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(Investigative Ophthalmology and Visual Science. 2007;48:5591-5596.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-0780
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Nuclear and Mitochondrial Analysis of Patients with Primary Angle-Closure Glaucoma

Khaled K. Abu-Amero,1,2 Jose Morales,3 Mazen N. Osman,1,2 and Thomas M. Bosley4,5

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


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Certain types of glaucoma are linked to nuclear genetic mutations or to mitochondrial disturbances. In this study, patients with primary angle-closure glaucoma (PACG) were examined for mutations in nuclear genes reported to be associated with glaucoma and for possible mitochondrial abnormalities.

METHODS. In patients with PACG, the nuclear genes MYOC, OPTN, CYP1B1, WDR36, OPA1, and OPA3 were sequenced, the entire mitochondrial (mt)DNA coding region was sequenced, relative mtDNA content was measured, and mitochondrial respiratory activity (MRA) was assessed.

RESULTS. No novel or previously reported mutations were present in the nuclear genes MYOC, OPTN, CYP1B1, WDR36, OPA1, and OPA3 in 29 patients with PACG. Four (13.8%) patients had potentially pathologic mtDNA nucleotide changes not found in control subjects. The patients with PACG did not differ significantly from the control subjects in relative mitochondrial content and had only a small decrease in MRA (2.4%) of indeterminate significance.

CONCLUSIONS. These Middle Eastern patients with PACG had no mutations in nuclear genes associated with other types of glaucoma or inherited optic neuropathies. Mitochondrial abnormalities were minimal, and the overall pattern of those abnormalities was distinctly different from that of Leber hereditary optic neuropathy, nonarteritic ischemic optic neuropathy, primary open-angle glaucoma, and optic neuritis. These results are consistent with the hypothesis that anatomic factors may be more important determinants for PACG than the genetic and mitochondrial factors evaluated here.

The hallmark of primary angle closure glaucoma (PACG) is obstruction of the trabecular meshwork by iris tissue, which prevents exit of aqueous humor, elevates intraocular pressure (IOP), and often damages the optic nerve.1 Traditionally, PACG has been divided into acute, subacute, and chronic cases according to signs and symptoms at the time of diagnosis.1 Its prevalence varies greatly depending on the population involved, from a low of 0.1% among Europeans2 3 to a high of 2.65% among Eskimos older than 40 years,4 with Asian populations typically having an intermediate prevalence of approximately 0.8%.5 PACG is less common than primary open-angle glaucoma (POAG) in the Western hemisphere,6 but its prevalence in some parts of the globe is similar to that of POAG.7 8

More than 15 genetic loci and seven genes have been reported in association with POAG,9 the two most important of which are MYOC and OPTN.10 11 Only isolated patients with PACG have been reported to have MYOC mutations, including two with combined-mechanism glaucoma.12 13 A recent study screened 78 Taiwanese patients with acute PACG for occurrence of 67 single-nucleotide polymorphisms (SNPs) on 35 genes associated with various types of glaucoma and found a significant association with one SNP of the MMP-9 gene, which is important for remodeling of the extracellular matrix.14 No genetic mutations have been reported in chronic PACG15 even though anterior chamber depth may be an inherited characteristic16 and PACG occurs in 1% to 12% of relatives of patients with PACG.1 17

A recent study of Middle Eastern patients with POAG found evidence of mitochondrial abnormalities, including potentially pathologic mitochondrial (mt)DNA changes and decreased mitochondrial respiratory function.18 The purpose of this study was to evaluate patients with PACG in a similar fashion for the presence of mitochondrial abnormalities and for nuclear gene mutations associated with various types of glaucoma (MYOC, OPTN, WDR36, and CYP1B1) and certain inherited optic neuropathies (OPA1 and OPA3).19 20 21


