OPA3, mutated in 3-methylglutaconic aciduria type III, encodes two transcripts targeted primarily to mitochondria

Abstract

3-Methylglutaconic aciduria type III (3-MGCA type III), caused by recessive mutations in the 2-exon gene OPA3, is characterized by early-onset bilateral optic atrophy, later-onset extrapyramidal dysfunction, and increased urinary excretion of 3-methylglutaconic acid and 3-methylglutaric acid. Here we report the identification of a novel third OPA3 coding exon, the apparent product of a segmental duplication event, resulting in two gene transcripts, OPA3A and OPA3B. OPA3A deficiency (as in optic atrophy type 3) causes up-regulation of OPA3B. OPA3 protein function remains unknown, but it contains a putative mitochondrial leader sequence, mitochondrial sorting signal and a peroxisomal sorting signal. Our green fluorescent protein tagged OPA3 expression studies found its localization to be predominantly mitochondrial. These findings thus place the cellular metabolic defect of 3-MGCA type III in the mitochondrion rather than the peroxisome and implicate loss of OPA3A rather than gain of OPA3B in disease etiology.

Trial registration: ClinicalTrials.gov NCT00369421.

Figures

Fig. 1

OPA3A and OPA3B structure and expression. (A) Schematic of the OPA3 locus on chromosome 19q13.32 (not to scale). Intron and exon sizes are indicated. Neighboring upstream and downstream genes are GPR4 and VASP, respectively. A LINE-1 transposon (L1MC4), located about 24-kb upstream of exon 2, may have led to formation of exon 3 by segmental duplication. (B) Expression analysis of OPA3A (left) and OPA3B (right) using a multiple human tissue cDNA panel. (C) Expression of OPA3A and OPA3B in cDNA of normal (C1 and C2) and 3-MGCA type III fibroblasts P1 and P2, carrying homozygous c.143-1G>C and c.320-337del mutations, respectively. (D) Real-time quantitative PCR analysis of OPA3A and OPA3B transcripts in 3-MGCA type III (P1) fibroblasts compared to normal fibroblasts. Exon-specific primer-probe combinations showed a 5.8 fold (p<0.0001) up-regulation for exon 1 (ex1), a 2.7 fold (p=0.034) down-regulation of exon 2 (ex2; representing OPA3A), and a 4.8 fold (p<0.0001) up-regulation of exon 3 (ex3; representing OPA3B) (n=3). (E) Amino acid sequence alignment of OPA3A and OPA3B. The N-terminal amino acids encoded by exon 1 (bold) are shared by both proteins and contain a potential mitochondrial leader sequence (amino acids 1-18, underlined) and mitochondrial targeting signal NRIKE (amino acids 25-29, underlined). The potential C-terminal peroxisomal sorting signals SKK for OPA3A and SEK for OPA3B are gray shaded and underlined. Over the entire protein, OPA3A and OPA3B are 77.6% homologous; their C-terminal sequences contain 41 mismatches (printed in bold italic in the OPA3B sequence).

Fig. 2

OPA3A and OPA3B intracellular localization and mitochondrial and peroxisomal distribution in normal and 3-MGCA type III fibroblasts. OPA3A (A, B) and OPA3B (C) GFP-fusion proteins (green) were expressed in normal fibroblasts and co-stained with a mitochondrial marker (red, indicated in image as mito) or a peroxisomal marker (red, indicated in image as pex). Mutated sorting signals are indicated as - leader (disrupted mitochondrial leader sequence), -NRIKE (mutated mitochondrial targeting signal), or -SKK or -SEK (disrupted putative peroxisomal targeting signals). See text for detailed description of each image. Images in left panels are taken at low magnification (40x objective) and areas indicated by arrows are enlarged in right panels. (D) Mitochondria (green) and peroxisomes (red) were labeled with organelle-specific markers and imaged by confocal microscopy in a Z-stack, covering the entire cell in the Z dimension. Z-stacks were rendered in 3-D. Left panel, normal fibroblasts; right panel, 3-MGCA type III patient P1 fibroblasts (for animation, see Supplemental Fig. S3).

Fig. 2

OPA3A and OPA3B intracellular localization and mitochondrial and peroxisomal distribution in normal and 3-MGCA type III fibroblasts. OPA3A (A, B) and OPA3B (C) GFP-fusion proteins (green) were expressed in normal fibroblasts and co-stained with a mitochondrial marker (red, indicated in image as mito) or a peroxisomal marker (red, indicated in image as pex). Mutated sorting signals are indicated as - leader (disrupted mitochondrial leader sequence), -NRIKE (mutated mitochondrial targeting signal), or -SKK or -SEK (disrupted putative peroxisomal targeting signals). See text for detailed description of each image. Images in left panels are taken at low magnification (40x objective) and areas indicated by arrows are enlarged in right panels. (D) Mitochondria (green) and peroxisomes (red) were labeled with organelle-specific markers and imaged by confocal microscopy in a Z-stack, covering the entire cell in the Z dimension. Z-stacks were rendered in 3-D. Left panel, normal fibroblasts; right panel, 3-MGCA type III patient P1 fibroblasts (for animation, see Supplemental Fig. S3).