OPA1 mutations in Japanese patients suspected to have autosomal dominant optic atrophy
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- Hamahata, T., Fujimaki, T., Fujiki, K. et al. Jpn J Ophthalmol (2012) 56: 91. doi:10.1007/s10384-011-0096-1
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To report three types of heterozygous mutations in the OPA1 gene in five patients from three families with autosomal dominant optic atrophy (ADOA, MIM#165500).
DNA was extracted from the leukocytes of the peripheral blood. For mtDNA, mutations were examined at positions 11778, 3460 and 14484. For the OPA1 gene, the exons were amplified by PCR and mutations were detected by restriction enzymes or the dye terminator method.
We detected three types of OPA1 mutation but no mtDNA mutations. In the OPA1 gene, heterozygous frameshift mutations from codon 903 due to a four-base pair deletion in exon 27 were detected in three patients from one family (c.2708_2711delTTAG, p.V903GfsX905). A heterozygous mutation due to a three-base pair deletion in exon 17, leading to a one-amino acid deletion (c.1618_1620delACT, p.T540del), and a heterozygous mutation due to a one-base substitution in exon 11, leading to a stop codon (c.1084G>T, p.E362X), were detected in sporadic cases.
OPA1 mutations existed in three Japanese families with ADOA. After a detailed clinical assessment of the proband, the screening of the OPA1 gene may be helpful for precise diagnosis of ADOA, provided the relevant information of the family members is limited.
KeywordsOPA1Autosomal dominant optic atrophyMitochondriaMutation
Autosomal dominant optic atrophy (ADOA; MIM#165500), also known as Kjer’s disease , is a hereditary bilateral optic neuropathy. ADOA has quite a few characteristic indications: the age of onset is in the early decades of life; the symptoms are relatively insidious; visual acuity (VA) decrease is bilateral and progressive, but there is no acute vision loss; the visual field shows variable centrocecal, either central or paracentral visual field defects and central or centrocecal visual field scotoma is present [2, 3]; the optic nerve head shows either temporal or diffuse optic nerve pallor with optic disc excavation ; color vision indicates either blue-yellow dyschromatopsia or generalized color vision deficits. Intra- and interfamilial clinical variability and an incomplete penetrance are estimated to occur in about 90% of the familial forms of the disease . Extensive ophthalmic examination of the patients rules out any evidence of glaucomatous, inflammatory, ischemic, toxic or nutritional causes of bilateral optic neuropathy. Kjer first described 19 cases of ADOA in 1959 , and Delettre et al.  and Alexander et al.  reported OPA1 as a causative ADOA gene in 2000. To date, over 200 OPA1 mutations have been reported and submitted to the eOPA1 database . OPA1-related ADOA has incomplete penetrance, reported to range from 43 to 90% in several families [5, 9, 10]. The estimated ADOA prevalence is 1:12,300 for ADOA in Denmark  to 1:50,000 and is the most common hereditary optic neuropathy worldwide [12, 13]. On the other hand, Leber’s hereditary optic neuropathy (LHON)  is known as a sudden asynchronous onset of visual loss in both eyes and appears at a later age (18–35 years), although in the atrophic phase is it difficult to distinguish ADOA from LHON by clinical findings alone. We performed the OPA1 gene analysis in five patients from three families who were suspected of having ADOA from their clinical findings.
Patients and methods
The present study was designed by the Department of Ophthalmology of Juntendo University Hospital in order to investigate the clinical variability and the OPA1 genotype in five patients from three families who were suspected of having ADOA. The study was performed according to the tenets of the Declaration of Helsinki and approved by the Ethics Committee of Juntendo University School of Medicine.
Clinical characteristics in patients with OPA1 mutations
Age at examination (years)
Age of onset (years)
Visual acuity (right/left)a
Visual field (right/left)
Optic nerve head (right/left)
Case 1 (family 1, III-2)
Case 2 (family 1, IV-1)
A 14-year-old girl had suffered from a decrease in VA from age 6. Her VA, measured at a neighborhood medical clinic, was OD: 0.6 × −5.0 D and OS: 1.0 × −5.5 D, but the VA, when first measured in our hospital, was OD: 0.6 × soft contact lens with −0.25 D and OS: 0.7 × soft contact lens with −0.5 D cylinder −0.25 DAx 120°. The MD and PD of her visual fields were OD: −3.48 dB, OS: −4.09 dB with centrocecal scotoma. Her disc findings were slight excavation.
Case 3 (family 1, IV-2)
A 13-year-old girl had suffered from low VA from age 11. Upon first examination at our hospital, her VA was OD 0.2 × −2.75 D and OS 0.2 × −2.50 D. Fundus examination showed a pale optic disc on the temporal side, and the MD and PD of her visual fields were OD: −5.54 dB with centrocecal scotoma, OS: −5.28 dB with centrocecal scotoma.