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Patients
Patients were eligible for inclusion if they had evidence of glaucomatous optic nerve damage attributable to primary angle closure.5 Inclusion criteria included (1) clinical documentation of angle closure, defined as the presence of appositional or synechial closure of the anterior chamber angle involving at least 270° by gonioscopy in both eyes; (2) intraocular pressure elevated to a level ≥23 mm Hg measured before or after treatment by Goldmann applanation tonometry; (3) evidence of characteristic glaucomatous optic disc damage with excavation of the disc causing a cup-to-disc ratio (c/d) vertically of at least 0.70 in at least one eye; and (4) characteristic peripheral visual field loss including nerve fiber bundle defects (nasal step, arcuate scotoma, paracentral scotoma) or advanced visual field loss (central and/or temporal island of vision) as tested by a field perimeter (Humphrey Field Analyzer; Carl Zeiss Meditec, GmbH, Oberkochen, Germany), in those patients with vision better than 20/200, or Goldmann manual perimetry, in those with worse vision.

Exclusion criteria were (1) secondary angle closure glaucoma; (2) the presence of pseudoexfoliation syndrome even if coexistent with angle closure; (3) another cause of optic nerve injury affecting either eye; (4) significant visual loss in both eyes not associated with glaucoma; (5) inability to visualize the optic fundus for optic disc assessment; or (6) refusal to participate. Patients were Middle Eastern Arabs selected from the Glaucoma Clinic at King Khaled Eye Specialist Hospital (KKESH) after examination by a glaucoma specialist (JM) and informed consent approved by the KKESH Institutional Review Board. The research adhered to the tenets of the Declaration of Helsinki. Family members were not evaluated clinically or genetically.

Records were reviewed, and full ophthalmic examinations were performed. Every patient received laser iridotomy and antiglaucoma medications, and filtering surgery was performed when IOP remained elevated. Patients had either Goldmann manual kinetic perimetry (Haag Streit International, Köniz-Bern, Switzerland) or automated, white-on-white stimulus, static perimetry (Humphrey Field Analyzer II; Carl Zeiss Meditec, GmbH), or both. Optical coherence tomography (OCT) of the optic nerve including average nerve fiber thickness assessment and optic disc topography was performed (OCT3 System, Humphrey Systems (Carl Zeiss Meditec, GmbH) on some patients. Optic disc photographs were obtained by digital photography (FF 450 system; Carl Zeiss Meditec, GmbH).

Control Subjects
Control subjects were King Faisal Specialist Hospital and Research Centre blood donors 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. No ophthalmic examination was performed on these individuals. The control group for mtDNA sequencing consisted of 159 individuals (106 men and 53 women; mean age, 46.3 ± 3.8 years). For relative mtDNA content, 28 individuals (19 men and 9 women; mean age, 59.2 ± 3.3 years) and for mitochondrial respiration testing, 55 individuals (39 men and 16 women; mean age, 54.8 ± 4.6) were examined. Family information was obtained by history. All patients and control subjects were Middle Eastern Arabs.

Sample Collection and DNA Extraction
Single-density gradient (Ficoll-Paque-PLUS; Pharmacia Biotech AB, Uppsala, Sweden) was used for lymphocyte separation from peripheral blood as detailed previously.22 DNA was extracted from whole blood samples of all patients with PACG and control subjects (Puregene DNA isolation kit; Gentra Systems, Minneapolis, MN).

Sequence Analysis of Nuclear Genes
The coding exons, exon–intron boundaries, and promoter regions in the MYOC, OPTN, CYP1B1, and WDR36 genes were amplified by PCR from genomic DNA of all patients and control subjects and subjected to direct sequencing, as described previously.23 The 31 coding exons, exon–intron boundaries, and promoter regions of the OPA1 gene were amplified by PCR from genomic DNA for all patients and subjected to direct sequencing, as described previously.24 Similarly, the whole coding region and exon–intron boundaries for the OPA3 gene were sequenced in all patients by a protocol described previously.21

mtDNA Amplification and Sequencing
The entire coding region of the mitochondrial genome of all patients and control subjects was amplified in 24 separate polymerase chain reactions (PCRs) in single-set cycling conditions, as detailed elsewhere.25 Primers were designed to avoid amplifying mtDNA-like sequences in the nuclear genome. Each successfully amplified fragment was directly sequenced with a kit (BigDye Terminator, ver. 3.1 cycle sequencing kit; Applied Biosystems, Inc., Foster City, CA), and samples were run on a sequencer (Prism 3100; Applied Biosystems).

