Photoreceptor Compartment-Specific TULP1 Interactomes.

Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach in the current study was to physically isolate Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between Tulp1 and the scaffold protein Ribeye, and (3) an interaction between Tulp1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold.

A Novel Approach to Identify Photoreceptor Compartment-Specific Tulp1 Binding Partners.

Photoreceptors (PRs) are highly polarized and compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. The PR-specific protein, Tulp1, is localized to the IS and synapse and is hypothesized to be involved in protein trafficking. To better understand the molecular processes that regulate protein trafficking in PRs, we aimed to identify compartment-specific Tulp1 binding partners. Serial tangential sectioning of Long Evans rat retinas was utilized to isolate the IS and synaptic PR compartments. Tulp1 binding partners in each of these layers were identified using co-immunoprecipitation (co-IP) with Tulp1 antibodies. The co-IP eluates were separated by SDS-PAGE, trypsinized into peptide fragments, and proteins were identified by liquid chromatography tandem mass spectrometry. In the IS, potential Tulp1-binding partners included cytoskeletal scaffold proteins, protein trafficking molecules, as well as members of the phototransduction cascade. In the synaptic region, the majority of interacting proteins identified were cytoskeletal. A separate subset of proteins were identified in both the IS and synapse including chaperones and family members of the GTPase activating proteins. Tulp1 has two distinct PR compartment-specific interactomes. Our results support the hypothesis that Tulp1 is involved in the trafficking of proteins from the IS to the OS and the continuous membrane remodeling and vesicle cycling at the synaptic terminal.

Interaction of tubby-like protein-1 (Tulp1) and microtubule-associated protein (MAP) 1A and MAP1B in the mouse retina.

Tubby-like protein-1 (Tulp1) is a photoreceptor-specific protein involved in the transport of specific proteins from the inner segment (IS) to the outer segment (OS) in photoreceptor cells. Mutations in the human TULP1 gene cause an early onset form of retinitis pigmentosa. Our previous work has shown an association between Tulp1 and the microtubule-associated protein, MAP1B. An allele of Mtap1a, which encodes the MAP1A protein, significantly delays photoreceptor degeneration in Tulp1 mutant mice. MAP1 proteins are important in stabilizing microtubules in neuronal cells, but their role in photoreceptors remains obscure. To investigate the relationship between Tulp1 and MAP1 proteins, we performed western blots, immunoprecipitations (IP), immunohistochemistry and proximity ligand assays (PLA) in wild-type and tulp1-/- mouse retinas. Our IP experiments provide evidence that Tulp1 and MAP1B interact while PLA experiments localize their interaction to the outer nuclear layer and IS of photoreceptors. Although MAP1A and MAP1B protein levels are not affected in the tulp1-/- retina, they are no longer localized to the OS of photoreceptors. This may be the cause for disorganized OSs in tulp1-/- mice, and indicate that their transport to the OS is Tulp1-dependent.

VEGFA and VEGFR2 gene polymorphisms and response to anti-vascular endothelial growth factor therapy: comparison of age-related macular degeneration treatments trials (CATT).

