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(Investigative Ophthalmology and Visual Science. 2006;47:2533-2541.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-1639
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Mitochondrial Abnormalities in Patients with Primary Open-Angle Glaucoma

Khaled K. Abu-Amero,1 Jose Morales,2 and Thomas M. Bosley3,4

1From the Mitochondrial Research Laboratory of the Genetics Department and the 3Neuroscience Department, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia; and the 2Glaucoma Division and 4Neuro-ophthalmology Division, King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Primary open-angle glaucoma (POAG) is the second most common cause of blindness. It has been linked to mutations in the myocilin (MYOC) and optineurin (OPTN) genes, although mutations have been found in <5% of patients. The pathologic mechanism(s) of POAG remain unknown but may include retinal ganglion cell apoptosis, which causes progressive damage to axons at the optic nerve head.

METHODS. In 27 patients with definite POAG, the MYOC and OPTN genes were sequenced, the entire mitochondrial (mt)DNA coding region was sequenced, relative mtDNA content was investigated, and mitochondrial respiratory function was assessed.

RESULTS. Only three benign polymorphisms were identified in MYOC and OPTN in patients with POAG and in control subjects. Conversely, 27 different novel nonsynonymous mtDNA changes were found, only in patients with POAG (not control subjects), 22 of which (found in 14 patients) were potentially pathogenic. Unlike Leber hereditary optic neuropathy, most mtDNA sequence alterations in patients with POAG were transversions—sequence changes that alter the purine/pyrimidine orientation and imply oxidative stress. mtDNA content was relatively increased in 17 patients with POAG compared with age-matched control subjects, also implying a possible response to oxidative stress. Mean mitochondrial respiratory activity was decreased by 21% in patients with glaucoma compared with control subjects (P < 0.001).

CONCLUSIONS. These results reveal a spectrum of mitochondrial abnormalities in patients with POAG, implicating oxidative stress and implying that mitochondria dysfunction may be a risk factor for POAG. This concept may open up new experimental and therapeutic opportunities.

Glaucoma is the most common optic neuropathic process affecting humans and the second most common cause of blindness worldwide.1 Primary open-angle glaucoma (POAG) is the most frequent type of glaucoma, with risk factors that include elevated intraocular pressure (IOP), older age, family history, and black racial ancestry.2 However, these factors alone do not predict the presence or degree of visual loss.3 POAG has been linked to mutations in the myocilin4 (MYOC) and optineurin5 (OPTN) genes, although mutations have been found in less than 5% of patients.

The primary pathogenesis of POAG remains unknown but may include chronic retinal ganglion cell apoptosis6 7 causing progressive damage to axons at the optic nerve head.8 The subacute retinal ganglion cell apoptosis and axonal injury at the optic nerve head in Leber hereditary optic neuropathy (LHON) are associated with mitochondrial abnormalities.9 10 Given that all major risk factors for POAG have not yet been identified and that known nuclear genetic abnormalities are associated with only a small fraction of patients, we decided to investigate a possible link between mitochondrial abnormalities and patients with POAG by cataloging mtDNA nucleotide changes, quantifying relative mtDNA content (to evaluate mtDNA duplication or depletion), and assessing mitochondrial respiratory function.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and Control Subjects
Patients were eligible for inclusion in this study if they met standard clinical criteria for POAG, including age greater than 40 years, intraocular pressure (IOP) ≥ 21 mm Hg in at least one eye before treatment, open anterior chamber angles bilaterally on gonioscopy, and optic nerve injury characteristic of POAG, including cupping of the optic disc with cup-to-disc ratio ≥0.5 and/or nerve fiber bundle visual field loss on static and/or kinetic perimetry in at least one eye.

Exclusion criteria included historical, neuroimaging, or biochemical evidence of another possible optic neuropathic process affecting either eye, significant visual loss in both eyes not associated with glaucoma, or refusal to participate. Patients were selected from the Glaucoma Clinic at the King Khaled Eye Specialist Hospital after examination by a glaucoma specialist (JM) and obtaining informed consent approved by the institutional review board. This research adhered to the tenets of the Declaration of Helsinki. All patients and control subjects were Middle Eastern Arabs.

