Biochemical defects in ABCR protein variants associated with human retinopathies.

Mutations in the gene encoding ABCR (ABCA4), a photoreceptor-specific ATP-binding cassette (ABC) transporter, are responsible for autosomal recessive Stargardt disease (STGD), an early onset macular degeneration, and some forms of autosomal recessive cone-rod dystrophy and autosomal recessive retinitis pigmentosa. Heterozygosity for ABCA4 mutations may also represent a risk factor for age-related macular degeneration (AMD), although this idea is controversial. An ongoing challenge in the analysis of ABCA4-based retinopathies arises from the observation that most of the ABCA4 sequence variants identified so far are missense mutations that are rare in both patient and control populations. With the current sample size of most sequence variants, one cannot determine statistically whether a particular sequence variant is pathogenic or neutral. A related challenge is to determine the degree to which each pathogenic variant impairs ABCR function, as genotype-phenotype analyses indicate that age of onset and disease severity correlate with different ABCA4 alleles. To address these questions, we performed a functional analysis of human ABCR and its variants. These experiments reveal a wide spectrum of biochemical defects in these variants and provide insight into the transport mechanism of ABCR.

A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy.

Stargardt disease (STGD, also known as fundus flavimaculatus; FFM) is an autosomal recessive retinal disorder characterized by a juvenile-onset macular dystrophy, alterations of the peripheral retina, and subretinal deposition of lipofuscin-like material. A gene encoding an ATP-binding cassette (ABC) transporter was mapped to the 2-cM (centiMorgan) interval at 1p13-p21 previously shown by linkage analysis to harbour the STGD gene. This gene, ABCR, is expressed exclusively and at high levels in the retina, in rod but not cone photoreceptors, as detected by in situ hybridization. Mutational analysis of ABCR in STGD families revealed a total of 19 different mutations including homozygous mutations in two families with consanguineous parentage. These data indicate that ABCR is the causal gene of STGD/FFM.

Absorption spectra of the hybrid pigments responsible for anomalous color vision.

Unequal homologous recombination events between green and red cone pigment genes produce the red-green or green-red hybrid pigment genes found in many individuals with variant color vision. Photobleaching difference absorption spectroscopy of hybrid pigments produced in cultured cells shows that the spectral sensitivity of each hybrid pigment is intermediate between the parental green and red pigment sensitivities. Amino acids encoded by exons 2, 3, 4, and 5 produce spectral shifts at the wavelength of maximal absorbance of 0 to 4, 0 to 4, 3 to 4, and 15 to 21 nanometers, respectively, the exact value depending on the identities of amino acids elsewhere in the hybrid.

Molecular genetics of human color vision: the genes encoding blue, green, and red pigments.

Human color vision is based on three light-sensitive pigments. The isolation and sequencing of genomic and complementary DNA clones that encode the apoproteins of these three pigments are described. The deduced amino acid sequences show 41 +/- 1 percent identity with rhodopsin. The red and green pigments show 96 percent mutual identity but only 43 percent identity with the blue pigment. Green pigment genes vary in number among color-normal individuals and, together with a single red pigment gene, are proposed to reside in a head-to-tail tandem array within the X chromosome.

Tandem array of human visual pigment genes at Xq28.

Unequal crossing-over within a head-to-tail tandem array of the homologous red and green visual pigment genes has been proposed to explain the observed variation in green-pigment gene number among individuals and the prevalence of red-green fusion genes among color-blind subjects. This model was tested by probing the structure of the red and green pigment loci with long-range physical mapping techniques. The loci were found to constitute a gene array with an approximately 39-kilobase repeat length. The position of the red pigment gene at the 5' edge of the array explains its lack of variation in copy number. Restriction maps of the array in four individuals who differ in gene number are consistent with a head-to-tail configuration of the genes. These results provide physical evidence in support of the model and help to explain the high incidence of color blindness in the human population.

Molecular genetics of inherited variation in human color vision.

The hypothesis that red-green "color blindness" is caused by alterations in the genes encoding red and green visual pigments has been tested and shown to be correct. Genomic DNA's from 25 males with various red-green color vision deficiencies were analyzed by Southern blot hybridization with the cloned red and green pigment genes as probes. The observed genotypes appear to result from unequal recombination or gene conversion (or both). Together with chromosome mapping experiments, these data identify each of the cloned human visual pigment genes.

