The golden era of ocular disease gene discovery: race to the finish.

Within the last decade, technological advances have led to amazing genetic insights into Mendelian and multifactorial ocular diseases. We provide a perspective of the progress in gene discovery and discuss the implications. We believe that the time has come to redefine the goals and begin utilizing the genetic knowledge for clinical management and treatment design. The unbelievable opportunities now exist for those nimble enough to seize them.

Nrl is required for rod photoreceptor development.

The protein neural retina leucine zipper (Nrl) is a basic motif-leucine zipper transcription factor that is preferentially expressed in rod photoreceptors. It acts synergistically with Crx to regulate rhodopsin transcription. Missense mutations in human NRL have been associated with autosomal dominant retinitis pigmentosa. Here we report that deletion of Nrl in mice results in the complete loss of rod function and super-normal cone function, mediated by S cones. The photoreceptors in the Nrl-/- retina have cone-like nuclear morphology and short, sparse outer segments with abnormal disks. Analysis of retinal gene expression confirms the apparent functional transformation of rods into S cones in the Nrl-/- retina. On the basis of these findings, we postulate that Nrl acts as a 'molecular switch' during rod-cell development by directly modulating rod-specific genes while simultaneously inhibiting the S-cone pathway through the activation of Nr2e3.

Transcriptome analysis and molecular signature of human retinal pigment epithelium.

Retinal pigment epithelium (RPE) is a polarized cell layer critical for photoreceptor function and survival. The unique physiology and relationship to the photoreceptors make the RPE a critical determinant of human vision. Therefore, we performed a global expression profiling of native and cultured human fetal and adult RPE and determined a set of highly expressed 'signature' genes by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues. Using stringent selection criteria of at least 10-fold higher expression in three distinct preparations, we identified 154 RPE signature genes, which were validated by qRT-PCR analysis in RPE and in an independent set of 11 tissues. Several of the highly expressed signature genes encode proteins involved in visual cycle, melanogenesis and cell adhesion and Gene ontology analysis enabled the assignment of RPE signature genes to epithelial channels and transporters (ClCN4, BEST1, SLCA20) or matrix remodeling (TIMP3, COL8A2). Fifteen RPE signature genes were associated with known ophthalmic diseases, and 25 others were mapped to regions of disease loci. An evaluation of the RPE signature genes in a recently completed AMD genomewide association (GWA) data set revealed that TIMP3, GRAMD3, PITPNA and CHRNA3 signature genes may have potential roles in AMD pathogenesis and deserve further examination. We propose that RPE signature genes are excellent candidates for retinal diseases and for physiological investigations (e.g. dopachrome tautomerase in melanogenesis). The RPE signature gene set should allow the validation of RPE-like cells derived from human embryonic or induced pluripotent stem cells for cell-based therapies of degenerative retinal diseases.

Retinal repair by transplantation of photoreceptor precursors.

Photoreceptor loss causes irreversible blindness in many retinal diseases. Repair of such damage by cell transplantation is one of the most feasible types of central nervous system repair; photoreceptor degeneration initially leaves the inner retinal circuitry intact and new photoreceptors need only make single, short synaptic connections to contribute to the retinotopic map. So far, brain- and retina-derived stem cells transplanted into adult retina have shown little evidence of being able to integrate into the outer nuclear layer and differentiate into new photoreceptors. Furthermore, there has been no demonstration that transplanted cells form functional synaptic connections with other neurons in the recipient retina or restore visual function. This might be because the mature mammalian retina lacks the ability to accept and incorporate stem cells or to promote photoreceptor differentiation. We hypothesized that committed progenitor or precursor cells at later ontogenetic stages might have a higher probability of success upon transplantation. Here we show that donor cells can integrate into the adult or degenerating retina if they are taken from the developing retina at a time coincident with the peak of rod genesis. These transplanted cells integrate, differentiate into rod photoreceptors, form synaptic connections and improve visual function. Furthermore, we use genetically tagged post-mitotic rod precursors expressing the transcription factor Nrl (ref. 6) (neural retina leucine zipper) to show that successfully integrated rod photoreceptors are derived only from immature post-mitotic rod precursors and not from proliferating progenitor or stem cells. These findings define the ontogenetic stage of donor cells for successful rod photoreceptor transplantation.

