Molecular Vision 2006; 12:485-491 <>
Received 17 June 2005 | Accepted 28 April 2006 | Published 12 May 2006

Novel OPA1 mutations identified in Japanese pedigrees with optic atrophy

Minghui Qin,1 Hiroyuki Kondo,2 Hideaki Uno,2 Eriko Fujiwara,2 Eiichi Uchio,2 Tomoko Tahira,1 Kenshi Hayashi1
(The first two authors contributed equally to this publication)

1Division of Genome Analysis, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2Department of Ophthalmology, Fukuoka University School of Medicine, Fukuoka, Japan

Correspondence to: Hiroyuki Kondo, Department of Ophthalmology, Fukuoka University School of Medicine, Fukuoka, Japan; Phone: 81-92-801-1011; FAX: 81-92-865-4445; email:


Purpose: To determine whether mutations in the OPA1 gene were present in two Japanese families with optic atrophy.

Methods: Thirty exons and their boundaries of the OPA1 gene were amplified by PCR with genomic DNA as templates and directly sequenced. The detected sequence changes were confirmed to be mutations by examining whether they were present in normal control individuals. A splicing mutation was characterized by RT-PCR of total RNA of leukocytes obtained from patients and one normal individual. The mutant transcripts resulting from the splicing mutation were further confirmed and quantified by sequencing and identifying the denatured RT-PCR products by polyacrylamide electrophoresis.

Results: One novel splicing mutation of c.871-1G>T and one novel insertion mutation of c.579_580insTT (p.R194fsX228) were identified from two familial cases, respectively. Both mutations segregated within the family heterozygously and were not found in the 189 control individuals examined. Two mutant transcripts resulted from the splicing mutation were identified through amplified OPA1 cDNA prepared from the RNA of leukocytes of the patients. One had a 21 bp deletion at the beginning of the exon 9 leading to a 7 amino acid in-frame deletion of the protein. The expression level of this mutant transcript was similar to the transcript from the wild type allele of the patient. The other mutation was a 114 bp deletion, leading to a 38 amino acid in-frame deletion that skipped all of exon 9, and the expression of this mutant transcript was much lower than the 21 bp deletion.

Conclusions: The predicted consequence of both mutations is the loss of GTPase activity. Our findings further establish the involvement of OPA1 mutation in Japanese patients with optic atrophy and serve as supportive evidence that haploinsufficiency of the OPA1 gene is the cause of the optic atrophy.


Autosomal dominant optic atrophy (ADOA; OMIM 165500) is the most common hereditary optic neuropathy with an estimated prevalence of 1 in 10,000 [1] to 1 in 50,000 [2]. This hereditary optic atrophy is characterized by a gradual deterioration of vision in childhood but with little progression thereafter. The progressive decrease of visual acuity is accompanied by color vision defects, centrocecal scotomas, and optic nerve pallor. Mutations leading to this condition have been identified in the OPA1 gene on chromosome 3q28-q29 [3,4]. The OPA1 gene is composed of 30 exons and encodes a dynamin-related mitochondrial protein. The GTPase and central dynamin domains of the OPA1 gene are well conserved. The GTPase domain represents the most conserved part of the OPA1 protein compared with other members of the dynamin protein family. The OPA1 gene also has a basic N-terminal leader sequence which is required for its mitochondrial localization and a coiled-coil region at the C-terminal of the protein. It has been shown that the OPA1 protein is anchored in the mitochondrial inner membrane and is believed to be important for the biogenesis and maintenance of mitochondria in both mice and humans [5-7].

At present, the majority of the identified OPA1 mutations are clustered over the cDNA region corresponding to the GTPase domain and the 3' end of the coding region demonstrating the importance of these regions [3,4,8-13]. Most of the mutations were identified as family specific, and one frame shift mutation of c.2826delT was found in 14 Danish patients indicative of a founder effect, and one deletion mutation of c.2708_2711del4 was detected in patients from a different ethnic background suggesting that it is a mutational hot spot. As evidenced by a large number of null mutations and the report of a functional loss of a single allele, the cause of the disease is believed to be haploinsufficiency [10,11].

Among all the mutations identified to date, only four mutations (5%) have been reported in Japanese patients [14-16]. The purpose of this study was to confirm that mutations in the OPA1 gene are responsible for optic atrophy in the Japanese population. To accomplish this, we screened the OPA1 gene for mutations in two Japanese pedigrees with optic atrophy and have identified two novel mutations in the OPA1 gene. Additional studies were performed to determine how these mutations led to the optic atrophy.


