Association study of optineurin gene polymorphisms T34T and M98K with normal tension glaucoma in a Turkish cohort

Abstract

To investigate the relationship between normal tension glaucoma and Optineurin (OPTN) gene polymorphisms in the Turkish population. Two single nucleotide polymorphisms in the OPTN gene, T34T and M98K, were genotyped from 72 normal tension glaucoma patients and 100 control subjects and compared between the groups. The T34T polymorphism genotype frequencies were determined as 31/33/8 (wild type/heterozygous/mutant) and 42/46/12 in the normal tension glaucoma and control groups, respectively. The G/A allele frequencies were 95/49 in the normal tension glaucoma group and 130/70 in the control group. The M98K polymorphism genotype frequencies were 66/5/1 and 89/10/1, and the T/A allele frequencies were 137/7 and 188/12 in the normal tension glaucoma and control groups, respectively. The genotype and allele frequencies of the T34T and M98K polymorphisms were similar between the normal tension glaucoma and control groups (p > 0.05). These findings indicate that the T34T and M98K polymorphisms were not associated with the disease in this cohort. However, given the low frequency of the mutant genotypes and the modest sample size, these results do not definitively rule out a predisposition. Further large-scale studies with a broader molecular analysis spectrum will be beneficial to fully elucidate the underlying genetic etiology of normal tension glaucoma.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-43217-0.

Introduction

Glaucoma is the second most common cause of blindness in the world, and nearly 11.2 million people were estimated to be affected by it in 2020¹. Studies involving different populations have reported that at least 50% of glaucoma patients in Europe remain undiagnosed. It is predicted that there is a higher rate of undiagnosed glaucoma cases in Asia and Africa^2–4. Normal tension glaucoma (NTG), one of the subtypes of glaucoma, is recognized by the clinical triad of an intraocular pressure (IOP) of 21 mmHg or less, a glaucomatous optic disc, and visual field defects⁵. It is thought that NTG constitutes an exception to the general glaucoma spectrum, as it is not accompanied by a high IOP and may be associated with different pathogenetic mechanisms other than an elevated IOP⁶. NTG has been indicated as a variant of primary open-angle glaucoma (POAG). Large epidemiological studies have reported that approximately half of glaucoma patients have IOP values within normal limits⁷. Population-based studies have suggested that NTG is more prevalent than previously thought⁸. The presence of a family history in glaucoma patients, ranging from 20% to 59%, clearly indicates the genetic predisposition to glaucoma^9,10. Furthermore, some recent studies have indicated that nearly 21% of NTG patients have a positive family history. Recent developments in genomic and proteomic technologies have prompted researchers to delve into the genetic infrastructure of disease. Moreover, these studies will shed significant light on the elucidation of the pathogenesis of glaucoma and treatment approaches for it¹¹.

Particularly, causative genes and polymorphisms that confer susceptibility to the disease have been identified for many subtypes of POAG¹². Among these genes, the ‘optineurin’ (OPTN; OMIM:602432) gene, located on the short arm of chromosome 10, encodes ‘optineurin’ an adaptive protein with 16 exons and 577 amino acids. It has numerous cellular functions and interacts with many proteins. The OPTN protein is expressed in the trabecular meshwork, non-pigmented ciliary epithelium, retina, and brain, and is thought to play a neuroprotective role. After the OPTN gene was identified as responsible for open-angle glaucoma, population-based studies have accelerated to determine whether polymorphisms in this gene are a risk factor for NTG. As a result of these studies, it has been reported that many different variants in the OPTN gene, especially the T34T and M98K polymorphisms, may be risk factors for the disease in various ethnic groups^13–16.

Research has shown that the highest genetic relationship in NTG is with OPTN and TANK-binding kinase 1 (TBK1) genes. It has been shown that TBK1 actually leads cells to apoptosis via the OPTN gene¹⁷. Pan et al.¹⁸ reported that although different polymorphisms in the OPTN gene that were thought to be associated with NTG were detected in different populations, the strongest association with NTG was with the Thr34Thr (T34T) and Met98Lys (M98K) single nucleotide polymorphisms. Moreover, they presented strong evidence that these two polymorphisms cause NTG via the apoptosis pathway.