A member of the Frizzled protein family mediating axis induction by Wnt-5A.

In Xenopus laevis embryos, the Wingless/Wnt-1 subclass of Wnt molecules induces axis duplication, whereas the Wnt-5A subclass does not. This difference could be explained by distinct signal transduction pathways or by a lack of one or more Wnt-5A receptors during axis formation. Wnt-5A induced axis duplication and an ectopic Spemann organizer in the presence of hFz5, a member of the Frizzled family of seven-transmembrane receptors. Wnt-5A/hFz5 signaling was antagonized by glycogen synthase kinase-3 and by the amino-terminal ectodomain of hFz5. These results identify hFz5 as a receptor for Wnt-5A.

Molecular genetics of human blue cone monochromacy.

Blue cone monochromacy is a rare X-linked disorder of color vision characterized by the absence of both red and green cone sensitivities. In 12 of 12 families carrying this trait, alterations are observed in the red and green visual pigment gene cluster. The alterations fall into two classes. One class arose from the wild type by a two-step pathway consisting of unequal homologous recombination and point mutation. The second class arose by nonhomologous deletion of genomic DNA adjacent to the red and green pigment gene cluster. These deletions define a 579-base pair region that is located 4 kilobases upstream of the red pigment gene and 43 kilobases upstream of the nearest green pigment gene; this 579-base pair region is essential for the activity of both pigment genes.

Histidine residues regulate the transition of photoexcited rhodopsin to its active conformation, metarhodopsin II.

The biologically active photoproduct of rhodopsin, metarhodopsin II (M II), exists in a pH-sensitive equilibrium with its precursor, metarhodopsin I (M I). Increasing acidity favors M II, with the midpoint of the pH titration curve at pH 6.4. To test the long-standing proposal that histidine protonation regulates this conformational transition, we characterized mutant rhodopsins in which each of the 6 histidines was replaced by phenylalanine or cysteine. Only mutants substituted at the 3 conserved histidines showed abnormal M I-M II equilibria. Those in which His-211 was replaced by phenylalanine or cysteine formed little or no M II at either extreme of pH, whereas mutants substituted at His-65 or at His-152 showed enhanced sensitivity to protons. The simplest interpretation of these results is that His-211 is the site where protonation strongly stabilizes the M II conformation and that His-65 and His-152 are sites where protonation modestly destabilizes the M II conformation.

A novel signaling pathway from rod photoreceptors to ganglion cells in mammalian retina.

Current understanding suggests that mammalian rod photoreceptors connect only to an ON-type bipolar cell. This rod-specific bipolar cell excites the All amacrine cell, which makes connections to cone-specific bipolar cells of both ON and OFF type; these, in turn, synapse with ganglion cells. Recent work on rabbit retina has shown that rod signals can also reach ganglion cells without passing through the rod bipolar cell. This route was thought to be provided by electrical gap junctions, through which rods signal directly to cones and thence to cone bipolar cells. Here, we show that the mouse retina also provides a rod pathway bypassing the rod bipolar cell, suggesting that this is a common feature in mammals. However, this alternative pathway does not require cone photoreceptors; it is perfectly intact in a transgenic mouse whose retina lacks cones. Instead, the results can be explained if rods connect directly to OFF bipolar cells.

Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas.

To define the cis-acting DNA elements required for rhodopsin expression, we generated lines of transgenic mice carrying sequences upstream of the bovine rhodopsin gene fused to the E. coli beta-galactosidase gene (lacZ). Upstream sequences extending from -2174 to +70 bp, from -734 to +70 bp, and from -222 to +70 bp direct photoreceptor-specific expression. All three -2174 lines demonstrate a superior-temporal to inferior-nasal gradient of expression across the retina, whereas lines carrying the shorter constructs demonstrate either spatially continuous expression across the retina, discrete clusters of expression, or both. As a complementary approach to defining regulatory elements, we compared DNA sequences 5' of the murine, bovine, and human rhodopsin genes. Significant homology between all three species was found just upstream of the transcription start site and at approximately 1.5 kb upstream.

A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival.