Records were reviewed, and full ophthalmic examinations were performed. Patients had either Goldmann manual kinetic perimetry (Haag Streit International, Köniz-Bern, Switzerland) or Humphrey automated white-on-white stimulus static perimetry (Humphrey Field Analyzer II; Carl Zeiss Meditec, Inc., Dublin, CA), or both. Optical coherence tomography was performed (OCT3; Carl Zeiss Meditec, Inc.) on some patients. Fundus photographs were obtained (FF 450 system; Carl Zeiss Meditec, Inc.) on conventional film.

All control subjects were blood donors at the King Faisal Specialist Hospital and Research Centre, who represented the spectrum of Saudi Arabs and who reported no symptomatic metabolic, genetic, or ocular disorders on an extensive questionnaire regarding family history, past medical problems, and current health. Control subjects did not have ophthalmic examinations. The control group for MYOC and OPTN sequencing consisted of 96 individuals (64 men and 32 women; mean age, 47 ± 5.3 years); for mitochondrial (mt)DNA sequencing, 159 different individuals (106 men and 53 women; mean age, 46.3 ± 3.8 years); for relative mtDNA content, 30 different individuals (18 men and 12 women; mean age, 54 ± 7.2 years); and for mitochondrial functional testing, 64 different individuals (43 men and 21 women; mean age, 45.2 ± 7.5 years).

Sample Collection and DNA Extractions
A single-density gradient (Ficoll-Paque-PLUS; Pharmacia Biotech AB, Uppsala, Sweden) was used for lymphocyte separation and isolation from peripheral blood, as detailed previously.11 DNA was extracted from whole-blood samples of all patients with POAG and control subjects by using a DNA isolation kit (Purgene; Gentra Systems, Minneapolis, MN).

Sequence Analysis of MYOC and OPTN
The coding exons, exon–intron boundaries, and promoter regions in the MYOC and OPTN genes were amplified by PCR from genomic DNA for all patients and control subjects and subjected to direct sequencing, as described previously.12

DNA Amplification and Sequencing
The entire coding region of the mitochondrial genome was amplified in 24 separate polymerase chain reactions (PCRs), by using single-set cycling conditions, as detailed elsewhere13 for all patients and control subjects. Using a sequencing kit (BigDye Terminator ver. 3.1 Cycle Sequencing kit; Applied Biosystems [ABI], Foster City, CA), each successfully amplified fragment was directly sequenced (Prism 3100; ABI). All fragments were sequenced in both forward and reverse directions at least twice for confirmation of any detected variant.

Sequence Analysis of the Mitochondrial DNA Coding Region
Patient mtDNA sequences were compared with those from local control subjects, and all sequence variants were compared with the MitoMap database,14 the Human Mitochondrial Genome Database (http://www.genpat.uu.se/mtDB/ provided in the public domain by the Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden), GenBank (http://www.ncbi.nlm.nih.gov/GenBank/index.html/ provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), and Medline-listed publications (National Center for Biotechnology Information).

Prediction of Pathogenicity
Pathogenic characteristics of reported nonsynonymous mtDNA changes (those that change an amino acid in the resultant protein) were accepted from mitochondrial databases and Medline-listed literature. Pathogenic characteristics of a previously undescribed (novel) nucleotide change were determined according to a combination of standard criteria15 ; an evaluation of interspecies conservation using the Polyphen database (http://genetics.bwh.harvard.edu/pph/ provided in the public domain by Harvard University, Cambridge, MA), and when necessary, the Mamit-tRNA Web site (http://mamit-trna.u-strasbg.fr/index.html/ provided in the public domain by the University of Strasbourg, Strasbourg, France); an analysis of predicted changes in the Hydropathy Index, as measured by the Protean program (part of the Lasergene ver. 6 software; DNAStar, Inc., Madison, WI), according to the Kyte-Doolittle method, which predicts the regional hydropathy of proteins from their amino acid sequence (values were assigned for all amino acids and then averaged over a window size = 7); an assessment of the possible impact of an amino acid substitution on three-dimensional protein structure using Protean, which also predicts and displays secondary structural characteristics; and an assessment of the possible effect of the mtDNA change on protein function using PolyPhen16 and the SIFT (Sorting Intolerant From Tolerant) program (http://blocks.fhcrc.org/sift/SIFT.html/ provided in the public domain by the Fred Hutchinson Cancer Research Center, Seattle, WA), which predicts whether protein substitutions are tolerated.17

