Type III 3-methylglutaconic aciduria (optic atrophy plus syndrome, or Costeff optic atrophy syndrome): identification of the OPA3 gene and its founder mutation in Iraqi Jews

Abstract

Type III 3-methylglutaconic aciduria (MGA) (MIM 258501) is a neuro-ophthalmologic syndrome that consists of early-onset bilateral optic atrophy and later-onset spasticity, extrapyramidal dysfunction, and cognitive deficit. Urinary excretion of 3-methylglutaconic acid and of 3-methylglutaric acid is increased. The disorder has been reported in approximately 40 patients of Iraqi Jewish origin, allowing the mapping of the disease to chromosome 19q13.2-q13.3, by linkage analysis. To isolate the causative gene, OPA3, we sequenced four genes within the critical interval and identified, in the intronic sequence of a gene corresponding to cDNA clone FLJ22187, a point mutation that segregated with the type III MGA phenotype. The FLJ22187-cDNA clone, which we identified as the OPA3 gene, consists of two exons and encodes a peptide of 179 amino acid residues. Northern blot analysis revealed a primary transcript of approximately 5.0 kb that was ubiquitously expressed, most prominently in skeletal muscle and kidney. Within the brain, the cerebral cortex, the medulla, the cerebellum, and the frontal lobe, compared to other parts of the brain, had slightly increased expression. The intronic G-->C mutation abolished mRNA expression in fibroblasts from affected patients and was detected in 8 of 85 anonymous Israeli individuals of Iraqi Jewish origin. Milder mutations in OPA3 should be sought in patients with optic atrophy with later onset, even in the absence of additional neurological abnormalities.

Figure 1

Demonstration of founder mutation in OPA3 in Iraqi Jewish family. a, Sequence of intron 1/exon 2 boundary of OPA3, in normal control, affected patient, and three family members. The G→C mutation was present in the homozygous state in the daughter and in the heterozygous state in the mother and the father. b, Gel electrophoresis of PCR-amplification products cut with RsaI. A 540-bp fragment in the region of interest was amplified by primers R2 and MDP, the latter of which was specifically designed to introduce an additional RsaI cleavage site into the product formed using the mutant sequence as template. Treatment with RsaI produced a 280-bp band in all patients; in addition, a 180-bp fragment was visible in normal individuals, a 161-bp fragment was apparent in affected patients, and the 180-bp and 161-bp bands were both present for obligate heterozygotes.

Figure 2

Northern blot analyses by 619-bp probe to human OPA3 cDNA. a, Probe hybridized to 5.0-kb band and secondary 8.0-kb band in all tissues tested. Expression appeared greatest in skeletal muscle and kidney. The lower panel shows the band for β-actin, which served as a control for RNA loading. b, Same bands on human multiple-tissue northern blot, demonstrating expression in all parts of brain. c, 5.0-kb and 8.0-kb bands of RNA from normal fibroblasts (N-1 and N-2), which hybridized with OPA3-cDNA probe, whereas no bands appeared on use of RNA from fibroblasts from one Iraqi Jewish patient (Pt.).

Figure 3

OPA3 gene and protein sequence. Nucleotide 1 corresponds to nucleotide 22 of FLJ22187. The first three nucleotides, shown in boldface, comprise the nearest stop codon 5′ to the open reading frame. The arrow after nucleotide 142 denotes the border between exons 1 and 2.

Figure 4

Conserved motif of 22 amino acids within human, D. melanogaster, and yeast homologues of OPA3. Numbers indicate the position of the first amino acid in the conserved region; letters in boldface indicate amino acids conserved among all three species.