Case 4 (family 2, III-2)
A 32-year-old woman had been diagnosed with amblyopia when she was a schoolgirl, and both her mother and grandmother have visual impairment. Her first VA in our hospital was OD: 0.15 × −0.75 D cylinder −0.5 DAx 85°, and OS: 0.15 × +1.50 D cylinder −0.5 DAx 100°. Fundus examination showed bilateral whiteness and pallor of the optic disc on the temporal side. On Goldmann perimetry, her visual fields showed bilateral relative central scotoma with I/4e isopter (Fig. 2) OU.
Case 5 (family 3, II-1)
A 31-year-old man had been followed at his neighborhood clinic for low VA. His first VA measured in our hospital was OD: 0.3 × −1.5 D and OS: 0.3 × −1.0 D. Fundus examination showed bilateral pale optic disc temporal side, and the MD of his visual field was OD: −2.38 dB with centrocecal scotoma (Fig. 2), OS: −1.94 dB with blind spot enlargement.
Molecular genetic studies (OPA1 mutation analysis)
We first screened the OPA1 gene of the five patients who were suspected of having ADOA. Whenever the OPA1 mutation was found in these patients, the three primary LHON-causing mtDNA mutations were subsequently screened to confirm the absence of LHON.
Genomic DNA and mtDNA were extracted from leukocytes of the peripheral blood of these patients and of 100 unrelated subjects in a control group in accordance with the standard procedures after obtaining informed consent. The patient in case 1 agreed with the need to perform genetic analysis of himself, and we also obtained informed consent from the patients in case 2 and 3. We amplified all 28 exons of the OPA1 gene using polymerase chain reaction (PCR) with a Qiagen Multiplex PCR Kit (Qiagen, Hilden, Germany). The dye terminator method was used for sequencing. The primers used for PCR and sequencing were as described by Tooms et al. . PCR was carried out in a volume of 20 μL using a Gene Amp PCR system 9700 (Perkin Elmer Applied Biosystems, Foster City, CA, USA) with the following protocol: an initial cycle of 95°C for 15 min, followed by 35 cycles of 94°C for 45 s, 58°C for 45 s and 72°C for 60 s, and a final extension of 72°C for 10 min. The PCR products were purified with a High Pure PCR product Purification Kit (Roche Diagnostics, Mannheim, Germany), subjected to a dideoxy chain termination reaction using a BigDye Terminator v1.1 Cycle Sequencing Kit (Perkin Elmer Applied Biosystems) and run on an ABI PRISM 3130 Genetic Analyzer (Perkin Elmer Applied Biosystems) in both directions. Nucleotide sequences were compared with the published human mitochondrial DNA sequence and OPA1 cDNA sequence (GenBank entry AC_000021.2, NG_011605). The human OPA1 amino acid sequence was compared with that of other species using the NCBI amino acid sequence database.
We analyzed three primary LHON-causing mtDNA mutations of m.11778G>A, m.3460G>A and m.14484T>C with the same procedure to confirm the exception of LHON.
Partial alignment of the amino acid sequences of 5 eukaryotic OPA1 homologs selected using an online analysis tool (available at http://www.ncbi.nlm.nih.gov/homologene/) was constructed and analyzed. The website was accessed on 21 May 2011. The OPA1 proteins (GenBank accession numbers) used were Homosapiens (NP_570846.1), Mus musculus (NP_598513.1), Drosophila melanogaster (NP_725369.1), Anopheles gambiae (XP_309360.3) and Caenorhabditis elegans (NP_495986.3).
Autosomal dominant optic atrophy (ADOA; MIM#165500) is one hereditary bilateral optic neuropathy. The OPA1 gene is composed of 30 coding exons distributed across more than 90 kb of genomic DNA on chromosome 3q28–q29 [15, 16]. Alternative splicing of exons 4, 4b and 5b leads to eight transcript isoforms with open reading frames for polypeptides of 960–1,015 amino acids . OPA1 encodes a mitochondrial dynamin-related GTPase, which consists of an N-terminal mitochondrial leader sequence, a highly conserved GTPase domain, a central dynamin domain and a C-terminal coiled-coil domain [18, 19]. It is believed the protein is involved in the mitochondrial fusion process and in the maintenance of mitochondrial morphology . More than 200 OPA1 mutations have been reported . We performed a molecular screening of 5 patients from 3 pedigrees for the diagnosis of hereditary optic neuropathies. We identified a heterozygous frameshift mutation from codon 903 due to a four-base pair deletion in exon 27 in three patients from one family (c.2708-2711delTTAG, p.V903GfsX905), as well as a novel heterozygous mutation due to a three-base pair deletion in exon 17, leading to a one-amino acid deletion (c.1618_1620delACT, p.T540del), and a novel heterozygote mutation due to a one-base substitution in exon 11, leading to a stop codon (c.1084G>T, p.E362X) in two patients from other families.