The current study aimed to determine the association between T34T and M98K polymorphisms in the OPTN gene and NTG in the Turkish population.

Results

Baseline demographics were comparable between the NTG and control groups. No significant differences were observed in mean age (NTG: 56.3 ± 8.2 years; control: 54.1 ± 8.3 years; p > 0.05) or sex distribution (NTG: 31 males, 41 females; control: 46 males, 54 females; p > 0.05).

The T34T polymorphism genotype frequencies were determined as 31/33/8 (wild type/heterozygous/mutant) and 42/46/12 in the NTG and control groups, respectively (p > 0.05). Eight (11.1%) of the NTG patients and 12 (12.0%) of the controls were positive for the mutant genotype (T34T). The G/A allele frequencies were 95/49 and 130/70 in the NTG and control groups, respectively, and there was no significant difference between groups (p > 0.05). The T34T polymorphism results are shown in Table 1.

Table 1.

 Genotype and allele frequencies of the T34T (rs2234968) polymorphism in the NTG and control groups.

	NTG (n = 72)	Control (n = 100)
	n (%)		OR	*p-value
Genotype frequency
GG	31 (43.1)	42 (42.0)	1.044	0.890
GA	33 (45.8)	46 (46.0)	0.993	0.982
AA	8 (11.1)	12 (12.0)	0.916	0.857
Allele frequency
G	95 (66.0)	130 (65.0)	1.044	0.852
A	49 (34.0)	70 (35.0)	0.958	0.852

*The χ2 test; NTG, normal tension glaucoma; OR, Odds ratio; Significance level, p < 0.05; 

Confidence interval, 95%

The M98K polymorphism genotype frequencies were determined as 66/5/1 (wild type/heterozygous/mutant) and 89/10/1 in the NTG and control groups, respectively (p > 0.05). Only one (1.4%) of the patients had the mutant M98K genotype, as did only one (1.0%) of the controls. The T/A allele frequencies were 137/7 and 188/12 in the NTG and control groups, respectively, and there was no significant difference between the groups (p > 0.05). The M98K polymorphism results are shown in Table 2.

Table 2.

 Genotype and allele frequencies of the M98K (rs11258194) polymorphism in the NTG and control groups.

	NTG (n = 72)	Control (n = 100)
	n (%)		OR	*p-value
Genotype frequency
TT	66 (91.7)	89 (89.0)	1.359	0.765
TA	5 (6.9)	10 (10.0)	0.672	0.486
AA	1 (1.4)	1 (1.0)	1.394	0.815
Allele frequency
T	137 (95.1)	188 (94.0)	1.249	0.648
A	7 (4.9)	12 (6.0)	0.801	0.648

*The χ2 test; NTG, normal tension glaucoma; OR, Odds ratio; Significance level, p < 0.05; 

Confidence interval, 95%

Discussion

The possible role of the OPTN gene in NTG has long been considered, and several recent studies on different ethnicities have investigated the relationship between OPTN gene polymorphisms and glaucoma. The study by Rezaie et al.¹⁹ in 2002 was the first to show the association between the OPTN gene and familial glaucoma. They reported the M98K polymorphism in 23 glaucoma patients, 20 of whom had NTG. While the M98K polymorphism frequency was 2.1% in the control group, it was 13.6% in the glaucoma group (p < 0.05). They revealed that the M98K polymorphism in the OPTN gene was associated with NTG. In the study of Hauser et al.²⁰, in the USA, they reported that T34T and M98K polymorphisms in the OPTN gene in 67 NTG patients were found at rates similar to those in the control group, and the difference was not significant.

Two independent studies conducted in Japan during the same year reported a significant association between the M98K polymorphism and NTG (p < 0.05). Specifically, M98K frequencies were significantly higher in NTG patients compared to controls in both a 65-patient cohort (15.4% vs. 5.0%) and a 28-patient cohort (14.2% vs. 1.7%)^{13, 21}. However, the T34T polymorphism showed no such association in the study involving 28 NTG patients²¹.