Defects in neural stem cell proliferation and olfaction in Chd7 deficient mice indicate a mechanism for hyposmia in human CHARGE syndrome.

Mutations in CHD7, a chromodomain gene, are present in a majority of individuals with CHARGE syndrome, a multiple anomaly disorder characterized by ocular Coloboma, Heart defects, Atresia of the choanae, Retarded growth and development, Genital hypoplasia and Ear anomalies. The clinical features of CHARGE syndrome are highly variable and incompletely penetrant. Olfactory dysfunction is a common feature in CHARGE syndrome and has been potentially linked to primary olfactory bulb defects, but no data confirming this mechanistic link have been reported. On the basis of these observations, we hypothesized that loss of Chd7 disrupts mammalian olfactory tissue development and function. We found severe defects in olfaction in individuals with CHD7 mutations and CHARGE, and loss of odor evoked electro-olfactogram responses in Chd7 deficient mice, suggesting reduced olfaction is due to a dysfunctional olfactory epithelium. Chd7 expression was high in basal olfactory epithelial neural stem cells and down-regulated in mature olfactory sensory neurons. We observed smaller olfactory bulbs, reduced olfactory sensory neurons, and disorganized epithelial ultrastructure in Chd7 mutant mice, despite apparently normal functional cilia and sustentacular cells. Significant reductions in the proliferation of neural stem cells and regeneration of olfactory sensory neurons in the mature Chd7(Gt/+) olfactory epithelium indicate critical roles for Chd7 in regulating neurogenesis. These studies provide evidence that mammalian olfactory dysfunction due to Chd7 haploinsufficiency is linked to primary defects in olfactory neural stem cell proliferation and may influence olfactory bulb development.

Human SEC13Rp functions in yeast and is located on transport vesicles budding from the endoplasmic reticulum.

In the yeast Saccharomyces cerevisiae, Sec13p is required for intracellular protein transport from the ER to the Golgi apparatus, and has also been identified as a component of the COPII vesicle coat structure. Recently, a human cDNA encoding a protein 53% identical to yeast Sec13p has been isolated. In this report, we apply the genetic assays of complementation and synthetic lethality to demonstrate the conservation of function between this human protein, designated SEC13Rp, and yeast Sec13p. We show that two reciprocal human/yeast fusion constructs, encoding the NH2-terminal half of one protein and the COOH-terminal half of the other, can each complement the secretion defect of a sec13-1 mutant at 36 degrees C. The chimera encoding the NH2-terminal half of the yeast protein and the COOH-terminal half of the human protein is also able to complement a SEC13 deletion. Overexpression of either the entire human SEC13Rp protein or the chimera encoding the NH2-terminal half of the human protein and the COOH-terminal half of the yeast protein inhibits the growth of a sec13-1 mutant at 24 degrees C; this growth inhibition is not seen in a wild-type strain nor in other sec mutants, suggesting that the NH2-terminal half of SEC13Rp may compete with Sec13-1p for a common target. We show by immunoelectronmicroscopy of mammalian cells that SEC13Rp (like the putative mammalian homologues of the COPII subunits Sar1p and Sec23p) resides in the region of the transitional ER. We also show that the distribution of SEC13Rp is not affected by brefeldin A treatment. This report presents the first demonstration of a putative mammalian COPII component functioning in yeast, and highlights a potentially useful approach for the study of conserved mammalian proteins in a genetically tractable system.

Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa.