IMPORTANCE: Individual variation in response and duration of anti-vascular endothelial growth factor (VEGF) therapy is seen among patients with neovascular age-related macular degeneration. Identification of genetic markers that affect clinical response may result in optimization of anti-VEGF therapy. OBJECTIVE: To evaluate the pharmacogenetic relationship between genotypes of single-nucleotide polymorphisms (SNPs) in the VEGF signaling pathway and response to treatment with ranibizumab or bevacizumab for neovascular age-related macular degeneration. DESIGN, SETTING, AND PARTICIPANTS: In total, 835 of 1149 patients (72.7%) participating in the Comparison of Age-Related Macular Degeneration Treatments Trials (CATT) at 43 CATT clinical centers. INTERVENTION: Each patient was genotyped for 7 SNPs in VEGFA (rs699946, rs699947, rs833069, rs833070, rs1413711, rs2010963, and rs2146323) and 1 SNP in VEGFR2 (rs2071559) using TaqMan SNP genotyping assays. MAIN OUTCOMES AND MEASURES: Genotypic frequencies were compared with clinical measures of response to therapy at 1 year, including the mean visual acuity, mean change in visual acuity, at least a 15-letter increase, retinal thickness, mean change in total foveal thickness, presence of fluid on optical coherence tomography, presence of leakage on fluorescein angiography, mean change in lesion size, and mean number of injections administered. Differences in response by genotype were evaluated with tests of linear trend calculated from logistic regression models for categorical outcomes and linear regression models for continuous outcomes. The method of controlling the false discovery rate was used to adjust for multiple comparisons. RESULTS: For each of the measures of visual acuity evaluated, no association was observed with any of the genotypes or with the number of risk alleles. Four VEGFA SNPs demonstrated an association with retinal thickness: rs699947 (P = .03), rs833070 (P = .04), rs1413711 (P = .045), and rs2146323 (P = .006). However, adjusted P values for these associations were all statistically nonsignificant (range, P = .24 to P = .45). Among the participants in 2 as-needed groups, no association was found in the number of injections among the different genotypes or for the total number of risk alleles. The effect of risk alleles on each clinical measure did not differ by treatment group, drug, or dosing regimen (P > .01 for all). CONCLUSIONS AND RELEVANCE: This study provides evidence that no pharmacogenetic associations exist between the studied VEGFA and VEGFR2 SNPs and response to anti-VEGF therapy. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00593450.

Identification of three ABCA4 sequence variations exclusive to African American patients in a cohort of patients with Stargardt disease.

PURPOSE: To describe the clinical and molecular findings in ten unrelated African American patients with Stargardt disease. DESIGN: Retrospective, observational case series. METHODS: We reviewed the clinical histories, examinations, and genotypes of 85 patients with molecular diagnoses of Stargardt disease. Three ABCA4 sequence variations identified exclusively in African Americans were evaluated in 300 African American controls and by in silico analysis. RESULTS: ABCA4 sequence changes were identified in 85 patients from 80 families, of which 11 patients identified themselves as African American. Of these 11 patients, 10 unrelated patients shared 1 of 3 ABCA4 sequence variations: c.3602T>G (p.L1201R); c.3899G>A (p.R1300Q); or c.6320G>A (p.R2107H). The minor allele frequencies in the African American control population for each variation were 7.5%, 6.3%, and 2%, respectively. This is comparable to the allele frequency in African Americans in the Exome Variant Server. In contrast, the allele frequency of all three of these variations was less than or equal to 0.05% in European Americans. Although both c.3602T>G and c.3899G>A have been reported as likely disease-causing variations, one of our control patients was homozygous for each variant, suggesting that these are nonpathogenic. In contrast, the absence of c.6320G>A in the control population in the homozygous state, combined with the results of bioinformatics analysis, support its pathogenicity. CONCLUSIONS: Three ABCA4 sequence variations were identified exclusively in 10 unrelated African American patients: p.L1201R and p.R1300Q likely represent nonpathogenic sequence variants, whereas the p.R2107H substitution appears to be pathogenic. Characterization of population-specific disease alleles may have important implications for the development of genetic screening algorithms.

Retinal deimination and PAD2 levels in retinas from donors with age-related macular degeneration (AMD).

