Showing papers by "Anand Swaroop published in 2003"

Mutation in a short-chain collagen gene, CTRP5, results in extracellular deposit formation in late-onset retinal degeneration - a genetic model for age-related macular degeneration

Abstract: A primary feature of age-related macular degeneration (AMD) is the presence of extracellular deposits between the retinal pigment epithelium (RPE) and underlying Bruch’s membrane, leading to RPE dysfunction, photoreceptor death and severe visual loss. AMD accounts for about 50% of blind registrations in Western countries and is a common, genetically complex disorder. Very little is known regarding its molecular basis. Late-onset retinal degeneration (L-ORD) is an autosomal dominant disorder with striking clinical and pathological similarity to AMD. Here we show that L-ORD is genetically heterogeneous and that a proposed founder mutation in the CTRP5 (C1QTNF5) gene, which encodes a novel short-chain collagen, changes a highly conserved serine to arginine (Ser163Arg) in 7/14 L-ORD families and 0/1000 control individuals. The mutation occurs in the gC1q domain of CTRP5 and results in abnormal high molecular weight aggregate formation which may alter its higher-order structure and interactions. These results indicate a novel disease mechanism involving abnormal adhesion between RPE and Bruch’s membrane.

A comprehensive analysis of the expression of crystallins in mouse retina.

Abstract: Crystallins constitute a diverse group of proteins that areexpressed at high concentrations in the differentiated lens fi-ber cells and augment the refractive power of the transparentlens tissue [1,2]. In vertebrates, three major classes ofcrystallins, α, β, and γ, accumulate in the lens in a spatiallyand temporally regulated manner [3-5]. Their expression in-creases dramatically during differentiation of lens epithelialcells into fibers [1]. The two α-crystallins (αA and αB) be-long to the small heat shock protein family of molecular chap-erones and appear very early during mouse embryonic devel-opment [6,7]. Members of the β/γ-superfamily, which includeβ-crystallins (βA1/A3, βA2, βA4, βB1, βB2 and βB3) and γ-crystallins (γA-F, and γS, formerly βS), are related to micro-bial proteins induced by physiological stress [8,9]. In addi-tion, a growing number of crystallins (known as taxon-spe-cific enzyme-crystallins) are expressed at relatively high lev-els in the lens but only in selected species; these proteins in-clude µ, ζand λ-crystallins that are closely related to meta-bolic enzymes ornithine cyclodeaminase, NADPH:quinoneoxidoreductase and hydroxyl CoA dehydrogenase, respectively[3,10].Originally considered to be static, abundant proteins pro-viding transparency to the lens, it is now generally acceptedthat crystallins were selected from proteins with entirely dif-ferent non-lens roles and are retained in multiple tissues ofthe same organism [6]. Bhat and colleagues were the first todemonstrate the extra-lenticular expression of a crystallin [11].Further studies revealed the presence of αB-crystallin in nu-merous tissues and its increased accumulation in neurologicaldisorders [6,12-14]. Later, it was demonstrated that αA andαB-crystallins have chaperone-like activity [15], are phospho-rylated in vivo and possess autokinase activity [16,17], inter-act with cytoskeleton [18], and protect cells from thermal andmetabolic stress [19]. Furthermore, their ability to preventapoptosis by inhibiting caspases indicates that αA and αB-crystallins have more general physiological functions in non-lens tissues [20].

GRK1-Dependent Phosphorylation of S and M Opsins and Their Binding to Cone Arrestin during Cone Phototransduction in the Mouse Retina

Abstract: The shutoff mechanisms of the rod visual transduction cascade involve G-protein-coupled receptor (GPCR) kinase 1 (GRK1) phosphorylation of light-activated rhodopsin (R*) followed by rod arrestin binding. Deactivation of the cone phototransduction cascade in the mammalian retina is delineated poorly. In this study we sought to explore the potential mechanisms underlying the quenching of the phototransduction cascade in cone photoreceptors by using mouse models lacking rods and/or GRK1. Using the “pure-cone” retinas of the neural retina leucine zipper (Nrl) knock-out (KO, -/-) mice ([Mears et al., 2001][1]), we have demonstrated the light-dependent, multi-site phosphorylation of both S and M cone opsins by in situ phosphorylation and isoelectric focusing. Immunoprecipitation with affinity-purified polyclonal antibodies against either mouse cone arrestin (mCAR) or mouse S and M cone opsins revealed specific binding of mCAR to light-activated, phosphorylated cone opsins. To elucidate the potential role of GRK1 in cone opsin phosphorylation, we created Nrl and Grk1 double knock-out (Nrl-/-Grk1-/-) mice by crossing the Nrl-/- mice with Grk1-/- mice ([Chen et al., 1999][2]). We found that, in the retina of these mice, the light-activated cone opsins were neither phosphorylated nor bound with mCAR. Our results demonstrate, for the first time in a mammalian species, that cone opsins are phosphorylated and that CAR binds to phosphorylated cone opsins after light activation. [1]: #ref-29 [2]: #ref-8