Therefore, a novel nucleotide change was considered potentially pathogenic if (1) it was not reported in mitochondrial databases or Medline-listed literature as a confirmed polymorphism; (2) it was not present in local control subjects; (3) it changed a moderately or highly conserved amino acid; (4) it occurred in a region of high interspecies conservation; (5) Protean predicted an alteration of protein structure; (6) it was assessed as possibly or probably pathogenic by PolyPhen; and (7) it was predicted by SIFT to have an effect on protein function.

Quantification of Heteroplasmy
The heteroplasmy level was determined for each heteroplasmic sequence variant by primer extension assay, as described previously.18 The heteroplasmy level was quantified from fluorescence intensities associated with electrophoretically resolved mutant and wild-type peaks by using a computer software program (GeneScan ver. 3.7; ABI). The percentage of heteroplasmy was calculated with the equation: [fluorescent band intensity for the mutant/(fluorescent band intensity for the wild-type + fluorescent band intensity for the mutant)] x 100.

Determination of Relative mtDNA Content
Competitive multiplex PCR was performed with two simultaneous primer sets as described previously.19 One pair was designed to amplify a 450-bp fragment of the ND1 mitochondrial gene and the other pair to amplify a 315-bp fragment of the ß-actin nuclear gene, which served as an internal control. PCR products were separated on 1% agarose gel at 100 V for 1 hour, and the intensity of the two bands was quantified by the use of a gel imager (Typhoon 9410; GE Healthcare, Piscataway, NJ). The ratio of ND1 to ß-actin was determined for each patient and control subject by dividing the fluorescence intensity of the ND1 band by the intensity of the ß-actin band.

Mitochondrial Functional Testing
The resazurin assay for mitochondria respiratory function has been described elsewhere.11 Resazurin is a redox-active blue dye that becomes pink and highly fluorescent when reduced. It competes with oxygen for electrons in a standard preparation of circulating lymphocytes, and the change in fluorescence reflects respiration. Lymphocytes from each patient and control were incubated with 6 µM resazurin without and with mitochondrial inhibition by 200 µM amiodarone, and the fluorescence intensity resulting from resazurin reduction was monitored spectrofluorimetrically over time. Mitochondrial respiratory activity (MRA) was calculated as the difference between uninhibited and inhibited measurements at 240 minutes, taken in triplicate, averaged, and normalized for protein concentration and background activity.11


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Clinical Information
Table 1 details the clinical characteristics of 27 patients with POAG (mean age at onset, 57.9 ± 10.7 years; 17 men and 10 women) from 27 different families who met inclusion and exclusion criteria. They had been observed for an average of more than 5 years in a glaucoma clinic caring for patients with relatively advanced disease. Ten patients reported a family history of poor vision, but family members were not examined or evaluated genetically. No patient had type 1 diabetes mellitus, ptosis, restricted ocular motility, deafness, ataxia, weakness, myotonia, exercise intolerance, palpitations, or syncope.


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  		TABLE 1. Clinical Characteristics of Patients with POAG

 
Twelve patients had a history of glaucoma surgery, and nine had hand motions or worse visual acuity (VA) in one eye. VA did not correlate with maximum IOP, age, or sex within this group of patients. Results of optical coherence tomography testing and fundus photographs agreed well with clinical assessment of optic disc cupping (data not shown).

