Jpn J Ophthalmol 2006;50:128–134 © Japanese Ophthalmological Society 2006
DOI 10.1007/s10384-005-0290-0
LABORATORY INVESTIGATION
Mitochondrial DNA Mutations with Leber’s Hereditary Optic Neuropathy in Japanese Patients with Open-Angle Glaucoma Yoko Inagaki1, Yukihiko Mashima1, Nobuo Fuse2, Yuichiro Ohtake1, Takuro Fujimaki3, Takeo Fukuchi4, and the Glaucoma Gene Research Group 1
Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan; Department of Ophthalmology and Visual Sciences, Tohoku University Graduate School of Medicine, Sendai, Japan; 3 Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan; 4 Division of Ophthalmology and Visual Science, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan 2
Abstract Purpose: Abnormal optic disc excavations are found in patients with Leber’s hereditary optic neuropathy (LHON). The purpose of this study was to determine whether heteroplasmy for the major three LHON mutations or for the rare LHON mutations are risk factors for open-angle glaucoma. Methods: Blood samples from 835 Japanese subjects were screened with the Invader assay for ten LHON-associated mutations: three major mutations (G3460A, G11778A,T14484C) and seven rare mutations (T9101C, G9804A, C14482A, C14482G, G14459A, T14498C, and A14510G). Of the 835 subjects, 241 were patients with primary open-angle glaucoma (POAG), 310 were patients with normal-tension glaucoma (NTG), and 284 were healthy controls. Results: Five POAG patients and three NTG patients had one of five mutations, C9099A, T9101G, T9101C, G9804A, or G11778A, but none of these patients had LHON. The C9099A (Ile191Met) and T9101G (Ile192Ser) mutations were novel and identified within the probes by lack of signal in the assay. Two patients with the G11778A mutation showed heteroplasmy, with 15% mutant mtDNA in the male patient and 80% in the female patient. The remaining LHON-associated mutations were not detected in any of the subjects. A case-control study did not show a significant difference (P = 0.099): eight potentially disease-associated variants in 551 patients versus zero variants in the 284 controls. Conclusions: Rare LHON-associated mitochondrial DNA mutations were found in Japanese patients with open-angle glaucoma (OAG). However, whether mitochondrial DNA mutations are risk factors for OAG is still open to question. Jpn J Ophthalmol 2006;50:128–134 © Japanese Ophthalmological Society 2006 Key Words:
Leber’s hereditary optic neuropathy, mitochondrial DNA, mutation, open-angle glaucoma
Introduction Glaucoma is a major cause of blindness worldwide, and approximately 67 million individuals are estimated to have Received: November 22, 2004 / Accepted: June 28, 2005 Correspondence and reprint to: Yukihiko Mashima, Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan e-mail:
[email protected] The members of the Glaucoma Gene Research Group are listed at the end of this article.
some form of glaucoma.1 Most cases represent adult, lateonset open-angle glaucoma (OAG), and the overall prevalence of OAG in subjects >40 years of age in Japan has recently been estimated to be approximately 3.9%.2 Glaucoma, a multifactorial disorder characterized by progressive optic neuropathy with characteristic visual field loss, results from the interactions of multiple genes and environmental influences.3,4 An elevation of the intraocular pressure (IOP) is a major risk factor for OAG.5 Glaucoma-like morphological changes have been reported in patients with Leber’s hereditary optic neuropa-
Y. INAGAKI ET AL. MITOCHONDRIAL DNA MUTATIONS IN GLAUCOMA
thy (LHON) at the atrophic stage.6–11 Recently, we analyzed the optic disc excavations at the atrophic stage quantitatively in 15 Japanese LHON patients with the 11778 mutation, using Heidelberg retinal tomography (HRT).12 The optic discs at the atrophic stage of LHON were shown to have glaucoma-like morphological changes. The similarity in the optic disc findings in LHON and normal-tension glaucoma (NTG) suggests that alterations in mitochondrial function may be related to optic disc excavations. Patients with LHON usually have severe and permanent loss of vision with a visual acuity of less than 0.1. Central or cecocentral scotomas are the characteristic visual field defects.13 LHON is a maternally transmitted eye disease. About 70% of patients are male, mainly young adult men. Although more than 20 point mutations of mitochondrial DNA (mtDNA) have been reported in LHON patients worldwide,14 more than 80% of LHON patients carry only one of three mtDNA mutations at nucleotide positions 3460, 11778, or 14484.15 Large optic disc cups have also been reported in patients with dominant optic atrophy (DOA, Kjer type).9,16 The gene responsible for DOA is the OPA1 gene, which encodes a dynamin-related protein localized in mitochondria.17 Recently, polymorphisms in the OPA1 gene have been linked to NTG in England.18 When 62 NTG patients in the United States19 and Germany20 were tested for the three LHON mutations involving mtDNA nucleotide positions 3460, 11778, and 14484, no mutations were detected. As opposed to major mitochondrial defects, we have hypothesized that heteroplasmy for the three major LHON mutations or for the rare LHON mutations are risk factors for glaucoma. The purpose of this study was to detect and quantify heteroplasmy for the ten mtDNA mutations associated with LHON (G3460A, G11778A, and T14484C for the major mutations;15 T9101C,21 G9804A,22 G14459A,23 C14482A,24 C14482G,24 T14498C,25 and A14510G26 for the rare mutations) in 835 Japanese subjects, using the Invader assay. We recently developed a technique to quantify the degree of heteroplasmy of mtDNA mutations using the new biplex invader technique called the Invader assay (Third Wave Technologies, Madison, WI, USA).27 This method, which is not based on polymerase chain reaction (PCR), was developed to determine genotypes with single nucleotide polymorphisms (SNPs), and the assay has been used for high-throughput genotyping.28
Patients and Methods A total of 835 blood samples were collected at eight ophthalmological institutions in Japan. The subjects included 241 primary open-angle glaucoma (POAG) patients, 310 NTG patients, and 284 normal controls. None of the subjects were related to any of the others.The patients whose age at diagnosis was less than 40 years and patients with more than −5.5 D of myopia were excluded. Patients with myocilin29 or optineurin30 mutations were also excluded.
129
The procedures used in this research conformed to the tenets of the Declaration of Helsinki. Written informed consent was obtained after the nature and possible consequences of the study were explained. Where applicable, the research was approved by the appropriate institutional Human Experimentation Committee. The mean ± SD age at time of the blood sampling was 64.8 ± 12.1 years for POAG (n = 241), 59.3 ± 13.3 years for NTG patients (n = 310), and 67.3 ± 12.9 years for the control subjects (n = 284). The control subjects were significantly older than the POAG patients (P < 0.001) or the NTG patients (P < 0.001). We deliberately selected older control subjects to reduce the likelihood that a subset of the controls would eventually develop glaucoma. All patients received serial ophthalmic examinations including IOP measurements by Goldmann applanation tonometry, Humphrey perimetry (30-2) or Goldmann perimetry, gonioscopy, and optic disc examination, including fundus photography. All of the patients were diagnosed as having glaucoma according to the following criteria: the presence of typical optic disc damage with glaucomatous cupping (cup/disc ratio >0.7) and loss of the neuroretinal rim; reproducible visual field defects compatible with the glaucomatous cupping; and open angles on gonioscopy. POAG was diagnosed if the IOP was >21 mmHg at any time during the follow-up period. NTG was diagnosed if the untreated peak IOP was consistently