Deimination is a form of protein posttranslational modification carried out by the peptidyl arginine deiminases (PADs) enzymes. PAD2 is the principal deiminase expressed in the retina. Elevated levels of PAD2 and protein deimination are present in a number of human neurological diseases, with or without ocular manifestation. To define the association of deimination with the pathogenesis of age-related macular degeneration (AMD), we studied protein deimination and PAD2 levels in retinas of AMD donor eyes compared to age-matched non-AMD retinas. Eyes from non-AMD and AMD donors were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer. Retina and retinal pigment epithelium (RPE) from donor eyes were processed for immunohistochemical detection and western blotting using antibodies to PAD2 and citrulline residues. The ganglion cell, inner plexiform, inner nuclear and outer nuclear layers were labeled by both PAD2 and citrulline antibodies. Changes in the localization of deiminated residues and PAD2 were evident as the retinal layers were remodeled coincident with photoreceptor degeneration in AMD retinas. Immunodetection of either PAD2 or citrulline residues could not be evaluated in the RPE layer due to the high autofluorescence levels in this layer. Interestingly, higher deimination immunoreactivity was detected in AMD retinal lysates. However, no significant changes in PAD2 were detected in the AMD and non-AMD retinas and RPE lysates. Our observations show increased levels of protein deimination but not PAD2 in AMD retinas and RPE, suggesting a reduced rate of turnover of deiminated proteins in these AMD retinas.

Pharmacogenetics for genes associated with age-related macular degeneration in the Comparison of AMD Treatments Trials (CATT).

PURPOSE: To evaluate the pharmacogenetic relationship between genotypes of single nucleotide polymorphisms (SNPs) known to be associated with age-related macular degeneration (AMD) and response to treatment with ranibizumab (Lucentis; Genentech, South San Francisco, CA) or bevacizumab (Avastin; Genentech) for neovascular AMD. DESIGN: Clinical trial. PARTICIPANTS: Eight hundred thirty-four (73%) of 1149 patients participating in the Comparison of AMD Treatments Trials (CATT) were recruited through 43 CATT clinical centers. METHODS: Each patient was genotyped for SNPs rs1061170 (CFH), rs10490924 (ARMS2), rs11200638 (HTRA1), and rs2230199 (C3), using TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA). MAIN OUTCOMES MEASURES: Genotypic frequencies were compared with clinical measures of response to therapy at one year, including mean visual acuity (VA), mean change in VA, 15-letter or more increase in VA, retinal thickness, mean change in total foveal thickness, presence of fluid on OCT, presence of leakage on fluorescein angiography (FA), mean change in lesion size, and mean number of injections administered. Differences in response by genotype were evaluated with tests of linear trend calculated from logistic regression models for categorical outcomes and linear regression models for continuous outcomes. To adjust for multiple comparisons, P≤0.01 was considered statistically significant. RESULTS: No statistically significant differences in response by genotype were identified for any of the clinical measures studied. Specifically, there were no high-risk alleles that predicted final VA or change in VA, the degree of anatomic response (fluid on OCT or FA, retinal thickness, change in total foveal thickness, change in lesion size), or the number of injections. Furthermore, a stepwise analysis failed to show a significant epistatic interaction among the variants analyzed; that is, response did not vary by the number of risk alleles present. The lack of association was similar whether patients were treated with ranibizumab or bevacizumab or whether they received monthly or pro re nata dosing. CONCLUSIONS: Although specific alleles for CFH, ARMS2, HTRA1, and C3 may predict the development of AMD, they did not predict response to anti-vascular endothelial growth factor therapy.

Immunocytochemical evidence of Tulp1-dependent outer segment protein transport pathways in photoreceptor cells.