In three different studies in China, conducted in different years, the T34T polymorphism was analyzed in 48, 106, and 380 NTG patients, respectively, and in all of those studies, contrary to the current one, the T34T polymorphism was observed significantly more in the NTG group (p < 0.05). In the second and third studies, the M98K polymorphism was also analyzed, but was not associated with NTG (p> 0.05)^16,22,23.

Moreover, studies conducted in Canada, Germany, and Australia, in which the M98K polymorphism was analyzed in 62, 112, and 128 NTG patients, respectively, revealed results similar to the current study, in that there was no association with NTG^15,24,25. However, in the study conducted in Canada, the T34T polymorphism was also analyzed, but no significant association with NTG was found²⁴.

Sripriya et al.²⁶, in their study in India, examined the M98K polymorphism in 50 NTG patients and found it at a significantly higher rate in the patient group (6%) compared to the control group (0%). In the study of Park et al.²⁷ in Korea, the T34T and M98K polymorphisms were found in 2.63% of 76 NTG patients, while they were not observed in the control group. Although the difference was not statistically significant, they reported that there may be a weak relationship between these polymorphisms and NTG. The significance values ​​of the above-mentioned studies are summarized in Table 3.

Table 3.

Similar studies investigating the relationship between the T34T and M98K polymorphisms and NTG.

Study	Year	Country	T34T	M98K	NTG (n)	Control (n)
Raymond et al.	2003	Canada	p > 0.05	p > 0.05	62	56
Fuse et al.	2004	Japan	-	p < 0.05	65	100
Umeda et al.	2004	Japan	p > 0.05	p < 0.05	28	58
Fan et al.	2005	China	p < 0.05	p > 0.05	106	281
Weisschuh et al.	2005	Germany	-	p > 0.05	112	100
Craig et al.	2006	Australia	-	p > 0.05	128	218
Hauser et al.	2006	USA	p > 0.05	p > 0.05	67	100
Sripriya et al.	2006	India	-	p < 0.05	50	100
Yao et al.	2006	China	p < 0.05	-	48	77
Park et al.	2016	Korea	p > 0.05	p > 0.05	76	100
He et al.	2019	China	p < 0.05	p > 0.05	380	402
The current study	2024	Türkiye	p > 0.05	p > 0.05	72	100

NTG, normal tension glaucoma; Significance level, p < 0.05.

Given the complex and polygenic genetic background of NTG, the potential modifying effects of other glaucoma-associated variants on the investigated polymorphisms could not be evaluated within the scope of this study. Other or novel polymorphisms or mutations associated with NTG can be detected with OPTN gene sequence analyses with further studies. The fact that only the T34T and M98K polymorphisms in the OPTN gene were studied can be considered a limitation of the study. Genome-wide association studies (GWAS) may also be helpful to show the relationship between the OPTN gene and NTG. There are a limited number of similar studies in the literature, and no epidemiological studies specifically addressing NTG in the Turkish population. This is the first molecular study to examine the relationship between the OPTN gene and NTG in Türkiye.

Consequently, genetic studies are very valuable for elucidating disease pathogenesis and developing new treatment approaches; however, results often vary across different populations and studies. The current study indicates that the T34T and M98K polymorphisms, previously reported to have the strongest association with NTG, did not demonstrate a significant association with the disease in this Turkish cohort. The limited sample size and the low frequency of the mutant alleles may limit the statistical power to detect minor genetic effects. Future population-based studies with larger sample sizes are required to confirm these findings and to better understand the influence of genetic background, ethnic differences, and population stratification.

Methods

Ethics committee approval for this cross-sectional case-control study was obtained from Ankara Bilkent City Hospital Ethics Committee No. 1 (E1/3952/2023). The study was conducted in accordance with the principles of the Declaration of Helsinki, and informed consent was obtained from all participants.

The study population comprised 72 NTG patients, followed for at least one year at the Glaucoma Department of Ankara Bilkent City Hospital, and 100 control subjects over 40 years of age with no family history of glaucoma. NTG was diagnosed after excluding optic neuropathy and other secondary causes of glaucoma; patients were selected via consecutive sampling. To rule out subclinical glaucoma in the control group, all participants underwent a comprehensive ocular examination. This included visual acuity, slit-lamp examination, Goldmann applanation tonometry, fundus examination, and peripapillary retinal nerve fiber layer (pRNFL) thickness measurement using the DRI OCT-1 Triton (Topcon Corp., Tokyo, Japan) between November 2023 and February 2024. Additionally, 2–5 mL of peripheral blood was collected from all participants during the same visit. Visual field testing was performed for the NTG group using Humphrey 24-2 SITA-Fast perimetry (HFA-II 720i, Carl Zeiss Meditec, Dublin, CA).

