Showing papers by "Anand Swaroop published in 2010"

Transcriptional regulation of photoreceptor development and homeostasis in the mammalian retina.

Abstract: In the developing vertebrate retina, diverse neuronal subtypes originate from multipotent progenitors in a conserved order and are integrated into an intricate laminated architecture. Recent progress in mammalian photoreceptor development has identified a complex relationship between six key transcription-regulatory factors (RORβ, OTX2, NRL, CRX, NR2E3 and TRβ2) that determine rod versus M cone or S cone cell fate. We propose a step-wise 'transcriptional dominance' model of photoreceptor cell fate determination, with the S cone representing the default state of a generic photoreceptor precursor. Elucidation of gene-regulatory networks that dictate photoreceptor genesis and homeostasis will have wider implications for understanding the development of nervous system function and for the treatment of neurodegenerative diseases.

Genetic variants near TIMP3 and high-density lipoprotein–associated loci influence susceptibility to age-related macular degeneration

Abstract: We executed a genome-wide association scan for age-related macular degeneration (AMD) in 2,157 cases and 1,150 controls. Our results validate AMD susceptibility loci near CFH (P < 10−75), ARMS2 (P < 10−59), C2/CFB (P < 10−20), C3 (P < 10−9), and CFI (P < 10−6). We compared our top findings with the Tufts/Massachusetts General Hospital genome-wide association study of advanced AMD (821 cases, 1,709 controls) and genotyped 30 promising markers in additional individuals (up to 7,749 cases and 4,625 controls). With these data, we identified a susceptibility locus near TIMP3 (overall P = 1.1 × 10−11), a metalloproteinase involved in degradation of the extracellular matrix and previously implicated in early-onset maculopathy. In addition, our data revealed strong association signals with alleles at two loci (LIPC, P = 1.3 × 10−7; CETP, P = 7.4 × 10−7) that were previously associated with high-density lipoprotein cholesterol (HDL-c) levels in blood. Consistent with the hypothesis that HDL metabolism is associated with AMD pathogenesis, we also observed association with AMD of HDL-c—associated alleles near LPL (P = 3.0 × 10−3) and ABCA1 (P = 5.6 × 10−4). Multilocus analysis including all susceptibility loci showed that 329 of 331 individuals (99%) with the highest-risk genotypes were cases, and 85% of these had advanced AMD. Our studies extend the catalog of AMD associated loci, help identify individuals at high risk of disease, and provide clues about underlying cellular pathways that should eventually lead to new therapies.

Transcriptome analysis and molecular signature of human retinal pigment epithelium

Abstract: 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.

E2-2 Protein and Fuchs's Corneal Dystrophy

Abstract: Background Fuchs’s corneal dystrophy (FCD) is a leading cause of corneal transplantation and affects 5% of persons in the United States who are over the age of 40 years. Clinically visible deposits called guttae develop under the corneal endothelium in patients with FCD. A loss of endothelial cells and deposition of an abnormal extracellular matrix are observed microscopically. In advanced disease, the cornea swells and becomes cloudy because the remaining endothelial cells are not sufficient to keep the cornea dehydrated and clear. Although rare genetic variation that contributes to both early-onset and typical late-onset forms of FCD has been identified, to our knowledge, no common variants have been reported. Methods We performed a genomewide association study and replicated the most significant observations in a second, independent group of subjects. Results Alleles in the transcription factor 4 gene (TCF4), encoding a member of the E-protein family (E2-2), were associated with typical FCD (P = 2.3×10 −26 ). The association increased the odds of having FCD by a factor of 30 for persons with two copies of the disease variants (homozygotes) and discriminated between case subjects and control subjects with about 76% accuracy. At least two regions of the TCF4 locus were associated independently with FCD. Alleles in the gene encoding protein tyrosine phosphatase receptor type G (PTPRG) were associated with FCD (P = 4.0×10 −7 ), but the association did not reach genomewide significance. Conclusions Genetic variation in TCF4 contributes to the development of FCD. (Funded by the National Eye Institute and others.)