Clinical and immunohistochemical evidence for an X linked retinitis pigmentosa syndrome with recurrent infections and hearing loss in association with an RPGR mutation

Abstract: Retinitis pigmentosa (RP) is the most common form of hereditary retinal degeneration, affecting approximately 1 in 3500 individuals.1,2 Classical RP is characterised by progressive night blindness and constriction of the peripheral visual fields, ultimately causing deterioration of the central vision in many patients. These symptoms are accompanied by ophthalmoscopically detectable degenerative and pigmentary changes of the retinal tissue and by reductions of the electrical retinal response to flashes of light using an electroretinogram (ERG). ERG abnormalities are typically present before any detectable retinal change becomes visible to clinical examination. RP can be transmitted by all inheritance modes, with X linked recessive RP (XLRP) accounting for 10–20% of genetically identifiable cases and being reportedly among the most severe forms.1,2 To date, two of the genes responsible for XLRP have been cloned: RP2 3 and the retinitis pigmentosa GTPase regulator, RPGR .4,5 RPGR accounts for the majority of XLRP.6,7 RP or RP-like retinopathies can also be part of syndromic conditions—that is, associated with extra-ocular manifestations. The most common are Bardet-Biedl and Usher syndromes.1,8–11 Usher syndrome is characterised by the association of RP with sensorineural hearing loss of variable severity, and has three broad clinical phenotypes, with types I and II being the most common.9,11 Type I is characterised by profound congenital non-progressive hearing loss, marked speech impediment, and vestibular dysfunction. The hearing loss of Usher syndrome type II is typically mild to moderate, limited to high frequencies, non-progressive in nature, associated with speech abnormalities commensurate to the hearing defect, and with normal or minimally abnormal vestibular function. Usher syndrome is inherited as an autosomal recessive trait, although a pseudo-dominant Usher syndrome-like phenotype due to a mutation in the mitochondrial MTTS2 gene has been described.12,13 Some reports have …

Annotation and analysis of 10,000 expressed sequence tags from developing mouse eye and adult retina

Abstract: Background: As a biomarker of cellular activities, the transcriptome of a specific tissue or cell type during development and disease is of great biomedical interest. We have generated and analyzed 10,000 expressed sequence tags (ESTs) from three mouse eye tissue cDNA libraries: embryonic day 15.5 (M15E) eye, postnatal day 2 (M2PN) eye and adult retina (MRA). Results: Annotation of 8,633 non-mitochondrial and non-ribosomal high-quality ESTs revealed that 57% of the sequences represent known genes and 43% are unknown or novel ESTs, with M15E having the highest percentage of novel ESTs. Of these, 2,361 ESTs correspond to 747 unique genes and the remaining 6,272 are represented only once. Phototransduction genes are preferentially identified in MRA, whereas transcripts for cell structure and regulatory proteins are highly expressed in the developing eye. Map locations of human orthologs of known genes uncovered a high density of ocular genes on chromosome 17, and identified 277 genes in the critical regions of 37 retinal disease loci. In silico expression profiling identified 210 genes and/or ESTs over-expressed in the eye; of these, more than 26 are known to have vital retinal function. Comparisons between libraries provided a list of temporally regulated genes and/or ESTs. A few of these were validated by qRT-PCR analysis. Conclusions: Our studies present a large number of potentially interesting genes for biological investigation, and the annotated EST set provides a useful resource for microarray and functional genomic studies.

Interaction of retinal bZIP transcription factor NRL with Flt3-interacting zinc-finger protein Fiz1: possible role of Fiz1 as a transcriptional repressor

Abstract: NRL (neural retina leucine zipper) is a basic motif leucine zipper transcription factor of the Maf-subfamily. Multiple phosphorylated isoforms of NRL are detected specifically in rod photoreceptors. NRL regulates the expression of several rod-specific genes, including rhodopsin and cGMP phosphodiesterase b-subunit, in synergy with other transcription factors (e.g. the homeodomain protein CRX). Missense mutations in the human NRL gene are associated with autosomal dominant retinitis pigmentosa, whereas the loss of its function leads to rodless retina in Nrl-knockout mice that exhibit enhanced S-cone function. To further elucidate the molecular mechanism(s) underlying NRL-mediated transcriptional regulation, we used yeast two-hybrid screening to isolate NRL-interacting proteins in the retina and report the identification of Flt3interacting zinc-finger protein, Fiz1. Interaction of Fiz1 and NRL-leucine zipper was validated by GST pulldown assays and co-immunoprecipitation from bovine retinal nuclear extracts. Fiz1 suppressed NRL- but not CRXmediated transactivation of rhodopsin promoter activity in transiently transfected CV1 cells. The mRNA and the protein for both Fiz1 and its only other known interacting protein Flt3, a receptor tyrosine kinase, are expressed in the retina. Our results indicate potential cross-talk among signaling pathways in the retina and suggest that the function of NRL is modulated by its interaction with specific repressor proteins.

