Purpose To characterize retinal structure and function in achromatopsia (ACHM) in preparation for clinical trials of gene therapy. Design Cross-sectional study. Participants Forty subjects with ACHM. Methods All subjects underwent spectral domain optical coherence tomography (SD-OCT), microperimetry, and molecular genetic testing. Foveal structure on SD-OCT was graded into 5 distinct categories: (i) continuous inner segment ellipsoid (ISe), (ii) ISe disruption, (iii) ISe absence, (iv) presence of a hyporeflective zone (HRZ), and (v) outer retinal atrophy including retinal pigment epithelial (RPE) loss. Foveal and outer nuclear layer (ONL) thickness was measured, and presence of hypoplasia determined. Main Outcome Measures Photoreceptor appearance on SD-OCT imaging; foveal and ONL thickness; presence of foveal hypoplasia; retinal sensitivity and fixation stability; and association of these parameters with age and genotype. Results Forty subjects with mean age of 24.9 years (range 6 to 52) were included. Disease-causing variants were found in CNGA3 (n=18), CNGB3 (n=15), GNAT2 (n=4), and PDE6C (n=1). No variants were found in 2 individuals. 22.5% of subjects had a continuous ISe layer at the fovea; 27.5% had ISe disruption; 20% had an absent ISe layer; 22.5% had a HRZ; and 7.5% had outer retinal atrophy. No significant differences in age (p=0.77), mean retinal sensitivity (p=0.21) or fixation stability (p=0.34) across the 5 SD-OCT categories were evident. No significant correlation was found between age and foveal thickness (p=0.84), or between age and foveal ONL thickness (p=0.12). Conclusions The lack of clear association of disruption of retinal structure or function in ACHM with age suggests that the window of opportunity for intervention by gene therapy is wider in some individuals than previously indicated. Therefore the potential benefit for a given subject is likely to be better predicted by specific measurement of photoreceptor structure rather than simply by age. The ability to directly assess cone photoreceptor preservation with SD-OCT and/or adaptive optics imaging is likely to prove invaluable in selecting subjects for future trials and measuring their impact.

Variation in the types and spectral characteristics of visual pigments is a common mechanism for the adaptation of the vertebrate visual system to prevailing light conditions. The extent of this diversity in mammals and birds is discussed in detail in this review, alongside an in-depth consideration of the molecular changes involved. In mammals, a nocturnal stage in early evolution is thought to underlie the reduction in the number of classes of cone visual pigment genes from four to only two, with the secondary loss of one of these genes in many monochromatic nocturnal and marine species. The trichromacy seen in many primates arises from either a polymorphism or duplication of one of these genes. In contrast, birds have retained the four ancestral cone visual pigment genes, with a generally conserved expression in either single or double cone classes. The loss of sensitivity to ultraviolet (UV) irradiation is a feature of both mammalian and avian visual evolution, with UV sensitivity retained among mammals by only a subset of rodents and marsupials. Where it is found in birds, it is not ancestral but newly acquired.

The short-wave-sensitive (SWS) visual pigments of vertebrate cone photoreceptors are divided into two classes on the basis of molecular identity, SWS1 and SWS2. Only the SWS1 class are present in mammals. The SWS1 pigments can be further subdivided into violet-sensitive (VS), with lambda(max) (the peak of maximal absorbance) values generally between 400 and 430 nm, and ultraviolet-sensitive (UVS), with a lambda(max)<380 nm. Phylogenetic evidence indicates that the ancestral pigment was UVS and that VS pigments have evolved separately from UVS pigments in the different vertebrate lineages. In this study, we have examined the mechanism of evolution of VS pigments in the mammalian lineage leading to present day ungulates (cow and pig). Amino acid sequence comparisons of the UVS pigments of teleost fish, amphibia, reptiles and rodents show that site 86 is invariably occupied by Phe but is replaced in bovine and porcine VS pigments by Tyr. Using site-directed mutagenesis of goldfish UVS opsin, we have shown that a Phe-86-->Tyr substitution is sufficient by itself to shift the lambda(max) of the goldfish pigment from a wild-type value of 360 nm to around 420 nm, and the reverse substitution of Tyr-86-Phe into bovine VS opsin produces a similar shift in the opposite direction. The substitution of this single amino acid is sufficient to account therefore for the evolution of bovine and porcine VS pigments. The replacement of Phe with polar Tyr at site 86 is consistent with the stabilization of Schiff-base protonation in VS pigments and the absence of protonation in UVS pigments.