The preponderance of OPA1 mutations leading to premature translation terminations and null mutations strongly suggest that haploinsufficiency is the main pathogenic mechanism from 204 pathogenic mutation analyses .
Some specific mutations like p.R445H cause ADOA and deafness (ADOAD) . This form of ADOA is linked to missense mutations affecting the GTPase domain , suggesting that a dominant negative effect contributes to the pathogenesis in these severe forms of the disease. There were no additional severe forms like ADOA in our cases. Thus, the three types of mutations that we detected might arise with a haploinsufficiency mechanism similar to the preponderance of OPA1 mutations.
The heterozygous frameshift mutation c.2708_2711delTTAG is one common mutation in the OPA1 gene . The haplotype analysis with the same mutation suggests that it is a mutation hot spot . Although we did not analyze the haplotypes around this mutation in family 1, the fact that the same mutation was identified in our Japanese family, a long distance from Europe and of a different race would also suggest that the c.2708_2711delTTAG is a mutation hotspot and not an ancient mutation [9, 24].
At the OPA1 protein level, 40% of mutations are premature translation terminations, 27% are missense mutations, 27% are in-frame splice variants and 6% are either deletions or duplications . The novel one-amino acid deletion p.T540del was interpreted as likely to be pathogenic because the threonine at residue 540 is highly conserved among all 5 close eukaryotic homologs ranging from H. sapiens to C. elegans (Fig. 3). Each amino acid sequence relative position is conserved around T540, for example Q538, R542, N543, S545 and L546 in Fig. 3. The mutation of S545R, which is near T540, is reported as a pathogenic amino acid substitution by two investigators [21, 24]. Additionally, an amino acid deletion of p.E521del is reported upstream and of p.C551del downstream of T540 [21, 26]. The mutation of p.T540del might break the fine effective amino acid positioning of the conserved OPA1 function as other amino acid deletions do. Case 4 (family 2, III-2) with the novel one-amino acid deletion mutation p.T540del was quite severely affected in our cases. Although we have not confirmed it by biochemical analysis, this would indicate that residue 540 plays an important function in the dynamin central region, such as involvement in the self-assembly of the OPA1 protein  or interaction with other proteins.
The novel nonsense mutation (c.1084G>T, p.E362X) detected in case 5 caused premature termination of the OPA1 translation. As the premature stop codons are not located in the last exon of the OPA1 gene, the possibility exists that nonsense-mediated mRNA decay (NMD) might be involved in abnormal RNA processing. NMD is a widespread cellular process that proofreads nascent mRNA transcripts and destroys those with premature termination codons before they are actually translated in truncated and potentially harmful proteins. In a recent study, Schimpf et al.  demonstrated that the majority of nonsense OPA1 mutations underwent NMD. They found that mutant transcript levels were reduced between 1.25- and 2.5-fold, and varied among premature termination codons containing mutations. The OPA1 protein is necessary for the mitochondrial fusion process and located at the external face of the mitochondrial inner membrane. Mitochondrial fragmentation has been observed in HeLa cells with downregulated OPA1 using specific small interfering RNA (siRNA)  and also in fibroblasts from patients with OPA1 mutations . The OPA1 gene is expressed in every cell, but the retinal ganglion cells become especially dysfunctional in ADOA patients because OPA1 is abundantly expressed in retinal ganglion cells, and the optic nerve requires more energy than other tissues. The mitochondrial disturbance arising from the OPA1 mutation leads to energy supply defects. Increase of sensitivity of apoptosis was also observed in fibroblasts from patients carrying OPA1 mutations [25, 28]. Retinal ganglion cells are particularly sensitive to cell death, and its dysfunction or reduced expression of OPA1 exacerbates the degeneration of these cells. The OPA1 mutant models [29, 30] are valuable for the study of this neurodegenerative process and for the development and evaluation of future therapeutic strategies for ADOA. In case 5, our results indicate that sporadically appearing cases of optic atrophy may be caused by OPA1 mutations. Ferre et al.  report finding an OPA1 mutation in 295 of their patients; 153 patients (52%) had a familial history and 142 patients (48%) were apparently sporadic cases. Thus, it is important to perform a detailed family examination to determine the hereditary patterns in cases of optic atrophy, even when they are considered to be sporadic cases without precedent in their family histories. Nonetheless, only molecular genetic examination can provide an exact determination of the hereditary patterns in these cases. If the information of a family member is limited after the detailed clinical assessment of the proband, the screening of the OPA1 gene may be helpful for precise diagnosis of ADOA.
This study was supported in part by a Grant in Aid, no. 20261901, from the Research Grant from the Study Group on Chorioretinal Degeneration and Optic Atrophy, The Ministry of Health, Labor and Welfare, Japan. The authors indicate no financial conflict of interest. The authors thank Dr. Miyuki Yoshikawa for the patients she introduced to this study.