FOXC1 variant in primary open-angle glaucoma patients

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National Pirogov Memorial Medical University, Vinnytsya, Ukraine

FOXC1 variant in primary open-angle glaucoma patients

Malachkova N.V. Department of Eye diseases,

Veretelnyk S.P. Department of Eye diseases

Abstract

As a working hypothesis, the assumption is made that point mutations of non-coding regions of developmental genes, including FOXC1, contribute significantly to the onset and progression of the primary open- angle glaucoma (POAG). The study included 93 POAG patients (185 eyes) and 89 volunteers (178 eyes) control subjects. Analysis of the FOXC1 gene was carried out in a real time polymerase chain reaction (PCR) process using TaqMan Mutation Detection Assays Thermo Fisher Scientific (USA). For statistical analysis of the results, MedStat and MedCalc v.15.1 (MedCalc Software bvba) were used. The rs984253 polymorphism of the FOXC1 gene had a strong association with the development of POAG (for genotypes x2 = 65.83; p = 0.0E-1, for alleles x2 = 46.52; p = 0.0E-1), but not related to it progression through the stages of the pathological process. The heterozygote T / A increased the chances of development of the POAG by 4.2 times (OR = 4.16; 95% CI 2.66-6.51), while the homozygote A / A was 1.7 times (OR = 1.69; 95% CI 0.87-3.28). Thus, the possible pathogenic role of polymorphism in the occurrence of POAG was proved. The carriers of the mutant allele A had significantly increased chance of occurrence of POAG.

Keywords: primary open-angle glaucoma, polymorphism rs984253 of FOXC1 gene.

Introduction

Another gene for testing as the genetic marker of the primary open-angle glaucoma (POAG) has become the developmental gene: forkhead box C1 (FOXC1) [1, 2]. This gene plays an important role in the normal morphogenesis of the anterior segment of the eye and is involved in the regulation of intraocular pressure and the function of the trabecular mesh [3]. The disembryogenesis of the anterior segment of the eye may lead to incomplete development or dysfunction of the structures that form the water drainage pathway, which can lead to increased pressure and development of glaucoma [4, 5].

The FOXC1 gene (previously FKHL7) is one of the six known genes of glaucoma [2]. Genetic loci responsible for several predominantly hereditary anomalies of the anterior segment are located on the chromosome 6p25 in the FOXC1 region [4, 5]. Mutations in FOXC1 and / or abnormal regulation of FOXC1 expression are responsible for most types of glaucoma observed in mutations in the chromosome 6p25 [6, 7]. Although FOXC1 expression has not been studied in adults yet, it is possible that prolonging the expression of an abnormal gene product (from age-related, sub- clinical mutations) throughout life, or altering the expression level of FOXC1 may affect the normal function of the trabecular mesh, thereby leading to an increased risk for the development of POAG through difficulty in drainage and increased intraocular pressure [8]. This assumption is confirmed by the fact that glaucoma, which is associated with mutations in the developmental gene, can begin to develop at any time from birth to adult life, and in some cases - more than 70 years [9].

Thus, as a working hypothesis, it can be assumed that point mutations of non-coding regions of developmental genes and, including FOXC1, make a significant contribution to the POAG. This can occur with age, since the structures of ocular drainage may not have been diagnosed to "cover up" anomalies, which subsequently and under the influence of external factors lead to increase of intraocular pressure [8, 9].

The connection of the polymorphism of the FOXC1 gene with the development of POAG has been studied in individual researches. In a study of British scientists [3] four polymorphisms were studied, among which was rs984253, which is located in a non-coding intron of the FOXC1 gene (1 613 294). The indicated polymorphism is not strictly pathogenic, but it is located in the regulatory region of the gene and can determine the genetic predisposition to the development of POAG. The influence of molecular genetic factors in Ukraine was studied in individual works [10, 11], but similar developments with the developmental gene in Ukraine have not been carried out at this time.

The aim of the study was to determine the relation between the rs984253 polymorphism of FOXC1 gene and POAG in patients from the Ukrainian population.

1. Materials and methods

The research was performed at the Department of Eye Diseases of the National Pirogov Memorial Medical University and the department of ophthalmology of Vinnitsa Regional Clinical Hospital Named After N. I. Pirogov.