Usher syndrome type IIa (OMIM 276901), an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa, maps to the long arm of human chromosome 1q41 between markers AFM268ZD1 and AFM144XF2. Three biologically important mutations in Usher syndrome type IIa patients were identified in a gene (USH2A) isolated from this critical region. The USH2A gene encodes a protein with a predicted size of 171.5 kilodaltons that has laminin epidermal growth factor and fibronectin type III motifs; these motifs are most commonly observed in proteins comprising components of the basal lamina and extracellular matrixes and in cell adhesion molecules.

Mutations in the cone-rod homeobox gene are associated with the cone-rod dystrophy photoreceptor degeneration.

Crx is a novel paired-like homeodomain protein that is expressed predominantly in retinal photoreceptors and pinealocytes. Its gene has been mapped to chromosome 19q13.3, the site of a disease locus for autosomal dominant cone-rod dystrophy (CORDII). Analysis of the proband from a family with autosomal dominant CORD revealed an Arg41Trp substitution in the third residue of the CRX homeodomain. The sequence change cosegregated with the disease phenotype and was not detected in 247 normal controls. Recombinant CRX homeodomain containing the Arg41Trp substitution showed decreased DNA binding activity. Analysis of another 169 CORD probands identified three additional CRX sequence variations (Arg41Gln, Val242Met, and a 4 bp deletion in codons 196/7) that were not found among the controls. This data suggests that mutations in the CRX gene are associated with photoreceptor degeneration and that the Crx protein is necessary for the maintenance of normal cone and rod function.

Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics.

We have characterized comprehensive transcript and proteomic profiles of cell lines corresponding to normal breast (MCF10A), noninvasive breast cancer (MCF7) and invasive breast cancer (MDA-MB-231). The transcript profiles were first analysed by a modified protocol for representational difference analysis (RDA) of cDNAs between MCF7 and MDA-MB-231 cells. The majority of genes identified by RDA showed nearly complete concordance with microarray results, and also led to the identification of some differentially expressed genes such as lysyl oxidase, copper transporter ATP7A, EphB6, RUNX2 and a variant of RUNX2. The altered transcripts identified by microarray analysis were involved in cell-cell or cell-matrix interaction, Rho signaling, calcium homeostasis and copper-binding/sensitive activities. A set of nine genes that included GPCR11, cadherin 11, annexin A1, vimentin, lactate dehydrogenase B (upregulated in MDA-MB-231) and GREB1, S100A8, amyloid beta precursor protein, claudin 3 and cadherin 1 (downregulated in MDA-MB-231) were sufficient to distinguish MDA-MB-231 from MCF7 cells. The downregulation of a set of transcripts for proteins involved in cell-cell interaction indicated these transcripts as potential markers for invasiveness that can be detected by methylation-specific PCR. The proteomic profiles indicated altered abundance of fewer proteins as compared to transcript profiles. Antisense knockdown of selected transcripts led to inhibition of cell proliferation that was accompanied by altered proteomic profiles. The proteomic profiles of antisense transfectants suggest the involvement of peptidyl-prolyl isomerase, Raf kinase inhibitor and 80 kDa protein kinase C substrate in mediating the inhibition of cell proliferation.

Molecular organization and expression of the genetic locus glued in Drosophila melanogaster.