A cadherin family member, prCAD, was identified in retina cDNA by subtractive hybridization and high throughput sequencing. prCAD is expressed only in retinal photoreceptors, and the prCAD protein is localized to the base of the outer segment of both rods and cones. In prCAD(-/-) mice, outer segments are disorganized and fragmented, and there is progressive death of photoreceptor cells. prCAD is unlikely to be involved in protein trafficking between inner and outer segments, since phototransduction proteins appear to be correctly localized and the light responses of both rods and cones are only modestly compromised in prCAD(-/-) mice. These experiments imply a highly specialized cell biological function for prCAD and suggest that localized adhesion activity is essential for outer segment integrity.

Brn-3b: a POU domain gene expressed in a subset of retinal ganglion cells.

A search for POU domain transcription factors in human retina cDNA has led to the identification of Brn-3b, a class IV POU domain protein. Immunohistochemical experiments show that chicken, mouse, rabbit, monkey, and human retinas contain Brn-3b exclusively within a subpopulation of ganglion cells. In the adult mouse brain, Brn-3b is found only within cells in the deep layers of the superior colliculus, in the dorsal periaqueductal gray, and in a small cluster of cells in the brain stem near the area postrema. During the immediate postnatal period, cells containing Brn-3b are distributed in a number of regions within the brain stem and cerebellum. These data suggest that Brn-3b plays a role in determining and/or maintaining the identities of a small number of neurons, including a subset of visual system neurons.

A locus control region adjacent to the human red and green visual pigment genes.

Deletion of sequences 5' of the human red and green pigment gene array results in blue cone monochromacy, a disorder in which both red and green cone function are absent. To test whether these sequences are required for transcription of the adjacent visual pigment genes in cone photoreceptors, we produced transgenic mice carrying sequences upstream of the red and green pigment genes fused to a beta-galactosidase reporter. The patterns of transgene expression indicate that the human sequences direct expression to both long and short wave-sensitive cones in the mouse retina and that a region between 3.1 kb and 3.7 kb 5' of the red pigment gene transcription initiation site is essential for expression. Sequences within this region are highly conserved among humans, mice, and cattle, even though the latter two species have only a single visual pigment gene at this locus. These experiments suggest a model in which an interaction between the conserved 5' region and either the red or the green pigment gene promoter determines which of the two genes a given cone expresses.

Normal light response, photoreceptor integrity, and rhodopsin dephosphorylation in mice lacking both protein phosphatases with EF hands (PPEF-1 and PPEF-2).

Rhodopsin dephosphorylation in Drosophila is a calcium-dependent process that appears to be catalyzed by the protein product of the rdgC gene. Two vertebrate rdgC homologs, PPEF-1 and PPEF-2, have been identified. PPEF-1 transcripts are present at low levels in the retina, while PPEF-2 transcripts and PPEF-2 protein are abundant in photoreceptors. To determine if PPEF-2 alone or in combination with PPEF-1 plays a role in rhodopsin dephosphorylation and to determine if retinal degeneration accompanies mutation of PPEF-1 and/or PPEF-2, we have produced mice carrying targeted disruptions in the PPEF-1 and PPEF-2 genes. Loss of either or both PPEFs has little or no effect on rod function, as mice lacking both PPEF-1 and PPEF-2 show little or no changes in the electroretinogram and PPEF-2-/- mice show normal single-cell responses to light in suction pipette recordings. Light-dependent rhodopsin phosphorylation and dephosphorylation are also normal or nearly normal as determined by (i) immunostaining of PPEF-2-/- retinas with the phosphorhodopsin-specific antibody RT-97 and (ii) mass spectrometry of C-terminal rhodopsin peptides from mice lacking both PPEF-1 and PPEF-2. Finally, PPEF-2-/- retinas show normal histology at 1 year of age, and retinas from mice lacking both PPEF-1 and PPEF-2 show normal histology at 3 months of age, the latest time examined. These data indicate that, in contrast to loss of rdgC function in Drosophila, elimination of PPEF function does not cause retinal degeneration in vertebrates.

Progressive cerebellar, auditory, and esophageal dysfunction caused by targeted disruption of the frizzled-4 gene.

