OPA1 increases the risk of normal but not high tension glaucoma

Abstract

Background

Primary open angle glaucoma (POAG) is a progressive optic neuropathy characterised by the selective loss of retinal ganglion cells (RGCs), pathological optic disc cupping and visual field defects. The OPA1 gene encodes for an inner mitochondrial membrane protein crucial for normal mitochondrial function and pathogenic mutations cause autosomal dominant optic atrophy (DOA) by specifically targeting RGCs. This raises the distinct possibility that more subtle genetic variations in OPA1 could alter the risk of developing glaucoma.

Methods

We studied 137 POAG patients, 67 patients with high tension glaucoma (HTG), 70 patients with normal tension glaucoma (NTG), and 75 controls from the North East of England. Three single nucleotide polymorphisms (SNPs) in intron 8 (IVS8+4c>t and IVS8+32t>c) and exon 4 (c.473A>G) of the OPA1 gene were genotyped in our study group. In addition, the entire OPA1 coding region was sequenced in 24 individuals with the CT/TT compound genotype using standard BigDye™ chemistries.

Results

There was no difference in either allele or genotype frequency for the IVS8+32t>c SNP between patients and controls, but there was a significant association between the T allele at IVS8+4c>t and the risk of developing NTG (Odds ratio (OR) = 2.04, 95% confidence interval (CI) = 1.10-3.81, P = 0.004), but not HTG. Logistic regression analysis also confirmed a strong association between the CT/TT compound genotype at IVS8+4 and IVS8+32 with NTG (OR = 29.75, 95% CI = 3.83-231.21, P < 0.001).

Conclusions

The CT/TT compound genotype at IVS8+4 and IVS8+32 is a strong genetic risk determinant for NTG but not HTG.

Introduction

Primary open angle glaucoma (POAG) is the second most common cause of blindness in developed countries and accounts for about 10% of all blind registration in the United Kingdom.¹ It affects over 60 million people worldwide and with an ageing population, the prevalence of POAG is expected to increase by 30% in the next 20 years.² POAG is characterised by the selective, progressive loss of retinal ganglion cells (RGCs), leading to structural changes at the optic nerve head in the form of pathological cupping and visual field loss. The most significant risk factors for the development of glaucoma are raised intraocular pressure (IOP), increasing age, a positive family history in a first degree relative and ethnicity (Black > Hispanic > Caucasian > Asian).^3,4 In the non-glaucomatous population, IOP follows a normal distribution with a mean of 16.0 mmHg and a standard deviation (SD) of 2.5 mmHg, giving a statistical upper limit of 21.0 mmHg. About two thirds of POAG patients have IOPs > 21.0 mmHg at initial presentation i.e. high tension glaucoma (HTG), whereas the remainder with IOPs ≤ 21.0 mmHg are classified as normal tension glaucoma (NTG).^3,4

POAG is a complex disease with a strong genetic component and multiple susceptibility loci have been identified in populations from different ethnic backgrounds.^3,5 Four causative genes have been identified so far: optineurin (OPTN, OMIM 602432) on chromosome 10p14-15, myocilin (MYOC, OMIM 610652) on chromosome 1q24-25, CYP1B1 (OMIM 601771) on chromosome 2p21-22, and WDR36 (OMIM 609669) on chromosome 5q21-22, but these account for less than 5-10% of patients with sporadic, adult-onset POAG. Interestingly, a maternal family history of POAG is 6-8 times more likely than a paternal family history, which suggests a possible mitochondrial genetic influence.^6-8 Furthermore, mitochondrial abnormalities have been identified in patients with POAG, with an increase in mitochondrial DNA (mtDNA) content and a reduction in mitochondrial respiratory chain activities.⁹ The preferential loss of RGCs in glaucoma is also a key pathological feature seen in Leber hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (DOA), the two most common inherited optic neuropathies. Both these conditions are the result of mitochondrial dysfunction, LHON from primary mtDNA mutations affecting the respiratory chain complexes and the majority of DOA families have mutations in the OPA1 gene (3q28-q29), which codes for an inner mitochondrial membrane protein critical for mitochondrial maintenance, oxidative phosphorylation (OXPHOS) and regulation of apoptosis.¹⁰

Two single nucleotide polymorphisms (SNPs) within intron 8 of the OPA1 gene (IVS8+4c>t and IVS8+32t>c) have recently been linked with an increased risk of developing POAG in some, but not all, populations studied.^11-15 To further clarify the influence of OPA1 in modulating susceptibility to glaucoma, we have performed an analysis of OPA1 SNPs in a well-characterised cohort of POAG patients, and in addition reviewed the literature to compare the strength of the association identified in other study groups.

