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.

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.

Long-term follow-up of a family with dominant X-linked retinitis pigmentosa.

PURPOSE: To document the progression of disease in male and female members of a previously described family with X-linked dominant retinitis pigmentosa (RP) caused by a de novo insertion after nucleotide 173 in exon ORF15 of RPGR. METHODS: The clinical records of 19 members of family UTAD054 were reviewed. Their evaluations consisted of confirmation of family history, standardised electroretinograms (ERGs), Goldmann visual fields, and periodic ophthalmological examinations over a 23-year period. RESULTS: Male members of family UTAD054 had non-recordable to barely recordable ERGs from early childhood. The males showed contracted central fields and developed more severe retinopathy than the females. The female members showed a disease onset delayed to teenage years, recordable but diminishing photopic and scotopic ERG amplitudes in a cone-rod pattern, progressive loss and often asymmetric visual fields, and diffuse atrophic retinopathy with fewer pigment deposits compared with males. CONCLUSIONS: This insertion mutation in the RPGR exon ORF15 is associated with a RP phenotype that severely affects males early and females by 30 years of age, and is highly penetrant in female members. Families with dominant-acting RPGR mutations may be mistaken to have an autosomal mode of inheritance resulting in an incorrect prediction of recurrence risk and prognosis. Broader recognition of X-linked RP forms with dominant inheritance is necessary to facilitate appropriate counselling of these patients.

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.

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.

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.

RPGR ORF15 isoform co-localizes with RPGRIP1 at centrioles and basal bodies and interacts with nucleophosmin.

The ORF15 isoform of RPGR (RPGR(ORF15)) and RPGR interacting protein 1 (RPGRIP1) are mutated in a variety of retinal dystrophies but their functions are poorly understood. Here, we show that in cultured mammalian cells both RPGR(ORF15) and RPGRIP1 localize to centrioles. These localizations are resistant to the microtubule destabilizing drug nocodazole and persist throughout the cell cycle. RPGR and RPGRIP1 also co-localize at basal bodies in cells with primary cilia. The C-terminal (C2) domain of RPGR(ORF15) (ORF15(C2)) is highly conserved across 13 mammalian species, suggesting that it is a functionally important domain. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we show that this domain interacts with a 40 kDa shuttling protein nucleophosmin (NPM). The RPGR(ORF15)-NPM interaction was confirmed by (i) yeast two-hybrid analyses; (ii) binding of both recombinant and native HeLa cell NPM to RPGR(ORF15) fusion proteins in vitro; (iii) co-immunoprecipitation of native NPM, RPGR(ORF15) and RPGRIP1 from bovine retinal extracts and of native HeLa cell NPM and transfected RPGR(ORF15) from cultured cells and (iv) co-localization of NPM and RPGR(ORF15) at metaphase centrosomes in cultured cells. NPM is a multifunctional protein chaperone that shuttles between the nucleoli and the cytoplasm and has been associated with licensing of centrosomal division. RPGR and RPGRIP1 join a growing number of centrosomal proteins involved in human disease.

Guided tactile probing: a modified blind orotracheal intubation technique for the problem-oriented difficult airway.

We present 'tactile probing', a guided approach to blind orotracheal intubation to secure a problem-oriented anticipated difficult airway in a 55-year-old male patient scheduled for elective surgical tracheostomy for a postradiotherapy translaryngeal carcinoma. Standard techniques to gain the airway were inapplicable in this case and awake flexible fiberoscopy-aided intubation had already failed.

From disease genes to cellular pathways: a progress report.

Mutations in a large number of retinal and retinal pigment epithelium (RPE) expressed genes can lead to the degeneration of photoreceptors and consequently the loss of vision. The genetic and phenotypic heterogeneity of retinal dystrophies poses a complex problem with respect to rational development of therapeutic strategies. Delineation of physiological functions of disease genes and identification of pathways that lead to disease pathogenesis represent essential goals towards developing a systematic and global approach to gene-based treatments. We are interested in identifying cellular pathways that are involved in photoreceptor differentiation, function and degeneration. We are, therefore, generating comprehensive gene expression profiles of retina and RPE of humans and mice using both cDNA- and oligonucleotide-based (Affymetrix) microarrays. Because of the under-representation of retinal/RPE genes in the public databases, we have constructed several unamplified cDNA libraries and produced almost twenty thousand expressed sequence tags (ESTs) that are being printed onto glass slides ('I-Gene' microarrays). In this presentation, we will report the microarray analysis of the rodless (and cone-enhanced) retina from the Nrl-knockout mouse as a paradigm to initiate the identification of cellular pathways involved in photoreceptor differentiation and function.

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.

Multiple phosphorylated isoforms of NRL are expressed in rod photoreceptors.

