Genomic rearrangements in OPA1 gene frequent in adOA

Key Points

Tuebingen, Germany-Using a relatively new technique for detection of genomic rearrangements, researchers in Germany have made important findings about the underlying pathomechanism in cases of autosomal dominant optic atrophy (adOA) related to the OPA1 gene. These findings suggest that the technique is a stable, reliable method for detection of copy number variations in this gene that eventually could be included in the diagnostic process for adOA, said Nico Fuhrmann, MS.

Fuhrmann is a doctoral candidate at the Molecular Genetics Laboratory, Institute for Ophthalmic Research, Centre for Ophthalmology, University Clinics Tuebingen, Tuebingen, Germany. uhrmann discussed the OPA1 research at the annual meeting of the Association for Research in Vision and Ophthalmology (ARVO). He was a 2008 ARVO travel grant recipient. He used multiplex ligation probe amplification (MLPA), a high-resolution technique for detecting copy number variations in genomic sequences, to study the OPA1 gene in families affected with adOA.

AdOA is the most frequent hereditary optic neuropathy, with prevalence estimates ranging from 1:12,000 to 1:50,000. The juvenile-onset disease presents with central or paracentral scotomas, loss of visual acuity, and color discrimination impairments. It also is characterized by strong intra- and interfamilial variability in progression and severity. Through the study of donor eyes, investigators have learned that substantial loss of retinal ganglion cells and atrophy of the optic nerve underlie adOA pathology.

To date, investigators have identified two genes associated with adOA, OPA1 and OPA3; the latter gene is rare, and affected individuals often develop early-onset cataract. OPA4 and OPA5 loci have been mapped and published, and a new OPA7 locus also has been identified at the Molecular Genetics Lab, Fuhrmann said.

In total, 117 mutations in the OPA1 gene have been published at this time; about two-thirds are substitution mutations. No true copy number variations have been detected, and only one report has found a complete deletion of the OPA1 gene.

To find candidates for genomic variation, Fuhrmann and colleagues used a kit for MLPA analysis (MRC Holland) that includes hybridization probe pairs with target-specific sequences that bind specifically to individual exons of the OPA1 gene. An attached stuffer sequence (variable in length and different for each probe) allows final discrimination of the amplified exons.

The probe also contains a pair of identical polymerase chain reaction (PCR) primer sequences that are tagged to the hybridization and stuffer sequences. The target probe pair sequences are split and have to be joined to each other, which is possible only if they can align in the presence of the DNA target sequence. Only the ligated sequence, rather than the original DNA sample, is amplified. Comparison of amplification product intensities with a control DNA sample allows detection of copy number variations.

Fuhrmann screened for copy number variations in OPA1 in 37 independent adOA families and identified genomic rearrangements in seven of them. He found heterozygous deletion of exon 9 throughout the pedigree of one large family as well as deletion of exon 24 in a separate family. Those findings were verified with PCR amplification.

He also found heterozygous deletions of the complete OPA1 gene in two different families, although the mutation was not identical because the deletion was larger in the second family. Through a combination of MLPA and PCR analyses, he furthermore found heterozygous duplication of exons 7 to 9 in three unrelated families. Based on these findings, he estimated that the prevalence of OPA1 rearrangements is 12.8%.

"We think it's quite frequent in patients with adOA," Fuhrmann said. This rate of rearrangement could be a significant factor in the prevalence and spectrum of OPA1 mutations in these individuals, he added.

In addition, the discovery of two different deletions of the complete OPA1 allele sustains the hypothesis that haploinsufficiency is the underlying pathomechanism in cases of adOA associated with OPA1.