SUMMARYThe correlation between ontogenetic changes in the spectral absorption characteristics of retinal photoreceptors and expression of visual pigment opsins was investigated in the black bream, Acanthopagrus butcheri. To establish whether the spectral qualities of environmental light affected the complement of visual pigments during ontogeny, comparisons were made between fishes reared in: (1) broad spectrum aquarium conditions; (2) short wavelength-reduced conditions similar to the natural environment; or (3) the natural environment (wild-caught). Microspectrophotometry was used to determine the wavelengths of spectral sensitivity of the photoreceptors at four developmental stages: larval, post-settlement, juvenile and adult. The molecular sequences of the rod (Rh1) and six cone (SWS1, SWS2A and B, Rh2Aα and β, and LWS) opsins were obtained and their expression levels in larval and adult stages examined using quantitative RT-PCR. The changes in spectral sensitivity of the cones were related to the differing levels of opsin expression during ontogeny. During the larval stage the predominantly expressed opsin classes were SWS1, SWS2B and Rh2Aα, contrasting with SWS2A, Rh2Aβ and LWS in the adult. An increased proportion of long wavelength-sensitive double cones was found in fishes reared in the short wavelength-reduced conditions and in wildcaught animals, indicating that the expression of cone opsin genes is also regulated by environmental light.

New-world primates such as the marmoset (Callithrix jacchus) show polymorphism for the middle- to long-wavelength sensitive cone pigments. Each X-chromosome carries a gene for only one of three possible pigments. All males are thus dichromats, but some females will be trichromats. We have investigated the responses of cells of the parvocellular (PC) and magnocellular (MC) systems within animals from a single marmoset family. The middle- to long-wavelength pigment of dichromats was identified physiologically. Trichromats could readily be distinguished from dichromats by the presence of a high proportion of red-green opponent PC-cells. The physiological classification of phenotypes was confirmed with genetic analysis. The pattern of inheritance was consistent with current genetic models. In trichromatic females, the properties of cells resembled in detail those of cells from the PC- and MC-pathways of the macaque. In dichromats, cell responses resembled those of trichromats (except for the lack of opponency in PC-cells); PC-cells showed sustained and MC-cells transient responses, with a lower contrast gain for the former type. One difference was that a proportion of PC-cells in dichromats showed strong rod input even at high levels of retinal illuminance. Thus, in trichromatic marmosets the presence of two middle- to long-wave pigments appears to permit the elaboration of all the physiological properties associated with trichromacy.

The biologist Gordon Walls proposed his "transmutation" theory through the 1930s and the 1940s to explain cone-like morphology of rods (and vice versa) in the duplex retinas of modern-day reptiles, with snakes regarded as the epitome of his hypothesis. Despite Walls' interest, the visual system of reptiles, and in particular snakes, has been widely neglected in favor of studies of fishes and mammals. By analyzing the visual pigments of two henophidian snakes, Xenopeltis unicolor and Python regius, we show that both species express two cone opsins, an ultraviolet-sensitive short-wavelength-sensitive 1 (SWS1) ( max ϭ 361 nm) pigment and a long-wavelength-sensitive (LWS) ( max ϭ 550 nm) pigment, providing the potential for dichromatic color vision. They also possess rod photoreceptors which express the usual rod opsin (Rh1) pigment with a max at 497 nm. This is the first molecular study of the visual pigments expressed in the photoreceptors of any snake species. The presence of a duplex retina and the characterization of LWS, SWS1, and Rh1 visual pigments in henophidian snakes implies that "lower" snakes do not provide support for Walls' transmutation theory, unlike some "higher" (caenophidian) snakes and other reptiles, such as geckos. More data from other snake lineages will be required to test this hypothesis further.

"Cone dystrophy with supernormal rod electroretinogram (ERG)" is an autosomal recessive disorder that causes lifelong visual loss combined with a supernormal ERG response to a bright flash of light. We have linked the disorder to a 0.98-cM (1.5-Mb) region on chromosome 9p24, flanked by rs1112534 and rs1074449, using homozygosity mapping in one large consanguineous pedigree. Analysis of one gene within this region, KCNV2, showed a homozygous nonsense mutation. Mutations were also found in 17 alleles of 10 other unrelated families with the same disorder. In situ hybridization demonstrated KCNV2 expression in human rod and cone photoreceptors. The precise function of KCNV2 in human photoreceptors remains to be determined, although this work suggests that mutations might perturb or abrogate I(KX), the potassium current within vertebrate photoreceptor inner segments, which has been shown to set their resting potential and voltage response.

Previously published cross-sectional studies have described conflicting findings with respect to the age-dependency of progression. This study, which constitutes the largest and longest prospective longitudinal study of ACHM to date, suggests that although ACHM may be progressive, any such progression is slow and subtle in most patients, and does not correlate with age or genotype. We also describe the first serial assessment of FAF, which is highly variable between individuals, even of similar age and genotype.