Interaction of Retinitis Pigmentosa GTPase Regulator (RPGR) with RAB8A GTPase: Implications for cilia dysfunction and photoreceptor degeneration

Abstract: Defects in biogenesis or function(s) of primary cilia are associated with numerous inherited disorders (called ciliopathies) that may include retinal degeneration phenotype. The cilia-expressed gene RPGR (retinitis pigmentosa GTPase regulator) is mutated in patients with X-linked retinitis pigmentosa (XLRP) and encodes multiple protein isoforms with a common N-terminal domain homologous to regulator of chromosome condensation 1 (RCC1), a guanine nucleotide exchange factor (GEF) for Ran GTPase. RPGR interacts with several ciliopathy proteins, such as RPGRIP1L and CEP290; however, its physiological role in cilia-associated functions has not been delineated. Here, we report that RPGR interacts with the small GTPase RAB8A, which participates in cilia biogenesis and maintenance. We show that RPGR primarily associates with the GDP-bound form of RAB8A and stimulates GDP/GTP nucleotide exchange. Disease-causing mutations in RPGR diminish its interaction with RAB8A and reduce the GEF activity. Depletion of RPGR in hTERT-RPE1 cells interferes with ciliary localization of RAB8A and results in shorter primary cilia. Our data suggest that RPGR modulates intracellular localization and function of RAB8A. We propose that perturbation of RPGR-RAB8A interaction, at least in part, underlies the pathogenesis of photoreceptor degeneration in XLRP caused by RPGR mutations.

MicroRNA profile of the developing mouse retina.

Abstract: MicroRNAs (miRNAs) are small, highly conserved, noncoding molecules of 18 to 24 nucleotides that regulate gene expression in a wide variety of tissues and cell types.1,2 The human and mouse genomes have been reported to have 885 and 689 miRNAs, respectively (miRBASE Release 13.0, March 2009; www.mirbase.org/ hosted in the public domain by the Faculty of Life Sciences, University of Manchester, Manchester, UK). miRNAs are encoded in either the introns of protein-coding genes or between genes as intergenic miRNAs. Bioinformatics studies have predicted that individual miRNAs can target hundreds of distinct mRNAs. It has been estimated that miRNAs may modulate the expression of 20% or more of the human genome.3 miRNAs are involved in a diverse set of cellular processes, ranging from proliferation, apoptosis, and malignant transformation4 to neuronal development and fate specification.5–7 Several groups have begun exploring the expression and function of miRNAs within the brain and retina. As one example, conditional inactivation of Dicer in Purkinje cells leads to a loss of miRNA expression and progressive cerebellar degeneration.8 Loss of Dicer in the retina, identified by using the floxed Dicer allele and a Chx10-driven Cre, leads initially to the formation of photoreceptor rosettes followed by progressive functional and structural degeneration.9 Dicer inactivation by morpholinos in Xenopus resulted in a more severe phenotype, with small retinas and associated lamination defects.10 A more recent study has identified miR-24a as a regulator of apoptosis during Xenopus eye development.10 To gain a more complete understanding, several groups have begun to investigate global patterns of miRNA expression in the retina, particularly in the adult, and have compared these patterns to those in other tissues, identifying a number of miRNAs enriched in retina.11,12 Other studies have focused on detailed analysis of the spatial and temporal expression patterns of a few selected miRNAs within the retina.13 Our goal in this report was to expand on this work by using microarray analysis to comprehensively characterize the retinal miRNA transcriptome during embryonic and postnatal development. In addition, we wanted to explore potential differences between the rod and cone miRNA transcriptomes. Therefore, we generated miRNA profiles of the developing retinas of wild-type (WT, C57BL/6) and Nrl−/− mice. Nrl is a basic motif-leucine zipper transcription factor that when knocked out leads to a retina in which essentially all photoreceptors are S-cones.14–16 In addition, we have compared these retinal expression patterns to miRNA profiles derived from embryonic day 15 and adult brain.