Methods

Patient and control samples

We investigated a well-characterised white Caucasian cohort consisting of 137 POAG patients (Mean age = 71.6 yrs, SD = 8.0 yrs) and 75 controls (Mean age = 79.3 yrs, SD = 4.4 yrs) from the North East of England, a region which has been relatively stable in terms of migratory flux.^16,17 The POAG group consisted of 67 HTG patients with a mean pre-treatment IOP of 23.1 mmHg (SD = 2.0 mmHg, range 22-28 mmHg) and 70 NTG patients with a mean pre-treatment IOP of 17.7 mmHg (SD = 2.2 mmHg, range 13-21 mmHg). Both cases and controls underwent a full ophthalmological examination including (i) IOP measurement by Goldmann applanation tonometry, (ii) gonioscopy to confirm open drainage angles, (iii) dilated fundal examination, and (iv) Humphrey full-threshold 24:2 visual field perimetry. All POAG patients had unequivocal evidence of glaucoma with both pathological optic disc cupping and characteristic visual field defects. HTG patients with intraocular pressures greater than 30mmHg were not included in this study. Control subjects were unaffected spouses of POAG patients and other ocular pathologies were carefully excluded. Genomic DNA was extracted from whole blood using established methods^16,17 and stored at −80°C for future molecular genetic investigations, with prior approval having been obtained from our local research ethics committee.

OPA1 genotyping

The two SNPs in intron 8 (IVS8+4c>t and IVS8+32t>c) were genotyped using the following primers to generate a 383 base pair polymerase chain reaction (PCR) product: (i) forward 5′-TGA AGT TCT TGA TGT TCT CTC TG-3′ and (ii) reverse 5′-ATG GCT AAT TTA ATC CAC TGT TC-3′. In the second phase of this study, the entire coding region of the OPA1 gene was amplified in 24 individuals with the CT/TT compound genotype (HTG N=10, NTG N=12, controls N=2), using a set of 27 M13-tagged primer pairs (available on request). Genotyping for the c.473A>G SNP located in exon 4 was also determined for the entire study cohort, using the following primers to generate a 341 base pair PCR product: (i) forward 5′-GGG TTG TCA TGA GGA TTA AAC AA-3′ and (ii) reverse 5′-AAA AAT GTC CTG TTT TTC ATT GG-3′. For all three SNPs (IVS8+4c>t, IVS8+32t>c and c.473A>G) and the entire OPA1 coding region, PCR products were purified and sequenced using BigDye™ terminator cycle chemistries on an ABI3100 Genetic Analyser (Applied Biosystems). Sequence results were then compared with the Genbank OPA1 reference sequence (Accession number AB011139) using SeqScape™ software v2.1 (Applied Biosystems).

MtDNA haplogroups

The mtDNA haplogroup status for both POAG patients and controls had previously been determined by restriction fragment analysis of amplified PCR fragments spanning specific informative sites within the mitochondrial genome.¹⁸ Haplogroups K, W, I, V, X and M were present in less than 5% of our control population and these were analysed as one single group.

Statistical analysis

The Hardy-Weinberg equilibrium for OPA1 genotypes was assessed for patients and controls (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) and statistical analyses were performed using SPSS™ v.15 statistical software (Chicago, Illinois). Allele and genotype SNP frequencies were compared with Fisher’s Exact Test and χ² analysis, with Bonferonni correction for multiple comparisons where applicable. Binary logistic regression was also used for multivariate analysis of other possible confounding factors that could influence the risk of developing glaucoma. This form of analysis assumes that the logarithm of the odds ratio is a linear function of the predictor variables included in the model:

$$\mathrm{Log}(\mathrm{P}∕1-\mathrm{P})={\mathrm{B}}_0+{\mathrm{B}}_1{X}_1+{\mathrm{B}}_2{X}_2+\dots {\mathrm{B}}_{\mathrm{n}}{X}_n$$

where P is the probability of developing glaucoma; X₁, X₂…X_n represent the chosen predictor variables; and B₀, B₁…B_n are coefficients reflecting the nature of each predictor.¹⁹ The independent variables used in our model were: (i) age, (ii) gender, (iii) maximum pre-treatment IOP, (iv) mtDNA haplogroup, and (v) IVS8+4 and IVS8+32 compound genotype.