Sequence Analysis of the mtDNA Coding Region
The full mtDNA genome was sequenced except for the D-loop, and sequencing results were compared with the corrected Cambridge reference sequence.26 All fragments were sequenced in both forward and reverse directions at least twice, for confirmation of any detected variant. All nucleotide variants from patients and control subjects were compared with the MITOMAP database (last updated August 2007),27 the Human Mitochondrial Genome Database (http://www.genpat.uu.se/mtdb; last updated March 2007; provided in the public domain by the Institute for Genetics and Pathology, Uppsala University, Uppsala, Sweden), GenBank (http://www.ncbi.nlm.nih.gov/GenBank/index.html; last updated July 2007), and the MedLine listed publications. Reported homoplasmic synonymous or nonsynonymous (NS) polymorphisms associated with mitochondrial haplogroups28 were excluded from further consideration.29

Prediction of Pathogenicity
Pathologic characteristics of each remaining nucleotide change in both patients with PACG and control subjects were estimated according to a combination of standard criteria,30 an evaluation of interspecies conservation according to the PolyPhen database (http://genetics.bwh.harvard.edu/pph/ Brigham and Women’s Hospital, Harvard Medical School, Boston, MA), and the Mamit-tRNA Web site (http://mamit-trna.u-strasbg.fr/index.html/ Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France) when necessary; assessment of the possible impact of an amino acid substitution on three-dimensional protein structure on computer (Protean program, part of the Lasergene V.6 software; DNAStar, Inc., Madison, WI), which predicts and displays secondary structural characteristics; and assessment of the possible effect of the mtDNA change on protein function using PolyPhen.31 Therefore, a nonsynonymous nucleotide change was considered potentially pathologic if it met standard criteria30 and all the following criteria (when adequate information was available for databases to make predictions): (1) It changed a moderately or highly conserved amino acid, (2) Protean predicted an alteration of protein structure, and (3) it was assessed as possibly or probably pathologic by PolyPhen.

Quantification of Heteroplasmy
Heteroplasmy level was determined for each heteroplasmic sequence variant by the primer extension assay described previously.32 The level was quantified from fluorescence intensities associated with electrophoretically resolved mutant and wild-type peaks (Genescan, ver. 3.7 software; ABI). The percentage of heteroplasmy was calculated with the following equation: [fluorescent band intensity for the mutant/(fluorescent band intensity for the wild-type + fluorescent band intensity for the mutant)] x 100. This assay reliably detects mutant alleles present at ratios as low as 1% and 3%. The variability of the assay is typically ≤5%.32

Determination of Relative mtDNA Content
Competitive multiplex PCR was performed with two simultaneous primer sets, as described previously,33 a technique that has been applied successfully to a variety of tissues,34 35 including the blood of patients with LHON (Leber hereditary optic neuropathy)8 and other optic neuropathies.18 36 37 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 β-actin nuclear gene, which served as an internal control. Control samples were run simultaneously with those of patients. 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, Schenectady, NY). The ratio of ND1 to β-actin was determined for each patient and control subject by dividing the fluorescence intensity of the ND1 band by the intensity of the β-actin band.

Measurement of Mitochondrial Respiration
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 change in fluorescence (corrected for background and protein concentration) reflects respiration. Lymphocytes from patients and control subjects were incubated with 6 µM resazurin, without and with mitochondrial inhibition by amiodarone 200 µM, 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, as described previously.22 Mitochondrial metabolic activity has been assessed using resazurin in synaptosomes from spinal cord–injured animals38 and neonatal rat cerebellum39 and in isolated yeast mitochondria.40 The current technique has been validated in systemic mitochondrial disorders22 including LHON-like optic neuropathies.36

Statistical Methods
All statistical analyses were performed with commercial software (SPSS for Windows ver. 15.0; SPSS Inc, Chicago, IL). Snellen visual acuities were converted to ordinal values. Statistical comparisons included bivariate correlation, independent-samples t-test, and the Fisher exact analysis.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Clinical Information
Table 1 details the clinical characteristics of 29 unrelated patients with PACG (17 women and 12 men; mean age, 62.45 ± 8.40 years) who met inclusion and exclusion criteria. Patients were enrolled between August 2005 and December 2006 from a glaucoma clinic caring for patients with relatively advanced disease. They had been observed for an average of more than 5 years. Family history was positive for glaucoma in 10 patients, although family members were not examined and the type of glaucoma was not ascertained.