Sequence Analysis of MYOC and OPTN
Table 2 shows the results of sequencing MYOC and OPTN in patients with POAG and control subjects. In the MYOC gene, only one sequence variant (the novel change 2259 G->T in Exon 3, which resulted in codon change G324V) was detected in seven patients with POAG (25.9%) and 28 control subjects (29.2%; P = 0.74), implying that it is likely a non–disease-causing polymorphism in this ethnic population.


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  		TABLE 2. MYOC and OPTN Variants in Patients with POAG and Control Subjects

 
In the OPTN gene, sequence variants 412 G->A in exon 4 and 469 G->C in exon 5 were each detected in one patient. The 412 G->A variant did not change an amino acid (T34T) and was a previously reported polymorphism.20 The novel 469 G->C sequence variant resulted in a codon change (Q53H) but was also found with a similar prevalence in control subjects, implying that it also was probably a non–disease-causing polymorphism in this population.

Sequence Analysis of the Mitochondrial Coding Region
Table 3 details all nonsynonymous mtDNA changes detected in patients with POAG that were not previously reported as polymorphisms in mitochondrial databases and Medline-listed literature. These 34 mtDNA sequence changes spanned the mitochondrial coding region; 17 (50%) were in complex I, 2 (5.9%) in complex III, 9 (26.5%) in complex IV, 5 (14.7%) in complex V, and 1 (2.9%) in tRNAglycine. Seven nonsynonymous mtDNA changes were detected in both patients with POAG and control subjects and were predicted to be benign. Twenty-seven of these changes were detected in patients with POAG but not in control subjects, and 22 altered moderately or highly conserved amino acids and were predicted to be damaging to the corresponding protein structure and/or function. The presence of these mtDNA sequence changes did not correlate with clinical characteristics (age, sex, IOP, VA, or degree of optic disc cupping).


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  		TABLE 3. Analysis of Nonsynonymous mtDNA Sequence Changes

 
More than 70% of the mtDNA sequence changes in Table 3 (25/34) were transversions. Significantly more patients with POAG had transversions (17/27) than transitions (8/27; {chi}2 P < 0.003). The presence of transversions, however, did not correlate with clinical characteristics.

Seven nonsynonymous mtDNA sequence changes were present in a heteroplasmic status (see Table 3 ). The heteroplasmy levels were 55% for nucleotide (nt) 4936, 68% for nt 8516, 21% for nt 8660, 70% for nt 10053, 70% for nt 10135, 65% for nt 10791, and 25% for nt 14580.

Table 4 lists the 52 synonymous mtDNA sequence changes detected in the patients with POAG, all of which were transitions and all of which were present in control subjects.


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  		TABLE 4. Synonymous mtDNA Sequence Changes in Patients with POAG

 
Relative Mitochondrial DNA Content
Relative mtDNA content was not significantly different between patients with POAG (mean, 0.81 ± 0.25 [SD]; 95% CI 0.70–0.91) and simultaneous control subjects (mean 0.73 ± 0.12; 95% CI 0.67–0.79; P < 0.25). However, the variation of relative mtDNA content was greater in patients with POAG (SD = 0.25) than in the control subjects (SD = 0.12). The optimum value of the ratio to distinguish between the two groups was 0.76 as calculated by receiver operator characteristic curve (ROC curve not shown; area under the ROC curve 0.685, 95% CI 0.525–0.844), and 17 patients with POAG had relative mtDNA content greater than this level. Relative mtDNA content did not correlate with clinical characteristics or with the presence of either mtDNA transitions or transversions.

Mitochondrial Functional Testing
Figure 1 displays the distribution of MRA levels for control subjects and patients with POAG in box plots. MRA in control subjects (mean 22.48 ± 0.88 [SD]; 95% CI 22.26–22.70; P < 0.001) was significantly greater than patients with POAG (mean 17.8 ± 2.78; 95% CI 16.69–18.89; P < .002). The optimum MRA value to distinguish between the twogroups was 21.39 (area under the ROC curve 0.948, 95% CI 0.891–1.004), and 24 patients with POAG had MRA levels less than this. MRA did not correlate with clinical characteristics and did not predict the presence of either mtDNA transitions or transversions.


