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

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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.

Introduction

3-Methylglutaconic aciduria type III (3-MGCA type III; MIM 258501), also called Costeff optic atrophy syndrome, or Optic atrophy type 3 (OPA3), is an autosomal recessive neuro-ophthalmologic syndrome that consists of early-onset bilateral optic atrophy and late development of spasticity, extrapyramidal dysfunction and occasionally cognitive deficit [1], [2], [3]. Urinary excretion of the branched-chain organic acids 3-methylglutaconic acid (3-MGC) and 3-methylglutaric acid (3-MGR) are increased in 3-MGCA type III patients [4].

The occurrence of 3-MGCA type III in approximately 40 patients of Iraqi–Jewish origin [3] assisted in mapping the disease to 19q13.2-q13.3 [5]. In 2001, we identified the causative gene, OPA3 [6]. OPA3 consists of two exons and encodes for a 179-amino acid protein (OPA3) of unknown function, containing putative mitochondrial N-terminal and peroxisomal C-terminal sorting signals [6]. All patients of Iraqi–Jewish origin are homozygous for a splice site founder mutation, c.143-1G>C (IVS1-1G>C), which abolishes mRNA expression in fibroblasts [6]. We subsequently identified another novel OPA3 mutation, an in-frame 18-bp deletion in exon 2, c.320_337del (p.Q108_E113del), in a Kurdish–Turkish patient [7]. Recently, a third OPA3 mutation was identified in a patient of Asian (Indian) origin; a nonsense c.415C>T (p.Q139X) mutation [8].

Two OPA3 mutations, G93S and Q105E, result in a rare dominant disorder (ADOAC; MIM 165300) involving optic atrophy, cataracts and extrapyramidal signs [9], [10]. The ADOAC phenotype may reflect a dominant negative effect, since heterozygous carriers of the Iraqi–Jewish loss of function founder mutation (c.143-1G>C) do not show a clinical phenotype. Similarly, a recently reported murine model harboring an L122P mutation in the heterozygous state appears normal [11].

The function of the OPA3 protein and how its deficiency causes the clinical symptoms and 3-methylglutaconic aciduria in 3-MGCA type III patients remain enigmatic. Here we report a comprehensive study of the OPA3 gene and its translated protein. We identified a third exon and an alternate transcript of OPA3 and determined its expression in various tissues and in 3-MGCA type III patients. We performed expression studies in fibroblasts with green fluorescent protein (GFP)-tagged OPA3 and explore mitochondrial and peroxisomal localization. We also imaged the localization, shape and inter-organellar interactions of mitochondria and peroxisomes in normal and 3-MGCA type III patients’ cells.

Section snippets

Patients and cells

Patients samples were enrolled under the NIH protocol “Diagnosis and Treatment of Patients with Inborn Errors of Metabolism” (www.clinicaltrials.gov, trial NCT00369421), approved by the National Human Genome Research Institute’s Institutional Review Board. Each patient gave written informed consent, in accordance with the Declaration of Helsinki. Skin fibroblasts were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum containing 100 U/ml penicillin and 0.1 mg/ml

OPA3 gene structure

Previously believed to consist of two exons and a single transcript, we now demonstrate that the OPA3 gene (Fig. 1A) consists of 3 exons and is expressed in two transcripts, OPA3A (GenBank NM_025136) and OPA3B (GenBank NM_001017989). Both transcripts contain exon 1, which is spliced to exon 2 in OPA3A and exon 3 in OPA3B. Although cDNA studies indicate ubiquitous expression of OPA3A and OPA3B, OPA3A has low expression in brain and OPA3B has high expression in testis (Fig. 1B). The nucleotide

Conclusion

Taken together, our findings indicate that the OPA3 gene produces two distinct RNA transcripts, OPA3A and OPA3B. OPA3B has lower expression levels than OPA3A, and may not yield a significant translation product in human cells, since OPA3B is not identified in proteomic databases and no human disease has been associated with mutations in the OPA3B-specific exon 3. In addition, OPA3A is expressed and conserved from fungi to primates, while OPA3B is uniquely found in mammals (Supplementary Fig.

Acknowledgments

We thank Ian Nouvel for skillful laboratory assistance. This study was supported by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA and by the Costeff Support Group Foundation (Y.A.).

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