Wnt signaling has been implicated in the control of cell proliferation and in synapse formation during neural development, and these actions are presumed to be mediated by frizzled receptors. In this paper we report the phenotype of mice carrying a targeted deletion of the frizzled-4 (fz4) gene. fz4(-/-) mice exhibit three distinct defects: (1) progressive cerebellar degeneration associated with severe ataxia, (2) absence of a skeletal muscle sheath around the lower esophagus associated with progressive esophageal distension and dysfunction, and (3) progressive deafness caused by a defect in the peripheral auditory system unaccompanied by loss of hair cells or other auditory neurons. As assayed using a lacZ knock-in reporter, fz4 is widely expressed within the CNS. In particular, fz4 is expressed in cerebellar Purkinje cells, esophageal skeletal muscle, and cochlear inner hair cells, and the absence of Fz4 in these cells is presumed to account for the fz4(-/-) phenotype. In contrast to the early cell proliferation and patterning effects classically ascribed to Wnts, the auditory and cerebellar phenotypes of fz4(-/-) mice implicate Frizzled signaling in maintaining the viability and integrity of the nervous system in later life.

Expression and regulation of chicken fibroblast growth factor homologous factor (FHF)-4 at the base of the developing limbs.

Fibroblast growth factor homologous factors (FHFs) have been implicated in limb and nervous system development. In this paper we describe the expression of the cFHF-4 gene during early chicken development. cFHF-4 is expressed in the paraxial mesoderm, lateral ridge, and, most prominently, in the posterior-dorsal side of the base of each limb bud. The expression pattern of cFHF-4 at the base of the limbs is not altered by tissue grafts containing the zone of polarizing activity (ZPA), by implants of Shh-expressing cells, or by implants of beads containing retinoic acid, nor does it depend on the distal growth of the limb as it is not altered in limb buds that are surgically truncated. In three chicken mutants affecting limb patterning - talpid(2), limbless, and wingless - altered patterns of cFHF-4 expression are correlated with abnormal nerve plexus formation and altered patterns of limb bud innervation. Similarly, ectopic expression of cFHF-4 is correlated with a local induction of limb-like innervation patterns when beads containing FGF-2 are implanted in the flank. In these experiments, both ectopic innervation and ectopic expression of cFHF-4 in the flank were observed regardless of the size of the FGF-2-induced outgrowths. By contrast, ectopic expression of Shh and HoxD13 are seen only in the larger FGF-2-induced outgrowths. Taken together, these data suggest that cFHF-4 regulates or is coregulated with early events related to innervation at the base of the limbs.

Four novel mutations in the RPE65 gene in patients with Leber congenital amaurosis.

Leber congenital amaurosis (LCArpar; is a heterogeneous disorder representing the congenital forms of retinitis pigmentosa accounting for about 5% of all retinal dystrophies. The RPE65 gene product is required for regeneration of the visual pigment for phototransduction. Defects in the RPE65 gene have so far been shown to account for approximately 10 % of known cases of LCA. Here we describe four additional novel mutations in the RPE65 gene (c.889delA, c.131G>A, c.1249G>C, c.430T>G) and several novel polymorphisms in a large series of LCA patients. Hum Mutat 18:164, 2001.

Organization of the ABCR gene: analysis of promoter and splice junction sequences.

Mutations in the human ABCR gene have been associated with the autosomal recessive Stargardt disease (STGD), retinitis pigmentosa (RP19), and cone-rod dystrophy (CRD) and have also been found in a fraction of age-related macular degeneration (AMD) patients. The ABCR gene is a member of the ATP-binding cassette (ABC) transporter superfamily and encodes a rod photoreceptor-specific membrane protein. The cytogenetic location of the ABCR gene was refined to 1p22.3-1p22.2. The intron/exon structure was determined for the ABCR gene from overlapping genomic clones. ABCR spans over 100kb and comprises 50 exons. Intron/exon splice site sequences are presented for all exons and analyzed for information content (Ri). Nine splice site sequence variants found in STGD and AMD patients are evaluated as potential mutations. The localization of splice sites reveals a high degree of conservation between other members of the ABC1 subfamily, e.g. the mouse Abc1 gene. Analysis of the 870-bp 5' upstream of the transcription start sequence reveals multiple putative photoreceptor-specific regulatory elements including a novel retina-specific transcription factor binding site. These results will be useful in further mutational screening of the ABCR gene in various retinopathies and for determining the substrate and/or function of this photoreceptor-specific ABC transporter.