Results

Intron 8 SNPs

Both POAG and control groups were in Hardy-Weinberg equilibrium at IVS8+4c>t and IVS8+32t>c. The T allele at IVS8+4c>t was over-represented among NTG patients compared to controls (Odds ratio (OR) = 2.04, 95% confidence interval (CI) = 1.10-3.81, P = 0.03), but there was no significant difference in allele frequency for the HTG and whole POAG groups. Similarly, there was a significant difference in the distribution of the three IVS8+4c>t genotypes for the NTG group (P = 0.025) but not for the HTG or whole POAG groups (Table 1A). There was no significant difference in either allele or genotype frequency for the IVS8+32t>c SNP when comparing all POAG cases, HTG and NTG subgroups to controls (Table 1B).

Table 1. Allele and genotype frequencies for the IVS8+4c>t and IVS8+32t>c OPA1 SNPs.

(A) IVS8+4c>t
	Controls (N=75)	Whole Group (N=137)		HTG (N=67)		NTG (N=70)
Allele			P value		P value		P value
C	131 (87.3%)	222 (81.0%)		114 (85.1%)		108 (77.1%)
T	19 (12.7%)	52 (19.0%)	0.096	20 (14.9%)	0.581	32 (22.9%)	0.030*
Genotype			P valuea		P valuea		P valuea
CC	59 (78.7%)	90 (65.7%)		49 (73.1%)		41 (58.6%)
CT	13 (17.3%)	42 (30.7%)		16 (23.9%)		26 (37.1%)
TT	3 (4.0%)	5 (3.6%)	0.106	2 (3.0%)	0.061	3 (4.3%)	0.025*

(B) IVS8+32t>c
	Controls (N=75)	Whole Group (N=137)		HTG (N=67)		NTG (N=70)
Allele			P value		P value		P value
T	79 (52.7%)	155 (56.6%)		60 (44.8%)		59 (42.1%)
C	71 (47.3%)	119 (43.4%)	0.440	74 (55.2%)	0.666	81 (57.9%)	0.375
Genotype			P valueb		P valueb		P valueb
TT	21 (28.0%)	41 (29.9%)		12 (17.9%)		11 (15.7%)
TC	37 (49.3%)	73 (53.3%)		36 (53.7%)		37 (52.9%)
CC	17 (22.7%)	23 (16.8%)	0.578	19 (28.4%)	0.769	22 (31.4%)	0.566

χ² analysis of all three possible genotypes at ^a IVS8+4C>T and ^bIVS8+32T>C;

^*Significant P value.

Compound genotype

Analysis of both IVS8+4c>t and IVS8+32t>c SNPs showed a statistically significant increased risk of glaucoma in subjects with the CT/TT compound genotype for the whole POAG group (OR = 6.98, 95% CI = 1.59-30.59, P = 0.003) and NTG group (OR = 7.55, 95% CI = 1.63-35.10, P = 0.004). Although there was a trend towards a higher risk of HTG with the CT/TT compound genotype (P=0.013), this was not significant following Bonferroni correction (Table 2). The CT/TT compound genotype was also not associated with higher pre-treatment IOPs or worse CDRs, in both the HTG and NTG groups (P > 0.05, data not shown).

Table 2. Compound genotype frequencies for the IVS8+4c>t and IVS8+32t>c OPA1 SNPs.

(A) Whole group
IVS8+4	IVS8+32	Patients	Controls	P value*	Odds ratio	95% CI
TT	TT	5 (3.6%)	3 (4.0%)	1.000*	0.91	0.21-3.92
CT	TT	22 (16.1%)	2 (2.7%)	0.003*	6.98	1.59-30.59
CT	TC	20 (14.6%)	11 (14.7%)	0.989*	0.99	0.45-2.21
CC	TT	14 (10.2%)	16 (21.3%)	0.026*	0.42	0.19-0.92
CC	TC	53 (38.7%)	26 (34.7%)	0.563*	1.19	0.66-2.14
CC	CC	23 (16.8%)	17 (22.7%)	0.296*	0.69	0.34-1.39