The study included 93 patients (185 eyes) with POAG I-IV stage and 89 volunteers (178 eyes) control subjects. The patients were divided into groups according to the degree of perimetric changes [12] (Table 1). All patients performed visometry, computer perimetry, tonometry, biomicroscopy, ophthalmoscopy, gonioscopy, keratopahymetry, optical coherent tomography of the optic nerve.

The analysis of rs984253 polymorphism of the FOXC1 gene was carried out in a real time polymerase chain reaction (PCR) method in the Gene Amp® PCR System 7500 (Applied Biosystems, USA) automatic amplifier. In the first stage of the study, the genomic DNA from whole venous blood was isolated using the standard reagents PureLink® Genomic DNA Kit for purification of genomic DNA, manufactured by INVITROGEN (USA). The analysis of polymorphism was carried out using unified test systems of TaqMan Mutation Detection Assays Life-Technology (USA). For statistical analysis of the results, MedStat and Med- Calc v.15.1 (MedCalc Software bvba) were used. Association of genotypes and alleles with the disease were determined by the odds ratio (OR); The limits of 95% of the credible interval (CI) were calculated by the method of J. Neyman. The differences were statistically significant at p <0.05. To test the probability of differences between groups, x2 was used and Fischer's exact criterion was used.

2. Results and discussion

The distribution of genotypes in the control group was comparable to the data provided in the Program 1000 Genomes Project Phase 3 (http: //www.internationalgenome.org/). The program for the determination of frequency of genotypes rs984253 of the FOXC1 gene involved 2504 people. The ancestral genotype T / T was determined with a frequency of 0.695 (in our studies - 0.53), heterozygote T / A - 0.246 (in our studies - 0.38), mutant homozygote A / A - 0.059 (in our studies - 0, 09), the mutant heterozygote T / S is 0.001 (in our studies - 0). The mutant heterozygote C / T was detected in two people from the population of South Asia. In the European population (n = 503), the rates were, respectively, 0.354; 0.465 and 0.181. The distribution of alleles in the control group was also compared with the data of the Program 1000 Genomes Project Phase 3. For all observations, the ancestral allele T was determined with a frequency of 0.818 (in our studies - 0,72), mutant allele A - 0.182 (in our studies - 0, 28), mutant allele C - 0.0004 (in our studies - 0); for the European population, the figures were, respectively, 0.586 and 0.414. Consequently, the data obtained in the control group, in general, corresponded to the data of the Program 1000 Genomes Project Phase 3.

In general, compared with control subjects, in groups of patients a clear decrease the frequency of the anterior homozygous genotype T / T was observed 2.510.6 times (for all patients - 4.1 times), which had a statistical probability at the level p = 0.0E-1. Frequencies of heterozygous T / A and minor A / A genotypes increased significantly - by 1.1-2.8 times (for all patients,respectively, 1.8 times and 2.1 times), which was also statistically significant (p = 0.0E-1). For clarity, these divergence are given in Figure 1.

Fig. 1 Distribution of genotypes rs984253 in the studied groups

The smallest degrees of severity this tendency was in the 4th group, where the frequency of genotype T / T among the other groups was highest (0.21 vs. 0.53 in control), as well as the frequency of the genotype A / A (0.25 versus 0.09 in control) Consequently, the general tendency of differences in the distribution of genotypes in the groups of patients was the same.

Discriminative analysis revealed a very significant contribution of genetic polymorphism rs984253 in the distribution of patients in groups (F = 15.02; p = 2.3E- 11), that is, indeed, one or another genotype determined the distribution of patients into groups. The dispersion analysis showed a high effect of polymorphism genotypes rs984253 on the groups (F = 26.36; p = 2.1E-11).

The analysis of the calculated indices showed the statistical significance of the differences in the control group as with all POAG patients, and with each group separately, which related to the genotypes (for all patients x2 = 65,83; p = 0,0E-1) and alleles (for all patients x2 = 46,52; p = 0,0E-1).

According to the results of the distribution of genotypes, the alleles of this polymorphism were distributed among the groups of patients. However, unlike genotypes, the distribution of alleles was almost the same in each group when compared to control and quantified the general data. This, in our opinion, already at this stage of the study showed that there is a direct relationship between the rs984253polymorphism and the occurrence of POAG, but without further influence on its development and progression.

Further evaluation of the statistical significance of differences between genotype and allele distribution between groups according to Pearson's x2 criterion is given in Table 1.