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  		TABLE 1. Clinical Characteristics of Patients

 
Five patients had acute angle-closure episodes and went on to develop the chronic form after customary treatment with laser iridotomy and glaucoma medications. Twenty-one had painless, chronic angle-closure glaucoma, and three had symptoms suggestive of intermittent angle closure with haloes, intermittent blurry vision, ocular pain, or headache. Eleven patients had a visual acuity (VA) of hand motions or worse in at least one eye. Mean maximum documented IOP was 38.8 mm Hg. All 29 patients had undergone laser iridotomy to treat pupillary block, and 13 had a filtering procedure, either alone or combined with cataract extraction. Results of OCT and fundus photography agreed well with clinical assessment of optic disc appearance (data not shown).

Sequence Analysis of MYOC, OPTN, WDR36, CYP1B1, OPA1, and OPA3
No novel or previously reported sequence mutation was found in MYOC, OPTN, WDR36, CYP1B1, OPA1, or OPA3 in patients with PACG or control subjects. Patients had no previously reported or novel polymorphisms in any of these nuclear genes, and control subjects had only polymorphisms reported previously.18

Sequence Analysis of the Mitochondrial Coding Region
The prevalence of synonymous and NS mtDNA nucleotide changes in patients with PACG was not different from that in control subjects (Fisher exact analysis, P = 0.24 for synonymous nucleotide changes and P = 0.55 for NS changes). Table 2 details the 11 NS mtDNA changes found in patients with PACG after excluding all synonymous mtDNA changes, established NS polymorphisms, and NS mtDNA sequence changes relevant primarily to haplogroup designation. None of these mtDNA sequence changes had been reported, and none was found in ethnicity-matched control subjects. Nine were transversions and two were transitions. One was heteroplasmic (nt 6880 with 20% heteroplasmy) and was considered nonpathologic. Four NS mtDNA sequence changes were considered probably pathologic according to the criteria described in the Methods section.


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  		TABLE 2. Nonsynonymous mtDNA Nucleotide Changes

 
Table 3 details mtDNA sequence changes by patient. Nineteen (patients 1–19) had none of the NS mtDNA sequence changes listed in Table 2 , whereas six (patients 20–26) had NS sequence changes predicted to be nonpathologic. The four potentially pathologic mtDNA sequence changes were present in four patients (26–29). Patient 26 had two NS mtDNA sequence changes from Table 2 : one predicted to be pathologic (nt 8806) and one predicted to be nonpathologic (nt 8660). All four patients with potentially pathologic mtDNA changes were male, but they did not differ from other patients in clinical characteristics such as age, family history, IOP, and VA.


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  		TABLE 3. Nonsynonymous mtDNA Sequence Changes, Relative mtDNA Content, and Mitochondrial Respiration Activity by Patient

 
Relative mtDNA Content and Mitochondrial Functional Testing
Table 3 also details relative mtDNA content and MRA by patient. Mean relative mtDNA content in patients with PACG (1.13 ± 0.20; 95% CI 1.06–1.21) was not significantly different from that in control subjects (1.16 ± 0.16; 95% CI 1.09–1.22; P = 0.67). MRA in patients with PACG (20.63 ± 1.14; 95% CI 20.20–21.07) was 2.4% lower than in control subjects (21.14 ± 1.03; 95% CI 20.86–21.41; P = 0.044), which probably is not clinically relevant. This difference is statistically nonsignificant after Bonferroni correction but is best interpreted as indeterminate, given post hoc indication of a minimal sample size approximately three times that studied to attain 80% power of avoiding false-negative interpretation.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
We evaluated 29 patients with PACG diagnosed by closure of the anterior chamber angle, history of elevated IOP, and funduscopic and visual field changes compatible with glaucomatous optic nerve damage. Because these patients were seen at a tertiary eye referral center, they had relatively advanced disease, with 14 being legally blind in one eye, two thirds with severe central or peripheral visual field loss, and almost half requiring a glaucoma surgical procedure. Compared with a group of patients with POAG evaluated previously in a similar fashion,18 these patients with PACG had slightly worse VA and slightly more surgical interventions. These patients did not meet criteria for POAG because of anterior chamber configuration, and they also did not have the clinical characteristics of other types of glaucoma or other spontaneous optic neuropathies, such as LHON or dominant optic atrophy.41