Tulp1 is a protein of unknown function exclusive to rod and cone photoreceptor cells. Mutations in the gene cause autosomal recessive retinitis pigmentosa in humans and photoreceptor degeneration in mice. In tulp1-/- mice, rod and cone opsins are mislocalized, and rhodopsin-bearing extracellular vesicles accumulate around the inner segment, indicating that Tulp1 is involved in protein transport from the inner segment to the outer segment. To investigate this further, we sought to define which outer segment transport pathways are Tulp1-dependent. We used immunohistochemistry to examine the localization of outer segment proteins in tulp1-/- photoreceptors, prior to retinal degeneration. We also surveyed the condition of inner segment organelles and rhodopsin transport machinery proteins. Herein, we show that guanylate cyclase 1 and guanylate cyclase activating proteins 1 and 2 are mislocalized in the absence of Tulp1. Furthermore, arrestin does not translocate to the outer segment in response to light stimulation. Additionally, data from the tulp1-/- retina adds to the understanding of peripheral membrane protein transport, indicating that rhodopsin kinase and transducin do not co-transport in rhodopsin carrier vesicles and phosphodiesterase does not co-transport in guanylate cyclase carrier vesicles. These data implicate Tulp1 in the transport of selective integral membrane outer segment proteins and their associated proteins, specifically, the opsin and guanylate cyclase carrier pathways. The exact role of Tulp1 in outer segment protein transport remains elusive. However, without Tulp1, two rhodopsin transport machinery proteins exhibit abnormal distribution, Rab8 and Rab11, suggesting a role for Tulp1 in vesicular docking and fusion at the plasma membrane near the connecting cilium.

Tubby-like protein 1 (Tulp1) is required for normal photoreceptor synaptic development.

Mutations in the photoreceptor-specific tubby-like protein 1 (TULP1) underlie a form of autosomal recessive retinitis pigmentosa in humans and photoreceptor degeneration in mice. In wild type (wt) mice, Tulp1 is localized to the photoreceptor inner segment, connecting cilium and synapse. To investigate the role of Tulp1 in the synapse, we examined the pre- and postsynaptic architecture in tulp1-/- mice. We used immunohistochemistry to examine tulp1-/- mice prior to retinal degeneration and made comparisons to wt littermates and rd10 mice. In the tulp1-/- synapse, the spatial relationship between the ribbon-associated proteins, Bassoon and Piccolo, are disrupted, and few intact ribbons are present. Furthermore, bipolar cell dendrites are stunted, most likely a direct consequence of the malformed photoreceptor synapses. Comparable abnormalities are not seen in rd10 mice. The association of early onset and severe photoreceptor degeneration, which is preceded by synaptic abnormalities, appears to represent a phenotype not previously described. Our new evidence indicates that Tulp1 is not only critical for photoreceptor function and survival, but is essential for the proper development of the photoreceptor synapse.

Proteomic and genomic biomarkers for age-related macular degeneration.

Toward early detection of susceptibility to age-related macular degeneration (AMD), we quantified plasma carboxyethylpyrrole (CEP) oxidative protein modifications and CEP autoantibodies by ELISA in 916 AMD and 488 control donors. Mean CEP adduct and autoantibody levels were elevated in AMD plasma by ∼60 and ∼30%, respectively, and the odds ratio for both CEP markers elevated was ∼3-fold greater in AMD than in control patients. Genotyping was performed for AMD risk polymorphisms associated with age-related maculopathy susceptibility 2 (ARMS2), high-temperature requirement factor A1 (HTRA1), complement factor H (CFH), and complement C3. The AMD risk predicted for those exhibiting elevated CEP markers and risk genotypes was 2- to 3-fold greater than the risk based on genotype alone. AMD donors carrying the ARMS2 and HTRA1 risk alleles were the most likely to exhibit elevated CEP markers. Receiver operating characteristic curves suggest that CEP markers alone can discriminate between AMD and control plasma donors with ∼76% accuracy and in combination with genomic markers, provide up to ∼80% discrimination accuracy. CEP plasma biomarkers, particularly in combination with genomic markers, offer a potential early warning system for predicting susceptibility to this blinding disease.