Figure 1
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  		FIGURE 1. MRA in control subjects and patients with POAG. Box plot shows median, interquartile range, and outliers of MRA for 64 control subjects and 27 patients with POAG.

 
Table 5 summarizes pertinent mitochondrial data by patient, including nonsynonymous mtDNA sequence changes, relative mtDNA content, and MRA values (in ascending order).


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  		TABLE 5. Mitochondrial Abnormalities in Patients with POAG

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We evaluated 27 patients who met age, funduscopic, visual field, and anterior segment criteria that define POAG in contrast to other causes of optic nerve disease and other types of glaucoma. All these patients had documentation of elevated IOP during follow-up. Their nerve fiber bundle visual field loss would be atypical for hereditary optic neuropathies, and these patients were unusually old for LHON and other hereditary optic neuropathies. They were in an appropriate age range for nonarteritic anterior ischemic optic neuropathy (NAION),13 but a swollen optic disc was never observed, and characteristic glaucomatous optic nerve cupping was diagnosed by a glaucoma specialist (JM). No patient reported a maternal family history of visual loss.

No patient with POAG had a nucleotide change in MYOC or OPTN that was likely to be pathologic. All three nucleotide changes in these nuclear genes were present in control subjects and were likely to be polymorphisms in this population. The prevalence of MYOC and OPTN mutations in Arabic patients with POAG has not been evaluated before, but these results are not surprising, given the reported low prevalence of MYOC and OPTN mutations in open-angle glaucoma in other populations.21

No patient with POAG had a primary LHON mutation. The presence of primary LHON mutations has been investigated previously in normal-tension glaucoma12 but not in POAG. In addition, no patient had a secondary, intermediate, or provisional LHON mutation. Of course, LHON, which involves the subacute injury of the majority of optic nerve fibers,9 often in a young adult male with a maternal family history of visual loss, is a very different optic neuropathy from POAG, which causes the gradual loss of optic nerve fiber bundles over a period of years in an older individual.

However, several other mitochondrial abnormalities were detected in patients with POAG. More than 60% (17/27) of patients with POAG had nonsynonymous mtDNA changes not considered LHON mutations and not found in control subjects, most of which were predicted to cause pathologic changes. Most (17/27) had relatively increased mtDNA content, which may suggest a compensatory response to oxidative stress.22 In addition, almost 90% (24/27) of patients with POAG had reduced MRA, a measure of mitochondrial respiration.11 The coincidence of changes in the mitochondrial genome and in mitochondrial respiration raises the argument that the optic neuropathy of POAG is broadly associated with mitochondrial abnormalities. In fact, mitochondrial disturbances were much more frequent than MYOC or OPTN mutations in this Arabic population.

The list of optic neuropathies currently associated with mitochondrial abnormalities includes LHON, certain patients with optic neuritis and multiple sclerosis, Wolfram’s syndrome, dominant optic atrophy (DOA),9 and NAION.13 The maternal inheritance pattern of LHON led to initial mtDNA investigations, but even primary LHON mutations are incompletely penetrant with erratic reporting of family history.23 The penetrance of mtDNA changes reported herein may be somewhat less than primary LHON mutations with later presentation of symptoms and more interaction with other risk factors such as nuclear mitochondrial changes, anatomy of the anterior globe, IOP, race, and age, which is itself related in part to mitochondrial changes.24 These factors may obscure maternal inheritance in our patients and other POAG populations.25

The mechanisms by which mitochondrial abnormalities may place the optic nerve at risk remain uncertain.9 The high concentration of mitochondria at the optic nerve head implies dependency on some aspect of mitochondrial function,26 and mitochondrial disease in LHON, for example, has been linked to abnormalities in complex I activity.23 In contrast, some patients with LHON do not have primary LHON mutations20 27 or, at times, any mtDNA change at all.28 Patients with Wolfram’s syndrome29 or NAION13 have widely distributed mtDNA changes, and patients with dominant optic atrophy have mutations of OPA1 affecting a dynamin-related mitochondrial wall protein rather than a component of the electron chain.30 One hypothesis suggests that progressive optic nerve damage in POAG is the result of optic nerve fiber apoptosis.6 Mitochondria-induced apoptosis, which may be a mechanism of injury in experimental glaucoma31 and other optic neuropathies,32 may also be a pathologic factor in POAG.