(B) HTG
IVS8+4	IVS8+32	Patients	Controls	P value	Odds ratio	95% CI
TT	TT	2 (3.0%)	3 (4.0%)	1.000	0.74	0.12-4.56
CT	TT	10 (14.9%)	2 (2.7%)	0.013	6.40	1.35-30.40
CT	TC	6 (9.0%)	11 (14.7%)	0.316	0.57	0.20-1.64
CC	TT	7 (10.4%)	16 (21.3%)	0.110	0.43	0.17-1.12
CC	TC	30 (44.8%)	26 (34.7%)	0.216	1.53	0.78-3.01
CC	CC	12 (17.9%)	17 (22.7%)	0.483	0.74	0.33-1.70

(C) NTG
IVS8+4	IVS8+32	Patients	Controls	P value*	Odds ratio	95% CI
TT	TT	3 (4.3%)	3 (4.0%)	1.000*	1.08	0.21-5.51
CT	TT	12 (17.1%)	2 (2.7%)	0.004*	7.55	1.63-35.10
CT	TC	14 (20.0%)	11 (14.7%)	0.396*	1.46	0.61-3.46
CC	TT	7 (10.0%)	16 (21.3%)	0.062*	0.41	0.16-1.07
CC	TC	23 (32.9%)	26 (34.7%)	0.818*	0.92	0.46-1.84
CC	CC	11 (15.7%)	17 (22.7%)	0.289*	0.64	0.27-1.48

^*Significant with Bonferroni correction: P < 0.008.

Logistic regression

Binary logistic regression confirmed a statistically significant increased risk of developing glaucoma with the CT/TT compound genotype for the whole patient group (OR = 56.52, 95% CI = 5.98-533.78, P < 0.001) and those with NTG (OR = 29.75, 95% CI = 3.83-231.21, P < 0.001), but not for the HTG group (Table 3). None of the mtDNA haplogroups were significant risk factors for glaucoma. Although haplogroup J was over-represented among NTG patients compared to controls (P=0.045), this fell below the level for statistical significance with Bonferroni correction.

Table 3. Logistic regression analysis comparing POAG patients with controls.

	Whole group		HTG		NTG
Predictor variables	Odds ratio (95% CI)	P value	Odds ratio (95% CI)	P value	Odds ratio (95% CI)	P value
MtDNA haplogroup a
H	0.62 (0.26-1.50)	0.292	0.97 (0.43-2.18)	0.934	0.51 (0.20-1.28)	0.151
T	0.42 (0.12-1.48)	0.176	0.65 (0.21-2.00)	0.457	0.37 (0.10-1.45)	0.154
J	3.79 (0.86-16.63)	0.077	1.64 (0.34-8.05)	0.540	4.42 (1.04-18.92)	0.045
U	1.68 (0.50-5.62)	0.401	2.02 (0.62-6.62)	0.246	1.85 (0.53-6.38)	0.332
Others	0.82 (0.24-2.82)	0.757	0.65 (0.19-2.21)	0.490	0.72 (0.21-2.52)	0.609
Compound genotype b
CT/TT	56.52 (5.98-533.78)	< 0.001*	9.91 (1.74-56.57)	0.010	29.75 (3.83-231.21)	< 0.001*

Our logistic regression models also incorporated the following predictor variables with the controls acting as the reference group: (i) age, (ii) gender, and (iii) maximum IOP, except in the HTG subgroup where maximum IOP was not included;

^*Significant with Bonferroni correction:

^aP < 0.01

^bP < 0.008.

OPA1 haplotype

We sequenced the entire OPA1 coding region of all 24 individuals with the CT/TT compound genotype at IVS8+4 and IVS8+32 (HTG N = 10, NTG N = 12, controls N = 2) and no previously described pathogenic mutations or polymorphisms were identified. However, all of them shared the same haplotype, with homozygosity at both the c.473A>G (p.N158S) and c.2109C>T (p.A703A) SNPs, except for one NTG patient (Figure 1A). The latter was heterozygous for the c.473A>G and c.2109C>T SNPs, and also had two additional heterozygous SNPs at c.575C>T (p.A192V) and c.1608A>C (p.A536A). Further analysis of the non-synonymous c.473A>G (p.N158S) SNP in our entire study cohort, did not show any statistically significant difference between our patient and control groups (Table 4).

Figure 1.

A: Shared OPA1 haplotype in individuals with the IVS8+4 and IVS8+32 CT/TT compound genotype.

B: Alignment of the 36 amino acids encoded by exon 4, indicating the poor evolutionary conservation of the codon altered by the c.473A>G SNP (p.N158S) in both lower vertebrates and invertebrates.