Human retinopathy-associated ciliary protein retinitis pigmentosa GTPase regulator mediates cilia-dependent vertebrate development

Abstract: Dysfunction of primary cilia is associated with tissue-specific or syndromic disorders. RPGR is a ciliary protein, mutations in which can lead to retinitis pigmentosa (RP), cone-rod degeneration, respiratory infections and hearing disorders. Though RPGR is implicated in ciliary transport, the pathogenicity of RPGR mutations and the mechanism of underlying phenotypic heterogeneity are still unclear. Here we have utilized genetic rescue studies in zebrafish to elucidate the effect of human disease-associated mutations on its function. We show that rpgr is expressed predominantly in the retina, brain and gut of zebrafish. In the retina, RPGR primarily localizes to the sensory cilium of photoreceptors. Antisense morpholino (MO)-mediated knockdown of rpgr function in zebrafish results in reduced length of Kupffer's vesicle (KV) cilia and is associated with ciliary anomalies including shortened body-axis, kinked tail, hydrocephaly and edema but does not affect retinal development. These phenotypes can be rescued by wild-type (WT) human RPGR. Several of the RPGR mutants can also reverse the MO-induced phenotype, suggesting their potential hypomorphic function. Notably, selected RPGR mutations observed in XLRP (T99N, E589X) or syndromic RP (T124fs, K190fs and L280fs) do not completely rescue the rpgr-MO phenotype, indicating a more deleterious effect of the mutation on the function of RPGR. We propose that RPGR is involved in cilia-dependent cascades during development in zebrafish. Our studies provide evidence for a heterogenic effect of the disease-causing mutations on the function of RPGR.

Autosomal Recessive Retinitis Pigmentosa with Early Macular Affectation Caused by Premature Truncation in PROM1

Abstract: Retinitis pigmentosa (RP [MIM268000]) is a genetically and clinically heterogeneous group of ocular diseases that cause rod and cone degeneration. It is characterized by night blindness, constriction of the visual field, and pigment spicule deposits in the mid periphery of the retina, which eventually lead to blindness. To date, it has been postulated that mutations in at least 60 genes may cause RP (see RetNet). RP is a major genetic cause of blindness in adults, with a worldwide prevalence of 1:3000 to 1:4000.1,2 Allelic heterogeneity stands out as a prominent feature of several RP genes, as exemplified by ABCA4,3–5 CRB1,2,6 NRL,7 RDS,8 KLHL7,9 and CEP290,10 where different mutations lead to distinct retinal disease phenotypes. In addition to RP, these genes are responsible for Stargardt disease, cone–rod dystrophy (CORD), macular degeneration, Leber congenital amaurosis (LCA), and pattern macular dystrophy, among other disorders. The wide range of clinical entities associated with these genetic variants support that the proteins encoded by many of these genes are essential for both cone and rod function, and yet each mutation produces a specific phenotypic effect. Prominin 1 (PROM1, accession number: {"type":"entrez-nucleotide","attrs":{"text":"AF027208","term_id":"2688948","term_text":"AF027208"}}AF027208, Gene ID: 8842, also known as PROML1, AC133, and CD133; GenBank; http://www.ncbi.nlm.nih.gov/Genbank/ NCBI) is located at 4p15.32 and at maximum length comprises 27 exons. The encoded protein, PROM1, is a five-transmembrane glycoprotein located at the plasma membrane protrusions, with two short N (extracellular)- and C (cytoplasmic)-terminal tails, and two large N-glycosylated extracellular loops (between TM2 and -3, and TM4 and -5). Seven PROM1 protein isoforms produced by alternative splicing have been reported in human tissues,11 although the alternatively spliced exons in the coding region only affect the short N- and the C-terminal domains. PROM1 is expressed in both rod and cone photoreceptors. Moreover, PROM1 expression has been detected in the cells of several other human tissues—among them CD34+ progenitor populations from adult blood and bone marrow cells—which has conferred on this protein the status of a valuable marker for human allogeneic transplantation.12,13 A paralogue of PROM1, PROM2, shares 60% of amino acid identity and displays the same characteristic of membrane topology.14 The pattern of PROM2 expression largely overlaps that of PROM1, except that there is no expression in the retina. PROM1 function in the retina is not known, although it is selectively associated with microvilli, making a relevant contribution to the generation of plasma membrane protrusions, their organization, and lipid composition, notably with respect to cholesterol.15 In rods, prominin appears to be concentrated in the plasma membrane evaginations at the nascent disc membranes at the base of the outer segments, which are essential structures in the biogenesis of photoreceptor discs and to which the contribution of PROM1 seems crucial.16 The gene and probably also its function are highly evolutionarily conserved. In the Drosophila melanogaster eye, prom (known as eyes closed or eyc) interacts with spacemaker (also known as spam, eyes shut, or eys) and chaoptin to regulate the assembly of microvilli, ensure the structural integrity of the rhabdomeres, and guarantee the proper construction of an open rhabdom system.17 The human homologue of spacemaker, EYS, has been characterized as responsible for autosomal recessive retinitis pigmentosa.18,19 In mice, the absence of Prom 1 provokes progressive degeneration and functional deterioration of photoreceptors, due to impaired morphogenesis of the discs at the outer segment.16,20 In humans, mutations in PROM1 have been associated with severe forms of retinal dystrophy. Missense mutations are associated with autosomal dominant Stargardt-like or bull's-eye macular dystrophy,16 whereas nonsense and frameshift mutations have been related to retinitis pigmentosa,21,22 and severe cone–rod dystrophy with macular degeneration and night blindness.23 Herein, we describe a novel recessive mutation in the PROM1 gene that is responsible for severe RP with macular degeneration and myopia in a consanguineous pedigree from Spain. The retinal degeneration in these patients seems to be associated with the loss of PROM1 function as the nonsense-mediated decay machinery leads to an almost complete depletion of the mutated transcripts.