Protective effect of paraoxonase 1 gene variant Gln192Arg in age-related macular degeneration.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of blindness among older adults, in which oxidative damage may play a pivotal role. Paraoxonase 1 (PON1) protects against oxidative damage and has been evaluated for its involvement in aging diseases including AMD. This study investigated whether PON1 gene polymorphisms associate with AMD. DESIGN: Case-control association study. METHODS: We studied 1037 individuals with AMD subcategorized using AREDS criteria and 370 control subjects without retinal disease. Participants were primarily Caucasian of European descent. All exons of PON1 were evaluated by single-strand conformation polymorphism and direct sequence analysis. RESULTS: Six missense changes (Leu55Met, Met127Arg, His155Arg, Gln192Arg, Gln192Glu, Ala252Gly) were identified in PON1. We observed a weak association of Leu55Met with an increased risk of wet AMD (P = .02), but not with dry AMD or when combining all patient categories. A significantly higher allele frequency for Gln192Arg was detected in controls than in the combined AMD patient population (P < .0001), and when category 2, 3, and 4 patients were separately considered (P = .004, P = .002, and P < .0001, respectively). For category 4 AMD, the Arg192 allele was significantly less prevalent in the wet form (P < .0001), but not in the dry form (P = .377). CONCLUSION: We report a weak association of PON1 Leu55Met with an increased risk of wet AMD, replicating previous reports. Our findings indicate a protective role for Gln192Arg, particularly for patients with the wet form. Gln192Glu warrants consideration, as this variant alters the same amino acid as Gln192Arg and was identified only in category 4 AMD patients. We believe that Met127Arg, His155Arg, and Ala252Gly play minor roles in AMD susceptibility because of their limited frequency and/or location within the PON1 gene. The functional and biological mechanism by which Gln192Arg is acting to decrease AMD susceptibility remains to be determined.

Genetic analysis of complement factor H related 5, CFHR5, in patients with age-related macular degeneration.

PURPOSE: To investigate the complement factor H related 5 (CFHR5) gene, encoding a member of the complement factor H family, for the presence of genetic polymorphisms or mutations associated with age-related macular degeneration (AMD). METHODS: We screened 639 unrelated patients with AMD and 663 age-matched normal controls using direct genomic sequencing of the ten coding exons, along with the immediately flanking intronic DNA. The pathologic impact of the identified sequence variants were analyzed by computational methods using PolyPhen and PMut algorithms. RESULTS: We identified five heterozygous sequence changes in CFHR5. Asp169Asp had a minor allele frequency of 0.001% in patients and 0.014% in controls (p<0.0001), while Arg356His had a minor allele frequency of 0.016% in patients and 0.007% in controls. Val379Leu, Met514Arg, and Cys568Ter were found only in normal controls. In silico analysis predicted Arg356His and Val379Leu to be neutral and benign. Met514Arg was predicted to be pathological and damaging to the function of the CFHR5 protein. CONCLUSIONS: No definitive pathogenic CFHR5 mutations have been found in any of 639 unrelated patients with AMD, indicating that sequence variations in CFHR5 do not play a major role in determining AMD susceptibility. However, our findings suggest a possible protective role for Asp169Asp. Further studies of different and larger populations of patient and control samples will be required to address this observation.

Assessing susceptibility to age-related macular degeneration with proteomic and genomic biomarkers.

Age-related macular degeneration (AMD) is a progressive disease and major cause of severe visual loss. Toward the discovery of tools for early identification of AMD susceptibility, we evaluated the combined predictive capability of proteomic and genomic AMD biomarkers. We quantified plasma carboxyethylpyrrole (CEP) oxidative protein modifications and CEP autoantibodies by ELISA in 916 AMD and 488 control donors. CEP adducts are uniquely generated from oxidation of docosahexaenoate-containing lipids that are abundant in the retina. Mean CEP adduct and autoantibody levels were found to be elevated in AMD plasma by approximately 60 and approximately 30%, respectively. The odds ratio for both CEP markers elevated was 3-fold greater or more in AMD than in control patients. Genotyping was performed for AMD risk polymorphisms associated with age-related maculopathy susceptibility 2 (ARMS2), high temperature requirement factor A1 (HTRA1), complement factor H, and complement C3, and the risk of AMD was predicted based on genotype alone or in combination with the CEP markers. The AMD risk predicted for those exhibiting elevated CEP markers and risk genotypes was 2-3-fold greater than the risk based on genotype alone. AMD donors carrying the ARMS2 and HTRA1 risk alleles were the most likely to exhibit elevated CEP markers. The results compellingly demonstrate higher mean CEP marker levels in AMD plasma over a broad age range. Receiver operating characteristic curves suggest that CEP markers alone can discriminate between AMD and control plasma donors with approximately 76% accuracy and in combination with genomic markers provide up to approximately 80% discrimination accuracy. Plasma CEP marker levels were altered slightly by several demographic and health factors that warrant further study. We conclude that CEP plasma biomarkers, particularly in combination with genomic markers, offer a potential early warning system for assessing susceptibility to this blinding, multifactorial disease.