mtDNA transition changes comprise almost 70% (23/33) of the primary, secondary, intermediate, and provisional LHON mutations reported currently in MitoMap.14 Similarly, 75% (12/16) of nonsynonymous mtDNA changes found in a group of 19 NAION patients were transitions.13 Unlike patients with LHON and NAION, patients with POAG had a high frequency of mtDNA transversion changes (25/34; 73.5%). The guanine base has the lowest oxidation potential of the four DNA nucleobases, making G:C->T:A or G:C->C:G transversion mutations frequent in the setting of oxidative stress.33 The presence of mtDNA transversions in POAG may be evidence that alternative mitochondrial damage and repair mechanisms are involved in the generation of mtDNA mutations in patients with POAG as part of a response to oxidative stress early in development. Transversions, in turn, may contribute to the unique optic nerve injury in POAG.

Mitochondrial pathology was variable in this POAG group, but mitochondrial abnormalities of one type or another were present in every individual. It is intriguing that potentially pathogenic mtDNA sequence changes (particularly transversions), increased relative mtDNA content, and reduced MRA levels all imply oxidative stress. Oxidative stress has also been reported to induce human trabecular meshwork degenerative changes that favor increased intraocular pressure.34 Therefore, oxidative stress early in development and/or throughout life could precipitate both metabolic and anatomic sequelae that increase the risk of optic nerve damage in POAG.

This report describes a relatively small number of patients with POAG from a restricted ethnic population. However, if these results are confirmed in other populations, knowledge that mtDNA mutations and/or mitochondrial dysfunction are present in POAG may lead to a better understanding of glaucoma pathophysiology.35 Clever approaches are now available for studying mitochondrial disease in the eye, and a novel in vitro treatment has already been devised for the metabolic defect of at least one mtDNA mutation.36 These tools might be applicable to investigating and treating POAG.


   Acknowledgements
 
The authors thank George Spaeth for reviewing the manuscript and the staff of the Research Department at King Khaled Eye Specialist Hospital for their assistance.


   Footnotes
 
Supported by the Research Department of King Faisal Specialist Hospital.

Submitted for publication December 23, 2005; revised January 28, 2006; accepted April 10, 2006.

Disclosure: K.K. Abu-Amero, None; J. Morales, None; T.M. Bosley, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Khaled K. Abu-Amero, Mitochondrial Research Laboratory, Department of Genetics, KFSHRC, PO Box 3354, MBC-03, Riyadh 11211, KSA; kamero{at}kfshrc.edu.sa.


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 Materials and Methods
 Results
 Discussion
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Y. He, K. W. Leung, Y.-H. Zhang, S. Duan, X.-F. Zhong, R.-Z. Jiang, Z. Peng, J. Tombran-Tink, and J. Ge
Mitochondrial Complex I Defect Induces ROS Release and Degeneration in Trabecular Meshwork Cells of POAG Patients: Protection by Antioxidants
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K. K. Abu-Amero, J. Morales, M. N. Osman, and T. M. Bosley
Nuclear and Mitochondrial Analysis of Patients with Primary Angle-Closure Glaucoma
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W.-K. Ju, Q. Liu, K.-Y. Kim, J. G. Crowston, J. D. Lindsey, N. Agarwal, M. H. Ellisman, G. A. Perkins, and R. N. Weinreb
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Invest. Ophthalmol. Vis. Sci., May 1, 2007; 48(5): 2145 - 2151.
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A. W. Hewitt, S. L. Bennett, J. H. Fingert, R. L. Cooper, E. M. Stone, J. E. Craig, and D. A. Mackey
The Optic Nerve Head in Myocilin Glaucoma
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