NRL, a bZIP transcription factor of the Maf subfamily, interacts with the homeodomain protein CRX and synergistically regulates rhodopsin expression. Here we report that six isoforms of NRL (29-35 kDa) are generated by phosphorylation and expressed specifically in the mammalian retina. The anti-NRL antibody also cross-reacts with a cytosolic 45-kDa protein, which is detected in neuronal tissues but is not encoded by the NRL gene. In both human retinal cell cultures and sections of fetal and adult human retina, NRL is present in the nuclei of developing and mature rods but not cones. We propose that NRL regulates rod photoreceptor-specific gene expression and is involved in rod differentiation.

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.

Evaluation of the ELOVL4 gene in patients with age-related macular degeneration.

Stargardt-like macular degeneration (STGD(3)) and autosomal dominant macular degeneration (adMD) share phenotypic characters with atrophic age-related macular degeneration (AMD). Mutations in a photoreceptor cell-specific factor involved in the elongation of very long chain fatty acids (ELOVL(4)) were shown to be associated with STGD(3), adMD, and pattern dystrophy. We screened 778 patients with AMD and 551 age-matched controls to define the role of sequence variants in the ELOVL(4) gene in age-related macular degeneration. We detected three sequence variants in the non-coding region and eight variants in the coding region. No statistically significant association was observed between sequence variants in the ELOVL(4) gene and susceptibility to AMD. However, for the detection of modest effects of multiple alleles in a complex disease, the analysis of larger cohorts of patients may be required.

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.

Remapping of the RP15 locus for X-linked cone-rod degeneration to Xp11.4-p21.1, and identification of a de novo insertion in the RPGR exon ORF15.

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe, because of their early onset, often leading to significant vision loss before the 4th decade. Previously, the RP15 locus was assigned to Xp22, by linkage analysis of a single pedigree with "X-linked dominant cone-rod degeneration." After clinical reevaluation of a female in this pedigree identified her as affected, we remapped the disease to a 19.5-cM interval (DXS1219-DXS993) at Xp11.4-p21.1. This new interval overlapped both RP3 (RPGR) and COD1. Sequencing of the previously published exons of RPGR revealed no mutations, but a de novo insertion was detected in the new RPGR exon, ORF15. The identification of an RPGR mutation in a family with a severe form of cone and rod degeneration suggests that RPGR mutations may encompass a broader phenotypic spectrum than has previously been recognized in "typical" retinitis pigmentosa.

Cloning and functional expression of human retinal kir2.4, a pH-sensitive inwardly rectifying K(+) channel.

To identify novel potassium channel genes expressed in the retina, we screened a human retina cDNA library with an EST sequence showing partial homology to inwardly rectifying potassium (Kir) channel genes. The isolated cDNA yielded a 2,961-base pair sequence with the predicted open reading frame showing strong homology to the rat Kir2. 4 (rKir2.4). Northern analysis of mRNA from human and bovine tissues showed preferential expression of Kir2.4 in the neural retina. In situ hybridization to sections of monkey retina detected Kir2.4 transcript in most retinal neurons. Somatic hybridization analysis and dual-color in situ hybridization to metaphase chromosomes mapped Kir2.4 to human chromosome 19 q13.1-q13.3. Expression of human Kir2. 4 cRNA in Xenopus oocytes generated strong, inwardly rectifying K(+) currents that were enhanced by extracellular alkalinization. We conclude that human Kir2.4 encodes an inwardly rectifying K(+) channel that is preferentially expressed in the neural retina and that is sensitive to physiological changes in extracellular pH.

The leucine zipper of NRL interacts with the CRX homeodomain. A possible mechanism of transcriptional synergy in rhodopsin regulation.

Photoreceptor-specific expression of rhodopsin is mediated by multiple cis-acting elements in the proximal promoter region. NRL (neural retina leucine zipper) and CRX (cone rod homeobox) proteins bind to the adjacent NRE and Ret-4 sites, respectively, within this region. Although NRL and CRX are each individually able to induce rhodopsin promoter activity, when expressed together they exhibit transcriptional synergy in rhodopsin promoter activation. Using the yeast two-hybrid method and glutathione S-transferase pull-down assays, we demonstrate that the leucine zipper of NRL can physically interact with CRX. Deletion analysis revealed that the CRX homeodomain (CRX-HD) plays an important role in the interaction with the NRL leucine zipper. Although binding with the CRX-HD alone was weak, a strong interaction was detected when flanking regions including the glutamine-rich and the basic regions that follow the HD were included. A reciprocal deletion analysis showed that the leucine zipper of NRL is required for interaction with CRX-HD. Two disease-causing mutations in CRX-HD (R41W and R90W) that exhibit reduced DNA binding and transcriptional synergy also decrease its interaction with NRL. These studies suggest novel possibilities for protein-protein interaction between two conserved DNA-binding motifs and imply that cross-talk among distinct regulatory pathways contributes to the establishment and maintenance of photoreceptor function.

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.