The Glued locus of Drosophila melanogaster is genetically defined as the functional unit which is affected by the dominant Glued mutation Gl. Genomic DNA was cloned from the region of the Glued locus, at 70C2 on chromosome 3, by using a P element insertion in the region as a molecular marker. Three genes encoding polyadenylated transcripts were detected within a 30-kilobase span of the cloned DNA. The gene nearest the P element insertion site was identified as a Glued gene on the basis of alterations in its DNA and encoded transcript associated with the Gl mutation and with reversions of Gl which eliminate the dominant effect by inactivation of the mutant allele. Expression of the wild-type Gl+ gene is temporally regulated during development; the amount of the encoded transcript is highest in the embryonic stage, decreasing in the first and second larval instars, and then increasing in the third instar and pupal stages. There is a maternal contribution of the Gl+ transcript to the embryo, which probably accounts for the maternal lethal effect of Glued mutations on early development. In situ hybridizations of Gl+ DNA to RNA in tissue sections showed that the Gl+ transcript is present in virtually all tissues of the embryo, late larva, and pupa. The general distribution of this transcript is consistent with genetic evidence indicating that the Glued locus controls a generally essential cell function (P. J. Harte and D. R. Kankel, Genetics 101:477-501, 1982). Different Glued mutations produce distinct phenotypic effects, including adults with severe visual defects, larvae lacking imaginal discs, and early lethality. These diverse mutant phenotypes are discussed in terms of quantitative changes in the Glued function. Closely adjacent to Gl+ is another gene which is transcribed in a divergent direction and expressed coordinately with Gl+ throughout Drosophila development. It remains to be determined whether this gene is also involved with the Glued function.

Lens-specific gene recruitment of zeta-crystallin through Pax6, Nrl-Maf, and brain suppressor sites.

Zeta-Crystallin is a taxon-specific crystallin, an enzyme which has undergone direct gene recruitment as a structural component of the guinea pig lens through a Pax6-dependent mechanism. Tissue specificity arises through a combination of effects involving three sites in the lens promoter. The Pax6 site (ZPE) itself shows specificity for an isoform of Pax6 preferentially expressed in lens cells. High-level expression of the promoter requires a second site, identical to an alphaCE2 site or half Maf response element (MARE), adjacent to the Pax6 site. A promoter fragment containing Pax6 and MARE sites gives lens-preferred induction of a heterologous promoter. Complexes binding the MARE in lens nuclear extracts are antigenically related to Nrl, and cotransfection with Nrl elevates zeta-crystallin promoter activity in lens cells. A truncated zeta promoter containing Nrl-MARE and Pax6 sites has a high level of expression in lens cells in transgenic mice but is also active in the brain. Suppression of the promoter in the brain requires sequences between -498 and -385, and a site in this region forms specific complexes in brain extract. A three-level model for lens-specific Pax6-dependent expression and gene recruitment is suggested: (i) binding of a specific isoform of Pax6; (ii) augmentation of expression through binding of Nrl or a related factor; and (iii) suppression of promoter activity in the central nervous system by an upstream negative element in the brain but not in the lens.

Expression of the bZIP transcription factor gene Nrl in the developing nervous system.

Proteins of the Maf/Nrl subfamily of bZIP transcription factors are involved in the regulation of tissue-specific gene expression. The Nrl gene, initially identified from a subtracted retinal library, is expressed in all cell layers of the adult retina, including photoreceptors. The Nrl protein has high sequence homology with Maf proteins, binds to an AP-1 like sequence element, and in photoreceptors appears to be involved in regulating the expression of rhodopsin. In the present study, we investigated the expression of Nrl in the developing and adult mouse using in situ hybridization and RT-PCR. We demonstrate that beginning at embryonic day 12.5 Nrl is expressed throughout the developing central and peripheral nervous system, with the exception of the nasal epithelium. The spatial pattern of hybridization suggests that Nrl is transcribed in post-mitotic, differentiating neurons, the developing cephalic mesenchyme and lens. Nrl expression is downregulated postnatally in the brain, and becomes restricted to neocortex and brainstem in the adult. High levels of Nrl transcripts, however, persist in the mature photoreceptors and other retinal neurons. Our studies suggest a role for the Nrl protein in neuronal differentiation and in mature neurons of the adult retina.

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase.

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish peroxidase and H2O2 and decreased the ratio of scopoletin (consumed):H2O2 (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH:O2 of 2:1. On step-wise addition of a small concentration of H2O2 a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H2O2 ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound accumulated during oxidation of NADH. The effectiveness of a small concentration of H2O2 in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H2O2 during scopoletin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.

Nucleotide sequence of a cDNA for the beta 2 subunit isoform of Na+,K(+)-ATPase from human retina.