The Retinitis Pigmentosa Protein RP2 Interacts With Polycystin 2 and Regulates Cilia-Mediated Vertebrate Development

Abstract: Ciliopathies represent a growing group of human genetic diseases whose etiology lies in defects in ciliogenesis or ciliary function Given the established entity of renal–retinal ciliopathies, we have been examining the role of cilia-localized proteins mutated in retinitis pigmentosa (RP) in regulating renal ciliogenesis or ciliadependent signaling cascades Specifically, this study examines the role of the RP2 gene product with an emphasis on renal and vertebrate development We demonstrate that in renal epithelia, RP2 localizes to the primary cilium through dual acylation of the amino-terminus We also show that RP2 forms a calcium-sensitive complex with the autosomal dominant polycystic kidney disease protein polycystin 2 Ablation of RP2 by shRNA promotes swelling of the cilia tip that may be a result of aberrant trafficking of polycystin 2 and other ciliary proteins Morpholino-mediated repression of RP2 expression in zebrafish results in multiple developmental defects that have been previously associated with ciliary dysfunction, such as hydrocephalus, kidney cysts and situs inversus Finally, we demonstrate that, in addition to our observed physical interaction between RP2 and polycystin 2, dual morpholino-mediated knockdown of polycystin 2 and RP2 results in enhanced situs inversus, indicating that these two genes also regulate a common developmental process This work suggests that RP2 may be an important regulator of ciliary function through its association with polycystin 2 and provides evidence of a further link between retinal and renal cilia function

