C. P. Pang

Effects of scan circle displacement in optical coherence tomography retinal nerve fibre layer thickness measurement: a RNFL modelling study

ObjectiveTo study the effect of optical coherence tomography (OCT) scan circle displacement on retinal nerve fibre layer (RNFL) measurement errors using cubic spline models.MethodsForty-nine normal subjects were included in the analysis. In one randomly selected eye in each subject, RNFL thickness around the optic disc was measured by taking 16 circular scans of different sizes (scan radius ranged from 1 to 2.5 mm). The RNFL profile in each eye was constructed with a mathematical model using a smoothing spline approximation. Scan circle (diameter 3.4 mm) RNFL measurements (total average, superior, nasal, inferior, and temporal RNFL thicknesses) obtained from eight directions (superior, superonasal, nasal, inferonasal, inferior, inferotemporal, temporal, and superotemporal) displaced at different distances (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mm) from the disc centre were then computed by a computer program and compared to the ‘reference standard’ where the scan circle is centred at the optic disc. RNFL measurement error was calculated as the absolute of (RNFL thickness (displaced) – RNFL thickness (reference standard)).ResultsThe respective mean average, superior, nasal, inferior, and temporal RNFL measurement errors were 2.3±2.0, 4.9±4.5, 4.1±3.8, 6.2±7.6, and 3.8±3.5 μm upon 0.1 mm scan circle displacement, and 12.1±11.4, 27.8±18.4, 21.7±18.6, 34.8±22.9, and 15.2±10.7 μm upon 0.7 mm scan circle displacement. Significant differences of average and quadrant RNFL thicknesses were evident between centred and displaced scan circle measurements (all with P<0.001). RNFL measurement error increased in a monotonic fashion with increasing distance away from the disc and the change was direction-dependent.ConclusionsRNFL measurement error varies with the direction and distance of scan displacement. The superior and the inferior RNFL measurements are most vulnerable to scan displacement errors, whereas the average RNFL thickness is the least susceptible. Obtaining a well-centred scan is essential for reliable RNFL measurement in OCT.

Differential roles of phosphatidylinositol 3-kinase/akt pathway in retinal ganglion cell survival in rats with or without acute ocular hypertension

Intraocular pressure (IOP) elevation has often been used as an experimental model to study mechanisms underlying retinal ganglion cell (RGC) death associated with ocular ischemic injury and glaucoma. The aim of the present study, using both in vitro and in vivo approaches, was to investigate the role of phosphatidylinositol 3-kinase (PI3K)/akt pathway in RGC viability in normal rats and rats following transient IOP elevation. For in vivo studies, pathway inhibitors were administered intravitreally on days 3, 9, and 15 post-2-h IOP elevation at 110 mm Hg. Toward the end of the 3-week examination period, the fluorescent dye Fluorogold was used to retrogradely label surviving RGCs. In order to examine the role of macrophages that were recruited into the eye following the pathway inhibition, clodronate liposomes were used to deplete phagocytic cells in the eye. PI3K/akt pathway activity and location in the retina were examined using Western blot and immunohistochemistry, respectively. Here we showed that PI3K/akt inhibitors 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and KY12420 at low concentrations (2 microM or 20 microM) did not influence RGC survival but caused RGC loss at high concentration (200 muM) in retinal explants derived from intact rats. In contrast, both LY294002 and KY12420 at 20 microM led to RGC loss in retinal explants derived from IOP-elevated eyes. A detrimental action of phagocytic cells on RGC survival was also seen in these retinas. In vivo results confirmed the detrimental actions of PI3K/akt inhibition and macrophages on RGC survival in IOP-elevated, but not intact eyes even with high concentration of LY294002. Low level of PI3K/akt activity was detected in the ganglion cell layer (GCL) in intact retina. Acute IOP elevation activated PI3K/akt pathway in the inner nuclear layer and GCL including RGCs. This study thus demonstrates that PI3K/akt pathway mediates RGC survival after IOP elevation but not under normal condition.