Using as probe the entire human liver cDNA clone coding for the beta 2 subunit isoform of the Na+,K(+)-ATPase, which lacks the initiation codon ATG, and the entire 5'-untranslated region (Martin-Vasallo, P., Dackowski, W., Emanuel, J.R. and Levenson, R. (1989) J. Biol. Chem. 264, 4613-4618), we isolated a larger clone from a directional human adult retina cDNA library (Swaroop, A. and Xu, J. (1993) Cytogenet. Cell Genet. 64, 292-294). This clone, pNH beta 2, shows 100% homology with the nucleotide sequence of the human liver cDNA clone and also contains additional 407 nucleotides in the 5'-untranslated region, the initiation codon and a poly(A) tail. Northern blot hybridization analysis reveals that the human mRNA (3.6 kb) is approx. 300 nucleotides larger than the major transcript size expressed in rat (3.3 kb). The larger human size mRNA for the human beta 2 Na+,K(+)-ATPase indicates species differences in gene processing.

Five novel RPGR mutations in families with X-linked retinitis pigmentosa.

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe because of their early onset, often leading to significant visual impairment before the fourth decade. RP3, genetically localized at Xp21.1, accounts for 70% of XLRP in different populations. The RPGR (Retinitis Pigmentosa GTPase Regulator) gene that was isolated from the RP3 region is mutated in 20% of North American families with XLRP. From mutation analysis of 27 independent XLRP families, we have identified five novel RPGR mutations in 5 of the families (160delA, 789 A>T, IVS8+1 G>C, 1147insT and 1366 G>A). One of these mutations was detected in a family from Chile. Hum Mutat 17:151, 2001.

NRL S50T mutation and the importance of 'founder effects' in inherited retinal dystrophies.

The aim of this work was to identify NRL mutations in a panel of 200 autosomal dominant retinitis pigmentosa (adRP) families. All samples were subjected to heteroduplex analysis of the three exons of the NRL gene, and HphI restriction digest analysis of exon 2 (to identify the S50T mutation). Families found to have the S50T mutation, and six additional larger pedigrees (which had previously been excluded from the other nine adRP loci) underwent linkage analysis using polymorphic markers located in the region of 14q11. HphI restriction analysis followed by direct sequencing of the amplified NRL exon 2 product demonstrated the presence of the NRL S50T sequence change in three adRP families. Comparison of marker haplotypes in affected individuals from these families with those of affected members of the original 14q11 linked family revealed a common disease haplotype for markers within the adRP locus. Recombination events observed in these families define an adRP critical interval of 14.9 cM between D13S72 and D14S1041. Linkage analysis enabled all six of the larger adRP pedigrees to be excluded from the 14q11 locus. The NRL S50T mutation represents another example of a 'founder effect' in a dominantly inherited retinal dystrophy. Identification of such 'founder effects' may greatly simplify diagnostic genetic screening and lead to better prognostic counselling. The exclusion of several adRP families from all ten adRP loci indicates that at least one further adRP locus remains to be found.

Expression profile and chromosomal location of cDNA clones, identified from an enriched adult retina library.

PURPOSE: To delineate the profile of genes expressed in the adult human retina and assign chromosomal location of cDNA clones. METHODS: The end-sequence of random clones from an enriched human retinal cDNA library was analyzed by NCBI database search. Expression profile was established by northern blot analysis, database search, or both. Selected cDNA clones were localized to human chromosomes by somatic cell hybrid analysis, in situ hybridization to metaphase chromosomes, or both. Chromosomal location was also obtained by searching the databases. RESULT: One hundred and thirty-seven clones were isolated from the subtracted retinal library. Fifty-one clones were identical with 35 known human genes in GenBank, and 24 clones corresponded to 23 uncharacterized human expressed sequenced tags (ESTs), novel genes, or both. The remaining 59 clones were not pursued further because they contained bacterial sequences or repetitive elements. Several clones indicated a restricted pattern of expression with high levels of transcripts in the retina. Chromosomal location of novel retinal ESTs is also reported. CONCLUSIONS: This study provides a profile of genes expressed in the adult human retina. One round of subtraction eliminated most constitutively expressed genes and permitted partial normalization of the retinal library. Twenty-three novel genes were identified. The combined information obtained from expression analysis and chromosomal localization of retinal cDNAs should be valuable in identifying candidate genes for diseases involving retinal dysfunction.