RP2 Phenotype and Pathogenetic Correlations in X-Linked Retinitis Pigmentosa

Abstract: Objectives To assess the phenotype of patients with X-linked retinitis pigmentosa (XLRP) withRP2mutations and to correlate the findings with their genotype. Methods Six hundred eleven patients with RP were screened forRP2mutations. From this screen, 18 patients withRP2mutations were evaluated clinically with standardized electroretinography, Goldmann visual fields, and ocular examinations. In addition, 7 well-documented cases from the literature were used to augment genotype-phenotype correlations. Results Of 11 boys younger than 12 years, 10 (91%) had macular involvement and 9 (82%) had best-corrected visual acuity worse than 20/50. Two boys from different families (aged 8 and 12 years) displayed a choroideremia-like fundus, and 9 boys (82%) were myopic (mean error, −7.97 diopters [D]). Of 10 patients with electroretinography data, 9 demonstrated severe rod-cone dysfunction. All 3 female carriers had macular atrophy in 1 or both eyes and were myopic (mean, −6.23 D). All 9 nonsense and frameshift and 5 of 7 missense mutations (71%) resulted in severe clinical presentations. Conclusions Screening of theRP2gene should be prioritized in patients younger than 16 years characterized by X-linked inheritance, decreased best-corrected visual acuity (eg, >20/40), high myopia, and early-onset macular atrophy. Patients exhibiting a choroideremia-like fundus without choroideremia gene mutations should also be screened forRP2mutations. Clinical Relevance An identifiable phenotype for RP2-XLRP aids in clinical diagnosis and targeted genetic screening.

Age-related macular degeneration-associated variants at chromosome 10q26 do not significantly alter ARMS2 and HTRA1 transcript levels in the human retina.

Abstract: PURPOSE Multiple studies demonstrate a strong association between three variants at chromosome 10q26 - rs10490924, del443ins54, and rs11200638 - near the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HTRA1) genes with susceptibility to age-related macular degeneration (AMD). In different reports, the del443ins54 and rs11200638 variants are suggested to affect ARMS2 mRNA stability and/or HTRA1 mRNA expression, respectively. The goal of this study is to examine whether these AMD-associated variants alter expression levels of ARMS2 and HTRA1 in human retina samples. METHODS Genomic DNA and total RNA were obtained from 35 human retinas (three young controls, average age=32 years; twenty aged controls, average age=72 years; and twelve AMD retinas, average age=77 years) using standard procedures. As ARMS2 exhibits higher expression in the human placenta, we also included eighteen placenta samples in our analysis. Four polymorphisms - rs2736911, rs10490924, del443ins54, and rs11200638 - were genotyped by PCR followed by sequencing. Expression of ARMS2, HTRA1 and three endogenous control genes (rRNA [rRNA], hypoxanthine phosphoribosyltransferase 1 [HPRT1], and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) was measured by real-time quantitative RT-PCR using Taqman gene expression or SYBR Green assays. RESULTS ARMS2 and HTRA1 mRNA levels did not show a significant difference in expression among the control (young and elderly) and AMD retinas. No association of del443ins54 and rs11200638 variants was detected with mRNA expression levels of ARMS2 or HTRA1 in the retina. Human placenta samples showed high variability in expression levels. CONCLUSIONS We did not find association between AMD susceptibility variants at 10q26 and steady-state expression levels of either ARMS2 or HTRA1 in the human retina.

Associations of CFHR1-CFHR3 deletion and a CFH SNP to age-related macular degeneration are not independent.

Abstract: Associations of CFHR1–CFHR3 deletion and a CFH SNP to age-related macular degeneration are not independent

Loss of lysophosphatidylcholine acyltransferase 1 leads to photoreceptor degeneration in rd11 mice

Abstract: Retinal degenerative diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are a leading cause of untreatable blindness with substantive impact on the quality of life of affected individuals and their families. Mouse mutants with retinal dystrophies have provided a valuable resource to discover human disease genes and helped uncover pathways critical for photoreceptor function. Here we show that the rd11 mouse mutant and its allelic strain, B6-JR2845, exhibit rapid photoreceptor dysfunction, followed by degeneration of both rods and cones. Using linkage analysis, we mapped the rd11 locus to mouse chromosome 13. We then identified a one-nucleotide insertion (c.420–421insG) in exon 3 of the Lpcat1 gene. Subsequent screening of this gene in the B6-JR2845 strain revealed a seven-nucleotide deletion (c.14–20delGCCGCGG) in exon 1. Both sequence changes are predicted to result in a frame-shift, leading to premature truncation of the lysophosphatidylcholine acyltransferase-1 (LPCAT1) protein. LPCAT1 (also called AYTL2) is a phospholipid biosynthesis/remodeling enzyme that facilitates the conversion of palmitoyl-lysophosphatidylcholine to dipalmitoylphosphatidylcholine (DPPC). The analysis of retinal lipids from rd11 and B6-JR2845 mice showed substantially reduced DPPC levels compared with C57BL/6J control mice, suggesting a causal link to photoreceptor dysfunction. A follow-up screening of LPCAT1 in retinitis pigmentosa and Leber congenital amaurosis patients did not reveal any obvious disease-causing mutations. Previously, LPCAT1 has been suggested to be critical for the production of lung surfactant phospholipids and biosynthesis of platelet-activating factor in noninflammatory remodeling pathway. Our studies add another dimension to an essential role for LPCAT1 in retinal photoreceptor homeostasis.

