Quantitative measurement of fixation stability during RareBit perimetry and Humphrey visual field testing.

PURPOSE: To compare fixation stability and fixation loss between the Humphrey Field Analyzer (HVF, static fixation target) and the RareBit computer-based perimeter (RBP, kinetic fixation target) during visual field testing. METHODS: Fourteen healthy volunteer subjects wore an ASL Mobile Gaze Tracker as they completed HVF 10-2 and RareBit central field tests in a random order. Fixation stability, defined as the average distance from the fixation target to the subject's gaze location, was calculated using data from the processed video capture. Fixation loss, defined as eye closure or a deviation of >20 degrees from the fixation target, was also measured. All subjects were surveyed regarding test preference. RESULTS: Use of the RBP kinetic target was associated with 18% improved fixation stability compared with the HVF static target (P=0.02). Nine of 14 study subjects demonstrated better fixation with RBP compared with HVF. Subjects demonstrated decreased fixation loss during RBP (0.9 s) compared with HVF (10.0 s) (P=0.002). Eighty-six percent of study subjects preferred RBP over HVF. CONCLUSIONS: Use of the RBP kinetic fixation target is associated with consistent fixation stability and decreased fixation loss compared with the HVF static target. This improvement in fixation stability may result from decreased perception interference (Ganzfeld, Troxler, and binocular rivalry effects), and may help account for the greater comfort reported with RBP compared with HVF.

The utility of next-generation sequencing in the evaluation of the posterior polymorphous corneal dystrophy 1 locus.

PURPOSE: To identify the genetic basis of posterior polymorphous corneal dystrophy 1 (PPCD1) using next-generation sequencing (NGS) of the common PPCD1 support interval, in which Sanger sequencing failed to identify a pathogenic mutation. METHODS: Enrichment of the portion of chromosome 20 containing the common PPCD1 interval was performed on DNA extracted from an affected and an unaffected member of a family previously linked to the PPCD1 locus. NGS using the Roche 454 Titanium platform was performed, followed by computational analysis using NextGENe Software. RESULTS: NGS of the selectively enriched chromosomal 20 region between markers D20S48 and D20S190 produced over 400,000 DNA sequence reads with an average of 350 bases for each of the two DNA samples. Alignment of the DNA sequence reads with the reference sequence from the National Center of Biotechnology Information (NCBI) resulted in over 119 million matched bases per sample. Approximately 68,000 DNA sequence variants were identified in the common PPCD1 support interval in the affected individual, which was approximately twice the number of sequence variants identified in the unaffected individual. In both individuals, approximately 0.5% of the identified variants mapped to the 13 known and 16 predicted genes in the PPCD1 support interval, including 16 of the 17 (94%) variants previously identified by Sanger sequencing in the 13 known genes. In both individuals, the variant not identified by NGS was located in a region of inadequate coverage. CONCLUSIONS: NGS identified all of the exonic sequence variants that were previously identified by Sanger sequencing in known genes in adequately covered regions of the common PPCD1 interval, although the pathogenic variant is yet to be discovered. Given adequate coverage of a selectively enriched chromosomal region of interest, NGS represents a useful technique to screen for sequence variants in candidate gene loci that has multiple advantages over previously employed techniques for mutation discovery.