Disease expression in X-linked retinitis pigmentosa caused by a putative null mutation in the RPGR gene.

PURPOSE: To determine the disease expression in X-linked retinitis pigmentosa (XLRP) caused by a putative null mutation in the RPGR (retinitis pigmentosa GTPase regulator) gene. METHODS: In a family with XLRP, haplotype analysis was performed with polymorphic microsatellite markers from the Xp chromosomal region, and genomic polymerase chain reaction sequencing was used to identify sequence variations in the RPGR gene. Hemizygotes and heterozygotes were evaluated clinically and with visual function tests. Optical coherence tomography (OCT) was performed on heterozygotes. Postmortem donor retinas from a heterozygote were examined by microscopy and immunocytochemistry. RESULTS: X-linked inheritance was confirmed by haplotype analysis using Xp markers. Sequence analysis of the RPGR gene identified a single base pair change, a G-->T transversion, that converts codon 52 GGA (Gly) to TGA (stop codon); the mutation segregates with the disease. A hemizygote in the third decade of life had barely measurable rod function and severely impaired cone function that diminished further over a 7-year interval. Heterozygotes varied in degree of disease expression from mild to severe. Perimetry showed loci with normal rod and cone sensitivity interspersed with loci having either equal rod and cone dysfunction or rod > cone dysfunction. Electroretinographic photoreceptor responses had equal reductions in rod and cone maximal amplitude. OCT cross sectional reflectance images of retinal regions with severe dysfunction showed reduced thickness of the retina and retinal pigment epithelium-choriocapillaris (RPE-CC) complex and increased reflections posteriorly. Regions with mild dysfunction showed similar OCT findings but with preserved retinal thickness. Retinal histopathology in a heterozygote revealed loss of photoreceptors throughout, with retention of only a few islands of cones with tiny or absent outer segments and rods lacking outer segments. CONCLUSIONS: This RPGR gene mutation, in its mildest expression in heterozygotes, causes a relatively equal disturbance of rod and cone photoreceptor function. Detectable structural change by OCT at the level of the RPE-CC can be present in patches of retina with minimal functional disturbance. More advanced disease stages in heterozygotes show greater rod than cone dysfunction, and the end stage in hemizygotes and heterozygotes is that of typical RP, with only barely detectable cone function from residual cones in a thinned retina with abnormal RPE and choriocapillaris.

Phenotype of an X-linked retinitis pigmentosa family with a novel splice defect in the RPGR gene.

PURPOSE: To assess the clinical phenotype in a Swedish family with X-linked retinitis pigmentosa (XLRP) resulting from a novel splice defect in the RPGR gene. METHODS: RPGR mutation analysis was performed in one family with XLRP, and several individuals from the family were examined clinically. RESULTS: The causative mutation in the family was demonstrated to be a single base-pair change at the splice donor site in intron 7 that resulted in skipping of the complete exon 7 in the mature RPGR transcript. The aberrant mRNA is predicted to produce an RPGR protein with an in-frame deletion of 53 amino acids, corresponding to an RCC1-homology repeat. Clinical studies that included ophthalmological examination and full-field electroretinography showed that this splice mutation resulted in a comparatively less severe form of RP. CONCLUSIONS: Correlation of a causative RPGR genotype with clinical findings in hemizygotes and carrier heterozygotes is an important step toward predictive diagnosis and should assist in the development of gene-based therapies in the future.