Predicting the pathogenicity of RPE65 mutations.

To assist in distinguishing disease-causing mutations from nonpathogenic polymorphisms, we developed an objective algorithm to calculate an "estimate of pathogenic probability" (EPP) based on the prevalence of a specific variation, its segregation within families, and its predicted effects on protein structure. Eleven missense variations in the RPE65 gene were evaluated in patients with Leber congenital amaurosis (LCA) using the EPP algorithm. The accuracy of the EPP algorithm was evaluated using a cell-culture assay of RPE65-isomerase activity The variations were engineered into plasmids containing a human RPE65 cDNA and the retinoid isomerase activity of each variant was determined in cultured cells. The EPP algorithm predicted eight substitution mutations to be disease-causing variants. The isomerase catalytic activities of these RPE65 variants were all less than 6% of wild-type. In contrast, the EPP algorithm predicted the other three substitutions to be non-disease-causing, with isomerase activities of 68%, 127%, and 110% of wild-type, respectively. We observed complete concordance between the predicted pathogenicities of missense variations in the RPE65 gene and retinoid isomerase activities measured in a functional assay. These results suggest that the EPP algorithm may be useful to evaluate the pathogenicity of missense variations in other disease genes where functional assays are not available.

Rod photopigment deficits in albinos are specific to mammals and arise during retinal development.

Adult albino mammals have specific retinal defects, including reduced numbers of rod photoreceptors. To examine when this rod deficit arises and whether it exists in nonmammalian albinos, we have used absorbance spectrophotometry to measure photopigment levels in dark-adapted eyes taken from three groups of pigmented and albino animals: adult rodents (rats and mice), developing rats, and mature Xenopus frogs. Rhodopsin concentrations were consistently and significantly reduced in mammalian albinos compared to their wild-type counterparts from before the time of eye opening, but photopigment levels were similar in frogs of both pigmentation phenotypes. The results strongly suggest that deficits in the rod cell population arise early in development of the mammalian albino retina, but do not generalize to nonmammalian mutants lacking retinal melanin.

Vertebrate ancient (VA) opsin and extraretinal photoreception in the Atlantic salmon (Salmo salar).

A member of a new photopigment family first isolated from teleost fish, vertebrate ancient (VA) opsin, has recently been shown to form a functional photopigment and to be expressed within a subset of horizontal and amacrine cells of the inner retina. These sites of expression (and structural features) of VA opsin suggest that this photopigment might mediate non-image-forming light-detection tasks. We attempted to gain support for this hypothesis by examining the expression of VA opsin within the central nervous system (CNS) (pineal and deep brain) of the Atlantic salmon Salmo salar. In addition, we examined the sites of rod-opsin, cone-opsin and &agr; -transducin expression within the salmon CNS to provide a more complete description of the extraretinal photoreceptors of a teleost vertebrate. We show that multiple populations of cells within the salmon CNS appear to contain photoreceptors: VA opsin was strongly expressed in the pineal organ and in bilateral columns of subependymal cells in the epithalamus; anti-cone-opsin antibodies labelled cells within the pineal and numerous cells in the anterior hypothalamus (suprachiasmatic nucleus, nucleus preopticus magnocellularis, nucleus preopticus parvocellularis); anti-rod-opsin antibodies labelled cells within the pineal but no other areas within the central brain; and anti- &agr; -transducin antibodies labelled cells within the pineal and the ventral telencephalon. Collectively, our results suggest that VA opsin is a photopigment specialised for irradiance detection tasks within the eye, pineal and central brain, and that the salmon has multiple and varied populations of photoreceptors within the CNS. We review the significance of these findings within the broad context of vertebrate extraretinal photoreception.

A novel rod-like opsin isolated from the extra-retinal photoreceptors of teleost fish.

We have isolated a novel opsin from the pineal complex of Atlantic salmon (Salmo salar) and from the brain of the puffer fish (Fugu rubripes). These extra-retinal opsins share approximately 74% identity at the nucleotide and amino acid level with rod-opsins from the retina of these species. By PCR, we have determined that the novel rod-like opsin is not expressed in the salmon retina, and the retinal rod-opsin is not expressed in the salmon pineal. Phylogenetic analysis suggests that the rod-like opsins arose from a gene duplication event approximately 205 million years ago, a time of considerable adaptive radiation of the bony fish. In view of the large differences in the coding sequences of the pineal/brain rod-like opsins, their extra-retinal sites of expression, and phylogenetic position we have termed these novel opsins 'extra-retinal rod-like opsins' (ERrod-like opsins). We speculate that the differences between retinal rod-opsins and ERrod-like opsins have arisen from their differing photosensory roles and/or genetic drift after the gene duplication event in the Triassic.