Table 4. Allele and genotype frequencies for the c.473A>G OPA1 SNP.

		Allele			Genotype
	N =	A	G	P value	AA	AG	GG	P valuea
Whole group	137	128 (46.7%)	146 (53.3%)		26 (19.0%)	76 (55.5%)	35 (25.5%)
Controls	75	75 (50.0%)	75 (50.0%)	0.543	19 (25.3%)	37 (49.3%)	19 (25.3%)	0.531
HTG	67	65 (48.5%)	69 (51.5%)		13 (19.4%)	39 (58.2%)	15 (22.4%)
Controls	75	75 (50.0%)	75 (50.0%)	0.813	19 (25.3%)	37 (49.3%)	19 (25.3%)	0.548
NTG	70	63 (45.0%)	77 (55.0%)		13 (18.6%)	37(52.9%)	20 (28.6%)
Controls	75	75 (50.0%)	75 (50.0%)	0.818	19 (25.3%)	37 (49.3%)	19 (25.3%)	0.613

^aχ² analysis of all three possible genotypes at c.473A>G.

Discussion

This study has identified a strong association between specific OPA1 polymorphisms at IVS8+4c>t and IVS8+32t>c and the risk of glaucoma. Subgroup analysis based upon pre-treatment IOPs further indicated that the susceptibility conferred by these two SNPs was largely restricted to the NTG group. This is compatible with the notion that non-IOP related factors are probably more important in the pathophysiology of NTG compared to HTG.^3,5 The T allele at IVS8+4 led to a two-fold increased risk of NTG and although the IVS8+32 SNP on its own was not a risk factor, compound genotype analysis suggested a synergistic influence with the IVS8+4 SNP and identified CT/TT as the high-risk compound genotype.

To confirm our initial findings, we performed a more rigorous logistic regression analysis to control for the possible influence of other variables that might affect the risk of developing glaucoma, thereby minimising the chance of identifying a spurious association. The odds ratio for the CT/TT compound genotype remained highly significant, and was associated with a 30-fold increased risk of NTG, independent of age, gender, maximum pre-treatment IOP and mtDNA haplogroup. However, the CT/TT compound genotype was not associated with markers of disease severity i.e. higher pre-treatment IOPs or worse optic disc cupping, supporting the conclusions of a previous report.²⁰ MtDNA haplogroup J was also over-represented among the NTG group compared to controls, although this became non-significant with Bonferonni correction. Nevertheless, this is an interesting observation given that the mtDNA background is thought to exert a direct effect on the assembly of the mitochondrial respiratory chain complexes²¹ and haplogroup J specifically has been linked with an increased risk of visual loss among Caucasian LHON mutation carriers²², the classic example of an inherited mitochondrial optic neuropathy.

We performed a literature review to identify all previous OPA1 glaucoma association studies, including unpublished meeting abstracts, and there is a consistent lack of association between the IVS8+4 and IVS+32 SNPs and HTG (Table 5). A high-risk compound genotype for developing NTG was found in two studies involving British Caucasian subjects^11,13 and one Japanese study²³, with the latter reporting a relatively weaker association. However, no association was identified in other populations of Asian^14,15,24 and African extraction^25,26, possibly indicating that the influence of OPA1 polymorphisms on NTG is limited to certain ethnic groups or it has a smaller effect (OR < 1.5), not detectable by the sample size (< 100) used in most of these studies. In this respect, it is worth noting that the control groups in these studies indicate a marked variation in the frequency of the minor T allele at IVS8+4c>t in different populations: Caucasian (7-18%), African (2-5%) and Asian (1-2%). Of note, the three other studies which have found a significant association between OPA1 and NTG have all identified different high-risk compound genotypes to the one identified in our study. As case-control studies are open to several sources of bias which can be difficult to control, this could therefore indicate a spurious population stratification effect.

Table 5. Summary of previous studies investigating the role of OPA1 polymorphisms in POAG.