Two novel CRX mutant proteins causing autosomal dominant Leber congenital amaurosis interact differently with NRL.

Abstract: Leber congenital amaurosis (LCA) is a congenital retinal dystrophy characterized by severe visual loss in infancy and nystagmus. Although most often inherited in an autosomal recessive fashion, rare individuals with mutations in the cone-rod homeobox gene, CRX, have dominant disease. CRX is critical for photoreceptor development and acts synergistically with the leucine-zipper transcription factor, NRL. We report on the phenotype of two individuals with LCA due to novel, de novo CRX mutations, c.G264T(p.K88N) and c.413delT(p.I138fs48), that reduce transactivation in vitro to 10% and 30% of control values, respectively. Whereas the c.413delT(p.I138fs48) mutant allows co-expressed NRL to transactivate independently at its normal, baseline level, the c.G264T(p.K88N) mutant reduces co-expressed NRL transactivation and reduces steady state levels of both proteins. Although both mutant proteins predominantly localize normally to the nucleus, they also both show variable cytoplasmic localization. These observations suggest that some CRX-mediated LCA may result from effects beyond haploinsufficiency, such as the mutant protein interefering with other transcription factors' function. Such patients would therefore not likely benefit from a simple, gene-replacement strategy for their disease. © 2010 Wiley-Liss, Inc.

Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases

Abstract: Background Advanced age contributes to clinical manifestations of many retinopathies and represents a major risk factor for age-related macular degeneration, a leading cause of visual impairment and blindness in the elderly. Rod photoreceptors are especially vulnerable to genetic defects and changes in microenvironment, and are among the first neurons to die in normal aging and in many retinal degenerative diseases. The molecular mechanisms underlying rod photoreceptor vulnerability and potential biomarkers of the aging process in this highly specialized cell type are unknown. Methodology/Principal Findings To discover aging-associated adaptations that may influence rod function, we have generated gene expression profiles of purified rod photoreceptors from mouse retina at young adult to early stages of aging (1.5, 5, and 12 month old mice). We identified 375 genes that showed differential expression in rods from 5 and 12 month old mouse retina compared to that of 1.5 month old retina. Quantitative RT-PCR experiments validated expression change for a majority of the 25 genes that were examined. Macroanalysis of differentially expressed genes using gene class testing and protein interaction networks revealed overrepresentation of cellular pathways that are potentially photoreceptor-specific (angiogenesis and lipid/retinoid metabolism), in addition to age-related pathways previously described in several tissue types (oxidative phosphorylation, stress and immune response). Conclusions/Significance Our study suggests a progressive shift in cellular homeostasis that may underlie aging-associated functional decline in rod photoreceptors and contribute to a more permissive state for pathological processes involved in retinal diseases.

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

Abstract: 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 novoinsertion after nucleotide 173 in exon ORF15 of RPGR. 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. 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. This insertion mutation in the RPGRexon 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 RPGRmutations 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.

Multiprotein complexes of Retinitis Pigmentosa GTPase regulator (RPGR), a ciliary protein mutated in X-linked Retinitis Pigmentosa (XLRP).