Two Japanese pedigrees with optic atrophy, both familial, were screened for OPA1 mutations. The ocular examinations included: refraction, best corrected visual acuity, intraocular pressure, slit-lamp biomicroscopy, funduscopy, and color discrimination test, and the diagnosis were made at Fukuoka University. Informed consent was obtained from all subjects after an explanation of the purpose and procedures to be used. The procedures conformed to the tenets of the Declaration of Helsinki, and the protocol was approved by the Ethics Review Board of Fukuoka University.

DNA samples were extracted from peripheral blood using a DNA extraction kit (QIAamp, Qiagen, Valencia, CA). The presence of sequence variants in the normal population was determined by examining 193 normal Japanese controls.

A reference genomic contig containing the sequence for OPA1 (NT_005612) was obtained from Genbank (NCBI). Primers bracketing all 30 exons of OPA1 were designed using the Primer3 program (Whitehead Institute for Biomedical Research/MIT Center for Genomic Research, Cambridge, MA). Details of the primer sequences are shown in Table 1.

PCR amplification and subsequent sequencing were performed with standard protocol using the DNA of normal subjects and patients as templates as previously described [17]. Sequence data were aligned with the reference sequence of NM_015560 using phred/phrap/polyphred software [18]. When nucleotide changes were detected, they were compared with the dbSNP data base (SNP). The +1 of the cDNA nucleotide numbering is referred as the first nucleotide "A" of the initial codon (ATG) of OPA1 isoform 1 (Genbank entry NM_015560). The sequence changes were confirmed to be mutations by their absence in control subjects. In both cases, the identified mutations were further verified through segregation analysis. A cDNA characterization was performed on splicing mutation.

The RNA of leukocytes was prepared using QIAamp RNA Blood Mini Kit (Qiagen, Valencia, CA) following the protocol suggested by the manufacturer. Total RNA (0.5 μg) from each patient and a normal control individual served as templates for reverse transcription (RT) reaction using the First-Strand cDNA Synthesis Kit (Amersham Biosciences Corp., Piscataway, NJ), following the instructions provided by the manufacturer. The synthesized first strand cDNA was used as the template to amplify the relevant regions. Primers used for RT-PCR were: F: 5'-ATT GCA GAA AGA TGA CAA AGG C-3'; R: 5'-GTT AAA TAG GGC CAC ATG GTG AG-3', which covered a region of 283 bp of OPA1 from exon 7 through exon 10. ATT or GTT was added to the 5' end of each primer for the convenience of end labeling.

RT-PCR products were separated on a 2% agarose gel, purified, and sequenced. The extraction was performed using GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences Corp., Piscataway, NJ). End labeling and denaturing capillary electrophoresis of the RT-PCR products were performed as previously described for identification and quantification purposes [19].


Family 1

The proband of Family 1 was a 42-year-old woman (Figure 1A). She was first found to have optic atrophy as a child but was not referred to our hospital until age 37 years. Her best corrected visual acuity was 0.03 with 10.0 D in both eyes. Fundus examination revealed bilateral optic disc pallor, central scotomas, and tritanopia by panel D-15 test. Her grandmother and a cousin were known to have severely reduced vision, although detailed diagnostic information was not available. The father of the proband had no symptom of visual disturbance related to optic atrophy and his visual acuities were 0.7 with 4.25 D in the right eye and 0.6 with 3.5 D after cataract surgery on the left eye.

Sequencing all 30 exons and their boundaries revealed one novel splicing mutation of c.871-1G>T in intron 8 (Figure 1A). Both the proband and her father carried the same mutation heterozygously, and this change was not found in the 193 normal individuals tested, suggesting the involvement of this mutation with the disease. Other members in this family were not genotyped as their DNA samples were not available.

To determine the consequences of this mutation, we examined total RNA of the patient's leukocytes as well as that from a control individual by RT-PCR followed by agarose gel electrophoresis and sequencing. A total of four bands were detected in the proband, which were designated as A, N, B, and C according to their size (Figure 1B). Only band N was present in the normal control RNA sample. The same results (the four bands) were obtained from her father's RNA sample.

The DNAs of band B, N, and C were purified and sequenced. The results revealed two mutant transcripts, which were 21 bp (7 residues) and 114 bp (38 residues) in-frame deletion, resulting in the loss of a part or the entire exon 9, respectively (Figure 1E).

The following experiments were performed to determine whether band A was a heteroduplex of band N and B. First, we mixed the gel-purified bands N, B, and C, and heat denatured the mixture at 95 °C for 5 min, re-natured at room temperature, and then loaded the mixture onto a 2% agaroge gel. Band A was detected (Figure 1C, right of the marker). Next, we labeled the RT-PCR products and analyzed them by denaturing capillary electrophoresis. As expected, three products corresponding to the lengths of N, B, and C were observed, but no DNA products corresponding to band A was detected (Figure 1D).

