Novel OPA1 mutations identified in Japanese pedigrees with optic atrophy.

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.

Reliability and significance of measurements of a-wave latency in rats.

PURPOSE: To determine whether measurements of the a-wave latency of the electroretinogram (ERG) can be made as reliably as that of the implicit time (IT) in rats. In addition, to determine the relationship between the potential level selected for the latency and the baseline potential level. METHODS: ERGs, elicited by different stimulus intensities, were recorded from Long-Evans rats. The a-wave latency was determined by measuring the time between the stimulus onset and the beginning of the negative-going a-wave, and the IT was measured as the time between the stimulus onset and the peak of the a-wave. To test the reliability of the measurements of the latency, the a-wave latency and the IT were measured by three independent observers for the same 15 ERGs. RESULTS: The mean a-wave latency was approximately 14 milliseconds, and the mean a-wave implicit time was approximately 36 milliseconds. The mean of the a-wave latency and the IT, as measured by the three observers, were within 1 millisecond of each other. The coefficient of variation was as good for the latency as for the IT of the a-wave. The potential level selected for the latency was lower than the mean baseline potential level by 1 to 2 standard deviations. CONCLUSIONS: Selection of the a-wave latencies can be made as reliably as that for the IT. Because the a-wave latency is not affected by the activity of the second order neurons, the latency is a better measure than the IT of the time course of the a-wave.

The a-wave latency in control subjects and patients with retinal diseases.

PURPOSE: To determine the a-wave latency of the electroretinograms (ERGs) recorded from control subjects and patients with retinal diseases. METHODS: The a-wave latency and implicit time (IT) were measured retrospectively from the ERGs of 40 control subjects and 99 patients. The patients included 9 with complete congenital stationary night blindness (cCSNB), 13 with achromatopsia or cone dystrophy, 5 with supernormal and delayed rod ERG syndrome, and 72 with retinitis pigmentosa (RP). To assess whether latency measurements can be obtained reliably by different observers from patients with smaller a-wave amplitudes and noisier baselines, the a-wave latency and IT of the ERG of the right eye of 10 control subjects and 10 patients with RP were measured by three observers. RESULTS: The mean a-wave latency measured for the same 10 control ERGs by three observers differed by less than 1 millisecond while the mean IT differed by 1.7 milliseconds. For 10 ERGs from RP patients, the mean for the a-wave latency measured by the three observers differed by less than 2.0 milliseconds and by 1.1 millisecond for the IT. The coefficient of variation varied from 24.8% to 36.7% for the latency and from 11.5% to 16.0% for the IT. The a-wave latencies elicited by the 0-dB stimulus under scotopic and photopic conditions from the 40 control subjects were not statistically different. The a-wave latency in patients with cCSNB did not differ significantly from that in control subjects. The longer a-wave latency in patients with achromatopsia suggested that the rods have a longer latency than cones. The scotopic and photopic a-wave latencies were significantly longer in RP patients. The longer latency in RP patients was not due to smaller a- or b-wave amplitudes. CONCLUSIONS: The a-wave latency can be measured as reliably as the IT in control subjects but the reliability is not as good for the latency as for the IT in RP patients. The larger coefficients of variation in RP patients were most likely due to the measurements being made from RP patients at different stages of their disease. Our results suggest that the a-wave latency in control subjects is determined by cones under both scotopic and photopic conditions. The longer a-wave latency in RP patients suggests that the rods and cones are altered over a significant area of the retina.

Evidence that a-wave latency of the electroretinogram is determined solely by photoreceptors.

PURPOSE: To identify the retinal cells that determine the a-wave latency of rats. METHODS: Electroretinograms (ERGs) were recorded from the rod-dominated (0.85% cones) retinas of Long-Evans rats following an intravitreal injection of 1 microL of 40 mM 2-amino-4-phosphonobutyric acid to block the activity of the ON pathway of the second order retinal neurons. ERGs were also recorded following an intraperitoneal injection of sodium iodate to destroy the retinal pigment epithelial (RPE) cells. Damage to a large area of the retina was produced by constant light exposure, and focal damage to the retina was induced by argon laser photocoagulation. The effects of age and anesthesia level on the a-wave latency were also determined. RESULTS: Blocking the activity of the ON pathway of the second order retinal neurons did not alter the a-wave latency, and destroying the RPE cells also did not alter the a-wave latency. Damage to a large area of the retina resulted in prolonging the latency but focal retinal damage did not alter the a-wave latency. The a-wave latency was longer in young rat pups but was adult-like by 18 days. The level of anesthesia had no effect on the latency except at very deep stages. CONCLUSIONS: The a-wave latency is determined solely by the activity of the photoreceptors. A prolonged latency would indicate that the photoreceptors are damaged over a large area of the retina.