To maintain a rigorous dataset, several exclusion criteria were applied: a refractive error spherical equivalent exceeding ± 3 diopters, or any retinal or vitreoretinal pathology affecting the macula, optic disc, or posterior pole. Participants were also excluded if they had a history of vascular, demyelinating, infiltrative, inflammatory, or compressive optic neuropathy, or any neurodegenerative, cerebrovascular, or systemic diseases known to influence test results. Regarding surgical history, individuals were excluded if they had undergone complicated cataract or glaucoma surgery; however, those with a history of uncomplicated procedures were included if at least six months had elapsed since the surgery.

Genomic DNA was isolated from whole peripheral blood samples collected in ethylene diamine tetraacetic acid tubes using a DiaRex blood DNA extraction kit (Diagen Biotechnological Systems Inc., Altındağ, Ankara, Türkiye). The nucleic acid loads (ng values) of the samples obtained from the total DNA extraction process were measured spectrophotometrically using a Colibri Microvolume Spectrometer (Titertek Berthold Technologies GmbH & Co.KG, Baden Württemberg, Germany) device for use later in the study.

Oligonucleotides were designed for the polymorphisms with rs2234968 (c.102G > A) and rs11258194 (c.293T > A) codes (Nucleotide Database Code numbers and variant positions for OPTN gene NM_001008212.2 transcript). Previously synthesized lyophilized primers were dissolved in nuclease-free distilled water to a volume of 100 µM. A SensiFAST Probe No-ROX Kit (Meridian Bioline, London, UK) was used in real-time polymerase chain reactions (PCR). Base changes were shown based on the allele-specific probe data obtained as a result of the real-time PCR process. Fluorescent dye-labeled probes were used for the detection of the T34T polymorphism, G and A alleles. Genotypes were recorded as GG (normal), GA (heterozygous), and AA (mutant). In the M98K polymorphism, genotypes for T and A alleles were recorded as TT (normal), TA (heterozygous), and AA (mutant).

The data were analyzed using IBM SPSS Statistics for Windows 26.0 (IBM Corp., Armonk, NY, USA). The Kolmogorov–Smirnov test was used to determine the distribution of the variables between the groups. The chi-squared test was used to assess the statistical significance of the T34T and the M98K polymorphisms in terms of the distribution of the allele frequencies between the groups. Genotype distributions in the control group were tested for Hardy–Weinberg Equilibrium (HWE); observed frequencies for both polymorphisms were consistent with expected HWE proportions (p > 0.05). Statistical significance was accepted as p < 0.05.

A post-hoc power analysis was conducted using G*Power 3.1 to determine the statistical adequacy of the sample size used in the study. The analysis used effect size (w) = 0.30, probability of error (α) = 0.05, degree of freedom (df) = 2, and total sample size (N) = 172. As a result of the power analysis conducted with these parameters, the statistical power (1–β) of the study was calculated as 95.0%. Consequently, these values ​​indicate that the study sample size was statistically sufficient to detect moderate effect sizes²⁸.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conception and design: CC, OGK Analysis and interpretation of the data: BC, AAY Drafting of the paper: YIE, CC Revising it critically for intellectual content: AAY, BC Final approval of the version to be published: OGK, YIE All authors agree to be accountable for all aspects of the work. All authors reviewed the manuscript.

Funding

This study was financially supported by University of Health Sciences, Department of Scientific Research Projects, İstanbul, Türkiye.

Data availability

The datasets analysed during the current study are available in the ClinVar repository, https://www.ncbi.nlm.nih.gov/clinvar/RCV000276775/ and https://www.ncbi.nlm.nih.gov/clinvar/RCV000324769/.

Declarations

Competing interests

The authors declare no competing interests.