No patients had a novel or previously described mutations in MYOC, OPTN, WDR36, CYP1B1, OPA1, and OPA3. These results extend previous negative studies of MYOC in Chinese patients with chronic PACG15 and of MYOC, OPTN, CYP1B1, and OPA1 in Chinese patients with acute PACG.14 MYOC, OPTN, WDR36, and CYP1B1 have been reported in only a small fraction of patients with POAG, and it remains possible that these genes are abnormal with a small prevalence in Arabic patients with PACG or in patients with PACG who are of other ethnicities. Also, other nuclear genes may be important in PACG. For example, a particular SNP of MMP-9 has been associated with acute PACG,14 and a genetic localization was reported almost a decade ago in an uncommon variant of angle closure glaucoma characterized by nanophthalmos,42 a condition marked by a very short globe, severe hyperopia, and high lens/globe volume ratio.

We found evidence of only mild mitochondrial abnormalities in these patients with PACG. Ten patients had a total of 11 novel mtDNA nucleotide changes, and four had mtDNA sequence changes that were predicted to be pathologic. Relative mtDNA content was unchanged from control subjects, and patients with PACG as a group had a small and possibly insignificant decrease in MRA. These results contrast with the substantial mitochondrial abnormalities documented in a group of patients with POAG,18 in which the frequency of NS mtDNA nucleotide changes that were novel and/or potentially pathologic was double that of the PACG group, and the defect in MRA was almost 10-fold greater. The absence of substantial mitochondrial abnormalities in PACG suggests that not all types of glaucoma are associated with mitochondrial changes.

Optic nerve injury in PACG has been attributed primarily to elevated IOP caused by anatomic changes in the anterior1 and posterior43 globe, in contrast with the molecular and biochemical abnormalities suspected in POAG.44 Perhaps for this reason, the role of nuclear genetic and mitochondrial abnormalities in PACG has received limited attention, even though PACG may be present in 3.9 million people around the world by 2010.45 If confirmed in other studies involving different ethnic groups, the results reported herein imply that these particular nuclear genetic and mitochondrial factors may indeed be less important than anatomic and dynamic factors that result in closure of the anterior chamber angle to determine who is at risk for visual loss in PACG. We report a relatively small number of Middle Eastern Arab patients, and these genetic and mitochondrial studies should be confirmed and extended, because ethnicity may be important in the clinical characteristics of PACG.46 47


   Acknowledgements
 
The authors thank the staff of the King Khaled Eye Specialist Hospital Research Department for assistance in enrolling patients; Vincente M. Cabrera, PhD, for help in identifying haplogroup-specific mtDNA nucleotide changes; and Barry Milcarek, PhD, for statistical assistance.


   Footnotes
 
2 Present affiliation: Shafallah Genetics Medical Center, Doha, Qatar; and the Back

5 Neurology Division, Cooper University Hospital, Camden, New Jersey. Back

Supported in part by the Research Department of King Faisal Specialist Hospital and by a grant from the Prince Salman Center for Disability Research.

Submitted for publication June 26, 2007; revised September 5, 2007; accepted October 16, 2007.

Disclosure: K.K. Abu-Amero, None; J. Morales, None; M.N. Osman, 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, Shafallah Genetics Medical Center, PO Box 4251, Doha, Qatar; abuamero{at}shafallahgenetics.org.


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 Discussion
 References
 

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