Table 1 The statistical significance of differences in the distribution of genotype frequencies and alleles rs984253 between groups

Notes: The POAG group combines all observations (1st to 4th groups);/2 - the Pearson 's %-square criterion; df - number of degrees offreedom; p - the significance of the differences.

Comparing groups among themselves, it was found that the probable differences were not determined in any case (p> 0,05).

Thus, it has been proved that the rs984253 polymorphism of the FOXC1 gene had a strong association with the development of POAG, but was not related to its progression in the stages of the pathological process.

The differences between the frequencies of the genotypes and rs984253 alleles in the control group and the patients with POAG were further analyzed.

Distribution of ancestral homozygote T / T and T / A heterozygotes in the control group and in POAG patients showed statistically significant differences (p = 0.0E-1 in both cases). At the same time, the frequency of homozygote T / T significantly decreased (by 4.1 times), and the frequency of heterozygotes - increased (by 1.8 times). Despite the fact that the frequency of minor homozygote A / A in patients increased (by 2.1 times), this shift was not statistically significant (p = 0.232).

According to this kind of redistribution of genotypes, a probable shift of the allele frequency in the direction of the minor allele A was noted. The frequency of the ancestral allele T in the POAG was 1.5 times lower (p = 0.0E-1), while the frequency of the minor allele T was 1, 9 times higher (p = 0.0E-1).

Thus, the dependence of the presence or absence of POAG according to the distribution of genotypes and alleles rs984253 of the FOXC1 gene was finally confirmed.

Forward, the influence of the distribution of the rs984253 polymorphism alleles on the POAG was revealed.

The analysis of the contingency table (3 x 3) indicated that the effect of the genotype rs984253 on the development of POAG (%2 = 65,83; p = 0,0E-1). The genotypes of risk were heterozygote T / A and minor homozygote A / A. The heterozygote T / A increased the chances of development of the POAG by 4.2 times (OR = 4.16; 95% CI 2.66-6.51), while the homozygote A / A was 1.7 times (OR = 1.69; 95% CI 0.87-3.28). The ancestral homozygote T / T significantly reduced the chance of development of POAG- by 7.1 times (OR = 0.14; 95% CI 0.08-0.24).

The comparison of the frequencies of alleles (the contingency table (2 x 2) showed that there was a significant difference between the patients and the control of the differences (%2 = 46,52; p = 0,0E-1). Minor allele A increased the chances of development of POAG by 2,6 times ( OR = 2.98; 95% CI 1.90-3.54), and the ancestral allele T reduced such chances by 2.6 times (OR = 0.39; 95% CI 0.28-0.53).

An analysis of the comparison of the control subjects with the individual groups of patients showed similar results of the distribution of genotypes and alleles, their association with the disease and the chances of the development of POAG.

Comparison of dominant and recessive patterns of inheritance of rs984253 genotypes showed a significant advantage of the dominant model.

The distribution of the genotypes rs984253 according to the dominant pattern of inheritance (T / T vs. T / A + A / A) was statistically significant by Pearson's x2 criterion (%2 = 60,61; p = 0,0E-1). The distribution of genotypes according to the recessive model of inheritance was not significant (%2 = 2,41; p = 0,120).

Consequently, it is possible to clearly determine that the presence of the allele predetermined the development of the POAG. The action of the protective allele T was manifested only in the homozygous state.

Conclusions

glaucoma mutation gene polymorphism

1. The rs984253 polymorphism of the FOXC1 gene had a strong association with development of the POAG (for genotypes x2 = 65.83; p = 0.0E-1, for alleles X2 = 46.52; p = 0.0E-1), but not relevant to its progression through the stages of the pathological process.

2. The heterozygote T / A increased the chances of development of the POAG by 4.2 times (OR = 4.16; 95% CI 2.66-6.51) and the homozygote A / A by 1.7 times (OR = 1, 69; 95% CI 0.87-3.28). The ancestral homozygote T / T significantly reduced the chance of development of the POAG - by 7.1 times (OR = 0.14; 95% CI 0.08-0.24). Minor allele A increased the chances of development of the POAG by 2.6 times (OR = 2.98; 95% CI 1.90-3.54), and the ancestral allele T reduced such chances by 2.6 times (OR = 0.39; 95% CI 0.28-0.53).

3. The significance of the dominant pattern of inheritance for rs984253 (x2 = 60,61; p = 0,0E-1) proved that the presence of the allele A caused the development of the POAG.

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