Predictors of atypical birefringence pattern in scanning laser polarimetry

Aim: To evaluate predictors of atypical birefringence patterns (ABP) observed in scanning laser polarimetry. Methods: A total of 179 eyes from 82 normal subjects and 97 glaucoma patients were included. The retinal nerve fibre layer in each eye was imaged sequentially with GDx variable corneal compensation (VCC) and GDx enhanced corneal compensation (ECC) (Carl Zeiss Meditec, Dublin, California). The associations between the typical scan score (TSS) and age, axial length, spherical error, parapapillary atrophy (PPA) and visual-field mean deviation (MD) were evaluated with univariate and multivariate regression analyses. Results: 23.5% (42/179) and 5.0% (9/179) of subjects had ABP (TSS<80) with GDx VCC and GDx ECC, respectively. For both GDx VCC and ECC, the TSS was significantly correlated with age, axial length, spherical error and PPA, but not with visual-field MD. After adjusting the effect of covariates, the axial length/spherical error and PPA were significantly associated with GDx VCC TSS, whereas the axial length/spherical error was the only predictor for GDx ECC TSS. Myopic eyes were more likely to develop ABP in both GDx VCC and ECC. Conclusions: Axial length or spherical error is a significant predictor for ABP with both GDx VCC and GDx ECC. Caution should be exercised in interpreting the results of scanning laser polarimetry in eyes with a long axial length or myopia.

A novel missense RP1 mutation in retinitis pigmentosa.

AimsMore than 20 mutations associated with retinitis pigmentosa (RP) have been identified in the retinitis pigmentosa 1 (RP1) gene, all of them leading to the production of a truncated protein without 50–70% of the C-terminal of the RP1 protein. RP1 was recently found to be a microtubule-associated protein (MAP) and responsible for the organisation of the photoreceptor outer segment. The N-terminal doublecortin (DCX) domain of RP1 is essential for its function. But how the C-terminal of the protein affects its function is still not known. This study aims to get a better understanding of the RP1 gene by mutation screening on RP patients.MethodsPeripheral blood was taken from 72 RP patients. Together with 101 RP patients and 190 control subjects previously reported, mutation screening was performed by polymerase chain reaction (PCR) and direct sequencing. Statistical analysis was performed using SPSS.ResultsTwo novel missense sequence changes, D984G and C727W, and one novel variant, 6492T>G, at the 3′ untranslated region were found. They were not found in 190 control subjects. D984G causes RP. It creates two possible N-myristoylation sites according to PROSITE. C727W does not segregate with RP in the family. It abolishes an N-myristoylation site. R872H, a previously reported polymorphism, was predominantly present in control subjects (P=0.001).ConclusionsOur results suggest that disruption of the C-terminal of RP1 may be associated with the development of RP, and the possible involvement of the RP1 polypeptide downstream of its DCX domain in normal RP1 function.

Rapid and cost-effective molecular diagnosis using exome sequencing of one proband with autosomal dominant congenital cataract

PurposeDue to high genetic heterogeneity, to exclude known mutations and map novel mutations in autosomal dominant congenital cataract (ADCC) using conventional candidate gene screening requires laborious laboratory work. We attempted to use a cost-effective exome sequencing strategy to identify disease-causing mutations in an ADCC pedigree.MethodsAn ADCC pedigree affected by nuclear cataract and 200 unrelated senile cataract controls were recruited and given comprehensive ophthalmic examination. Whole exome of the proband of the family was captured by the Illumina TruSeq Exome Enrichment Kit, followed by sequencing using Illumina HiSeq 2000 sequencer. Validation was performed by direct sequencing.ResultsThe whole exome, including all exons of known ADCC disease-causing genes, was screened for possible disease-causing mutations. A recurrent missense mutation c.773C>T (p.S258F) in exon 2 of the gap junction protein alpha 8 gene (GJA8) was identified in the proband with nuclear cataract. The result was confirmed by direct sequencing. The mutation showed complete co-segregation with the disease phenotype in the family but was not observed in unrelated unaffected controls.ConclusionBy successfully sequencing whole exome of only one proband and identifying a GJA8 mutation in one ADCC pedigree, the current study demonstrated that exome sequencing could serve as a rapid, robust, and cost-effective approach in clinical diagnosis and disease-causing gene discovery for ADCC.