We were able to quantify the expression level of each transcript detected by loading the RT-PCR products of different amplification number to ensure that it was in the linear range of the amplification (Figure 1C, left of the marker). Band C had only a trace amount compared with bands B and N, indicating the 21 bp deletion is the major mutated transcript besides the level of normal transcript derived from wild type allele of the patient.

Family 2

The proband of Family 2 was a 16-year-old boy who was referred to our hospital because of reduced central vision of 0.1 in both eyes. Fundus examination revealed no retinal abnormalities including the appearance of the optic disc, however, the retinal thickness around the optic disc was significantly reduced when analyzed by retinal tomography. Optic atrophy was suspected, but there was no family history, and no vision impairment was claimed by either of his parents and brothers. However, the ocular examinations of his mother revealed bilateral optic disc pallor, while other tests were completely normal including visual acuity, visual field and color discrimination test.

Sequencing all 30 exons and their boundaries revealed one insertion mutation of c.579_580insTT (p.R194fsX228) in exon 5 of the proband and his mother but not of his father (Figure 2A). To the best of our knowledge, this is the first mutation to be reported in exon 5 of OPA1 in patients with ADOA. This mutation caused a frame shift of 34 amino acids from codon 194 to 227 and ended at codon 228 by introducing a premature stop (Figure 2B). This led to the loss of 76% of the C-terminal of the protein, including the entire GTPase domain. Both mother and son carried the same mutation heterozygously and the absence of this sequence change in 189 normal control individuals (4 samples of the 193 normal control samples were not analyzed because of a failure of PCR amplification and/or subsequent sequencing reactions) indicated that it was a mutation although mother dose not have any symptoms.


OPA1 is composed of 30 exons, and it normally generates eight splicing isoforms. All isoforms contain exons 1, 2, 3, 5, and exon 6 to the last exon, with exon 4, 4b, and 5b as variable factors [10]. All eight isoforms were known to be expressed in most of the tissues, including leukocytes, brain, retina, colon, liver, ovary, kidney, thyroid, lung, skeletal muscle, and heart with variable expression levels. In retina, isoform 1 (missing exon 4b and 5b) and isoform 4 (missing exon 4 and 4b) were shown to have the highest level of expression [10]. To date, 83 mutations in the OPA1 gene have been reported to be responsible for ADOA [20]. Among these, 38 mutations were found to be located in the GTPase domain (exon 8-15), 21 mutations were identified in the central dynamin domain (exon 16-24), and 16 mutations were detected at the C-terminus of the protein (exon 25-28). Even in the remaining 8 mutations located at the N-terminal of the gene, 6 were null mutations leading to the loss of three domains of the gene. Therefore, it was hypothesized that at least two separate modifications of OPA1 functions can lead to optic atrophy: alterations in GTPase activity and loss of the last 7 C-terminal amino acids that putatively interact with other proteins [10].

Among the mutations reported to date, several are well-characterized splicing mutations. For instance, a c.983A>G, which modifies the consensus sequence of the 5' donor site of intron 9. Another example is c.984G>A which results in the substitution of the last nucleotide of exon 9. Both of these result in an in-frame skipping of the entire exon 9 [10,13]. These results highlight the importance of exon 9, which can be explained by the loss of the 38 aa in the GTPase domain.

The splicing mutation we identified in Family 1 was experimentally proven to result in two differentially expressed mutant transcripts, one a partial and the other an entire deletion of exon 9 of the OPA1 gene presumably due to the presence of a cryptic acceptor site shown in red in Figure 1E. We believe that the efficiency of this acceptor site is close to 100% as only a trace amount of the transcript, which had entire exon 9 been skipped, was actually detected. The insertion mutation in Family 2 case resulted in a truncated protein lacking the whole GTPase domain, or reduced translation product due to the nonsense-mediated mRNA decay. The outcome of either mutation should abolish GTPase activity and lead to a disease phenotype, thus supporting the idea that the haploinsufficiency is the cause of the disease.

Our findings have brought the number of OPA1 mutations responsible for hereditary optic atrophy in the Japanese population to six. Each mutation was presented as family specific, suggesting no major founder effect is involved in the Japanese population.


We thank patients and their family for the cooperation. This project is supported by a Grant-in-aid 15591883 for Scientific Research, Japan to HK and a Grant-in-Aid for Research Revolution 2002 from The Ministry of Education, Culture, Sports, Science and Technology, Japan to KH in the Division of Genome Analysis, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Japan.


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