Studya	Population	Significant association				High-risk compound genotype (NTG)
		HTG	N =	NTG	N =	IVS8+4/+32	Odds ratiob	95% CI
[11,12]	Caucasian	No	90	Yes	163	CT/TC	6.25	2.67-14.59
[13]	Caucasian	N/A	-	Yes	61	CC/CC	2.71	1.31-5.57
[14]	Japanese	N/A	-	No	337	-	-	-
[15]	Korean	N/A	-	No	65	-	-	-
[24]	Indian	N/A	-	No	50	-	-	-
[24]	Chinese	N/A	-	No	53	-	-	-
[24]	Japanese	N/A	-	No	42	-	-	-
[25]	African-Caribbean	No	48	No	61	-	-	-
[23]	Japanese	No	191	Yes	194	CC/TC	2.14	1.32-3.45
[26]	Caucasian	No	279	N/A	-	-	-	-
[26]	African-American	No	193	N/A	-	-	-	-
[26]	African	No	170	N/A	-	-	-	-

N/A: Not assessed; N: Number of patients analysed;

^aStudies in chronological order

^bOdds ratio were derived by χ² analysis using published figures and additional unpublished data provided by Dr Vincent Raymond (Personal communication) for study [14].

What are the possible explanations for the increased risk of NTG in individuals harbouring the CT/TT compound genotype? The majority of pathogenic OPA1 mutations are truncative (> 70%) and the reduction in protein level implicates haploinsufficiency as the underlying process leading to optic nerve degeneration in DOA.²⁷ Opa1 is a multi-functional protein and its crucial pro-fusion properties contribute to the maintenance of a highly interconnected mitochondrial network within cells.¹⁰ It is therefore not surprising that mitochondrial fragmentation is a prominent feature in fibroblasts cultured from DOA patients, with the release of cytochrome c from the mitochondrial compartment precipitating the onset of apoptotic cell death.^28,29 A recent study has also found murine RGC cells to be more sensitive to the downstream events of mitochondrial fragmentation and pro-apoptotic stimuli when compared to other neuronal populations. Since the IVS8+4 SNP is located within the donor splice site region, the CT/TT compound genotype could exert an effect, albeit unproven, on mRNA transcript levels, which would then potentiate RGC loss by disrupting the delicate balance between mitochondrial fusion and fission. However, the more pronounced effect seen in NTG compared to HTG clearly implicates other mechanisms and additional investigations are required.

Unlike other association studies, we also sequenced the whole OPA1 coding region, firstly to exclude the possibility of pathogenic mutations since DOA is not infrequently misdiagnosed as NTG in clinical practice³⁰, and secondly to explore the possibility that IVS8+4 and IVS8+32 are actually tagging SNPs in linkage disequilibrium with another functional OPA1 variant. Except for one NTG patient, all individuals carrying the CT/TT compound genotype shared the same OPA1 haplotype, with homozygosity at both the c.473A>G (p.N158S) and c.2109C>T (p.A703A) SNPs, suggesting a common ancestral event. Subsequent analysis of our entire cohort did not identify any significant difference in allele or genotype frequency at the poorly conserved, non-synonymous c.473A>G SNP in exon 4 (figure 1B). However, our study has not excluded the possibility that the IVS8+4 and IVS8+32 SNPs are in linkage disequilibrium with another causative gene in the vicinity of OPA1 or the presence of OPA1 intronic variants regulating mRNA splicing or transcriptional activity.

The finding of a significant association between specific combination of OPA1 polymorphisms and NTG does not imply causation and although biologically plausible, our results require additional functional confirmation, for example by comparing the bioenergetics and mitochondrial network morphology of fibroblasts obtained from patients with and without the high-risk CT/TT compound genotype. With a rapidly ageing population, glaucoma will remain an important cause of visual morbidity and a greater understanding of the complex genetic influences that lead to RGC loss will have important implications both for the identification and screening of high-risk groups, and in identifying pathophysiological pathways that could be amenable to therapeutic intervention.

Key Points.

• Primary open angle glaucoma (POAG) is the second leading cause of blindness in developed countries, and based upon their pre-treatment intraocular pressures (IOPs), patients are classified as having either high tension glaucoma (HTG, IOP > 21.0 mmHg) or normal tension glaucoma (NTG, IOP ≤ 21.0 mmHg).

• Although POAG is a late-onset acquired optic neuropathy, it has a major genetic basis and it shares striking clinical and pathological overlap with autosomal dominant optic atrophy (DOA), which is the result of mutations in the OPA1 gene.

• In this study, we demonstrate a strong association between two specific OPA1 SNPs (IVS8+4c>t and IVS8+32t>c) and the risk of developing NTG but not HTG. The high-risk CT/TT compound genotype conferred a 30-fold increased risk of disease, supporting a possible role of the Opa1 protein in the pathophysiology of NTG.