Abstract: Mutations in Retinitis Pigmentosa GTPase Regulator (RPGR) are a frequent cause of X-linked Retinitis Pigmentosa (XLRP). The RPGR gene undergoes extensive alternative splicing and encodes for distinct protein isoforms in the retina. Extensive studies using isoform-specific antibodies and mouse mutants have revealed that RPGR predominantly localizes to the transition zone to primary cilia and associates with selected ciliary and microtubule-associated assemblies in photoreceptors. In this chapter, we have summarized recent advances on understanding the role of RPGR in photoreceptor protein trafficking. We also provide new evidence that suggests the existence of discrete RPGR multiprotein complexes in photoreceptors. Piecing together the RPGR-interactome in different subcellular compartments should provide critical insights into the role of alternative RPGR isoforms in associated orphan and syndromic retinal degenerative diseases.

Phenotype associated with mutation in the recently identified autosomal dominant retinitis pigmentosa KLHL7 gene.

Abstract: OBJECTIVE: To characterize the clinical phenotype, with an emphasis on electrophysiologic findings, in a family with autosomal dominant retinitis pigmentosa caused by mutation in the recently identified KLHL7 gene. METHODS: Eleven patients from a single family were selected from the Swedish retinitis pigmentosa register. Four patients had been examined 13 to 17 years earlier and underwent further ophthalmologic examination, including visual acuity, fundus inspection, Goldmann perimetry, full-field electroretinography (ERG), multifocal ERG, and optical coherence tomography. KLHL7 mutation was identified by sequence analysis. RESULTS: In most examined family members, the fundus showed minor abnormalities. Full-field ERG demonstrated reduced cone and rod function, but rod responses were preserved in some patients late in life. Follow-up ( T) in 7 family members. CONCLUSIONS: Observed in 2 Scandinavian families to date, KLHL7 mutation has recently been associated with autosomal dominant retinitis pigmentosa. Clinical examination with long-term follow-up verified a phenotype with a varying degree of retinal photoreceptor dysfunction and, in some family members, with late onset and preserved rod function until late in life. Clinical Relevance Patients with minor retinal abnormalities and normal ERG findings early in life can harbor an autosomal dominant form of retinitis pigmentosa with a varying degree of visual impediment. Some patients with late onset may retain night vision for many years. (Less)

Compositions and methods for diagnosing and treating macular degeneration

Abstract: The present invention relates generally to biomarkers for macular degeneration. In particular, the present invention provides a plurality of biomarkers for monitoring and diagnosing macular degeneration. The compositions and methods of the present invention find use in diagnostic, therapeutic, research, and drug screening applications.

Two Novel CRX Mutant Proteins Causing Autosomal Dominant Leber Congenital Amaurosis (LCA) Interact Differently With NRL

Abstract: Leber congenital amaurosis (LCA) is a congenital retinal dystrophy characterized by severe visual loss in infancy and nystagmus. Although most often inherited in an autosomal recessive fashion, rare individuals with mutations in the cone‐rod homeobox gene, CRX, have dominant disease. CRX is critical for photoreceptor development and acts synergistically with the leucine‐zipper transcription factor, NRL. We report on the phenotype of two individuals with LCA due to novel, de novo CRX mutations, c.G264T(p.K88N) and c.413delT(p.I138fs48), that reduce transactivation in vitro to 10% and 30% of control values, respectively. Whereas the c.413delT(p.I138fs48) mutant allows co‐expressed NRL to transactivate independently at its normal, baseline level, the c.G264T(p.K88N) mutant reduces co‐expressed NRL transactivation and reduces steady state levels of both proteins. Although both mutant proteins predominantly localize normally to the nucleus, they also both show variable cytoplasmic localization. These observations suggest that some CRX‐mediated LCA may result from effects beyond haploinsufficiency, such as the mutant protein interefering with other transcription factors' function. Such patients would therefore not likely benefit from a simple, gene‐replacement strategy for their disease. © 2010 Wiley‐Liss, Inc.

Photoreceptor Development: Early Steps/Fate

Abstract: This article follows the developmental events in the mammalian retina leading to the generation of photoreceptors from distinct pools of retinal progenitor cells (RPCs). We describe cell-fate specification, determination, and maturation with a focus on human photoreceptors for their relevance to blinding diseases and on the mouse as a widely used model to investigate mammalian photoreceptor development.