors, β _i are regression coefficients. Next, the probability of developing normotensive glaucoma was calculated using the formula

р=1/(1+e ^-Z ),

where p is the probability, e is a mathematical constant approximately equal to 2.71828.

Regression coefficients, significance levels and collinearity statistics VIF [Salmerón R. et al. A note about the corrected VIF // Stat. Pap. 2017. T. 58. No. 3. pp. 929-945.] are listed in Table 2.

table 2 Values of the constant and regression coefficients for calculating the probability of developing GND Predictor Weight S.E. p VIF Constant -237.907 89,634 0.008 ADRB1 genotype GG:1 - 0 22,004 0.9995 0.028 1.05 Age (years) 0.1376 0.0431 0.001 1.18 Gender: Women - Men 22,736 0.8496 0.007 1.45 Weight, kg) -0.0441 0.0233 0.058 1.2 Glaucoma in relatives: Yes - No 24,056 0.7275 <0.001 1.07 Height (cm) 0.0859 0.045 0.056 1.5

ROC analysis revealed high specificity and sensitivity of the model, including the above indicators, in patients with normotensive glaucoma compared to the control group (Specificity 0.83, sensitivity 0.83, accuracy 0.83, AUC 0.89, with a cutoff value set by 0.26) (Fig. 2)

This indicates the importance of the genotype of the rs1801253 locus of the ADRB1 gene in combination with a number of other phenotypic indicators as a criterion for the early diagnosis of normotensive glaucoma.

Clinical examples.

Example 1.

Patient A. The following indicator values were determined: genetic variant for the ADRB1 gene locus - CC (0), height - 172 cm, weight - 80 kg, age - 74 years, family history - no (0), gender - m (0 ). After substituting the values into the formula, the following results were obtained:

Z=(-23.7907)+2.24⋅0+0.1376⋅74+2.2736⋅0+(-0.0441)⋅80+2.4056⋅0+0.0859⋅172=-2 ,3612.

p=1/(1+2.718282 ^-(-2.3612) )=0.09.

The resulting p value was less than 0.26, so the patient was not classified as at risk for normotensive glaucoma. A subsequent comprehensive ophthalmological examination did not reveal the presence of normotensive glaucoma.

Example 2.

Patient B. had the following values of indicators: genetic variant for the ADRB1 gene locus - GG (1), height - 162 cm, weight - 59 kg, age - 68 years, family history - yes (1), gender - female (1 ). After substituting the values into the formula, the following results were obtained:

Z=(-23.7907)+2.24⋅1+0.1376⋅68+2.2736⋅1+(-0.0441)⋅59+2.4056⋅1+0.0859⋅162=3, 7992.

p=1/(1+2.718282 ^-(3.7992) )=0.98.

The resulting p value was greater than 0.26, so the patient was classified as at risk for normotensive glaucoma. The diagnosis of normotensive glaucoma was confirmed by ophthalmological examination.

The use of this method will make it possible at the preclinical stage to form risk groups for normotensive glaucoma for more effective implementation of a complex of treatment and preventive measures.

Claims (11)

A method for predicting the risk of developing normal pressure glaucoma (NGL), including determining the age, weight, height, gender of the individual, the presence of a family history, taking buccal epithelium, isolating DNA from the buccal epithelium, analyzing the rs1801253 polymorphism of the ADRB1 gene, after which the value of the probability of developing NGL is determined by formula: p = 1/(1+e ^-Z ), where e is a mathematical constant approximately equal to 2.71828; Z = (-23.7907) + 2.24 ⋅ X1 + 0.1376 ⋅ X2 + 2.2736 ⋅ X3 + (-0.0441) ⋅ X4 + 2.4056 ⋅ X5 + 0.0859 ⋅ X6, where X1 is a genetic variant at the rs1801253 locus, where GG – X1 = 1; GC or CC – X1 = 0, X2 – age in years, X3 – gender, where men – 0; women – 1, X4 – weight in kg, X5 – burdened family history, where no – 0, yes – 1, X6 – height in cm, with a p value equal to or greater than 0.26, a high risk of developing normotensive glaucoma is predicted.