De novo mutation in the RP1 gene (Arg677ter) associated with retinitis pigmentosa.

Abstract: PURPOSE. The Arg677ter mutation in the RP1 gene is one of the most common causes of autosomal dominant retinitis pigmentosa (RP). In the current study, a de novo Arg677ter RP1 gene mutation was identified in a patient with RP. METHODS. RP1 gene mutation screening was performed in probands with simplex RP. In one proband with the RP1 mutation, paternity was established by analyzing 24 short tandem repeat polymorphisms. Additional candidate RP genes, including rhodopsin, RDS/peripherin, RP2, and RPGR, were also examined in this proband. Phenotype was characterized with psychophysics, electroretinography, and optical coherence tomography. RESULTS. An RP1 (Arg677ter) mutation was identified in one of the patients with simplex RP, but the sequence change was not detected in his parents. Parentage was confirmed, and other candidate genes were negative for mutations. Retinal function and cross-sectional imaging studies in the patient indicated greater rod than cone dysfunction with a photoreceptor basis for the abnormalities. CONCLUSIONS. The de novo origin of an RP1 (Arg677ter) mutation in a patient with simplex RP suggests that this common autosomal dominant RP mutation can arise independently in the population and supports the hypothesis of a mutational hotspot in the RP1 gene. (Invest Ophthalmol Vis Sci. 2003;44: 3593‐3597) DOI:10.1167/iovs.03-0155

Clinical studies of X-linked retinitis pigmentosa in three Swedish families with newly identified mutations in the RP2 and RPGR-ORF15 genes

Abstract: PURPOSE: To describe new disease-causing RP2 and RPGR-ORF15 mutations and their corresponding clinical phenotypes in Swedish families with X-linked retinitis pigmentosa (XLRP) and to establish genotype-phenotype correlations by studying the clinical spectrum of disease in families with a known molecular defect. METHODS: Seventeen unrelated families with RP and an apparent X-linked pattern of disease inheritance were identified from the Swedish RP registry and screened for mutations in the RP2 and RPGR (for the RP3 disease) genes. These families had been previously screened for the RPGR exons 1-19, and disease-causing mutations were identified in four of them. In the remaining 13 families, we sequenced the RP2 gene and the newly discovered RPGR-ORF exon. Detailed clinical evaluations were then obtained from individuals in the three families with identified mutations. RESULTS: Mutations in RP2 and RPGR-ORF15 were identified in three of the 13 families. Clinical evaluations of affected males and carrier females demonstrated varying degrees of retinal dysfunction and visual handicap, with early onset and severe disease in the families with mutations in the ORF15 exon of the RPGR gene. CONCLUSIONS: A total of seven mutations in the RP2 and RPGR genes have been discovered so far in Swedish XLRP families. All affected individuals express a severe form of retinal degeneration with visual handicap early in life, although the degree of retinal dysfunction varies both in hemizygous male patients and in heterozygous carrier females. Retinal disease phenotypes in patients with mutations in the RPGR-ORF15 were more severe than in patients with mutations in RP2 or other regions of the RPGR. (Less)

Mutation screening of patients with Leber Congenital Amaurosis or the enhanced S-Cone Syndrome reveals a lack of sequence variations in the NRL gene.

Abstract: Purpose: To determine if mutations in the retinal transcription factor gene NRL are associated with retinopathies other than autosomal dominant retinitis pigmentosa (adRP). Methods: Genomic DNA was isolated from blood samples obtained from 50 patients with Leber Congenital Amaurosis (LCA), 17 patients with the Enhanced S-Cone Syndrome (ESCS), and a patient with an atypical retinal degeneration that causes photoreceptor rosettes with blue cone opsin. The 5' upstream region (putative promoter), untranslated exon 1, coding exons 2 and 3, and exon-intron boundaries of the NRL gene were analyzed by direct sequencing of the PCR-amplified products. Results: Complete sequencing of the NRL gene in DNA samples from this cohort of patients revealed only one nucleotide change. The C->G transversion at nucleotide 711 of NRL exon 3 was detected in one LCA patient; however, this change did not alter the amino acid (L237L). Conclusions: No potential disease causing mutation was identified in the NRL gene in patients with LCA, ESCS, or the atypical retinal degeneration. Together with previous studies, our results demonstrate that mutations in the NRL gene are not a major cause of retinopathy. To date, only missense changes have been reported in adRP patients, and sequence variations are rare. It is possible that the loss of NRL function in humans is associated with a more complex clinical phenotype due to its expression in pineal gland in addition to rod photoreceptors. (Less)