Early synaptic defects in tulp1-/- mice.

PURPOSE: Mutations in the photoreceptor-specific tubby-like protein 1 (TULP1) underlie a form of autosomal recessive retinitis pigmentosa. To investigate the role of Tulp1 in the photoreceptor synapse, the authors examined the presynaptic and postsynaptic architecture and retinal function in tulp1(-/-) mice METHODS: The authors used immunohistochemistry to examine tulp1(-/-) mice before retinal degeneration and made comparisons with wild-type (wt) littermates and retinal degeneration 10 (rd10) mice, another model of photoreceptor degeneration that has a comparable rate of degeneration. Retinal function was characterized with the use of electroretinography. RESULTS: In wt mice, Tulp1 is localized to the photoreceptor synapse. In the tulp1(-/-) synapse, the spatial relationship between the ribbon-associated proteins Bassoon and Piccolo are disrupted, and few intact ribbons are present. Furthermore, bipolar cell dendrites are stunted. Comparable abnormalities are not seen in rd10 mice. The leading edge of the a-wave had normal kinetics in tulp1(-/-) mice but reduced gain in rd10 mice. The b-wave intensity-response functions of tulp1(-/-) mice are shifted to higher intensities than in wt mice, but those of rd10 mice are not. CONCLUSIONS: Photoreceptor synapses and bipolar cell dendrites in tulp1(-/-) mice display abnormal structure and function. A malformation of the photoreceptor synaptic ribbon is likely the cause of the dystrophy in bipolar cell dendrites. The association of early-onset, severe photoreceptor degeneration preceded by synaptic abnormalities appears to represent a phenotype not previously described. Not only is Tulp1 critical for photoreceptor function and survival, it is essential for the proper development of the photoreceptor synapse.

Interaction between the photoreceptor-specific tubby-like protein 1 and the neuronal-specific GTPase dynamin-1.

PURPOSE: Tubby-like proteins (TULPs) are a family of four proteins, two of which have been linked to neurosensory disease phenotypes. TULP1 is a photoreceptor-specific protein that is mutated in retinitis pigmentosa, an inherited retinal disease characterized by the degeneration of rod and cone photoreceptor cells. To investigate the function of TULP1 in maintaining the health of photoreceptors, the authors sought the identification of interacting proteins. METHODS: Immunoprecipitation from retinal lysates, followed by liquid chromatography tandem mass spectrometry and in vitro binding assays, were used to identify TULP1 binding partners. RT-PCR was performed on total RNA from wild-type mouse retina to identify the Dynamin-1 isoform expressed in the retina. Immunocytochemistry was used to determine the localization of TULP1 and Dynamin-1 in photoreceptor cells. Electroretinography (ERG) and light microscopy were used to phenotype tulp1-/- mice at a young age. RESULTS: Immunoprecipitation from retinal lysate identified Dynamin-1 as a possible TULP1 binding partner. GST pull-down assays further supported an interaction between TULP1 and Dynamin-1. In photoreceptor cells, Dynamin-1 and TULP1 colocalized primarily to the outer plexiform layer, where photoreceptor terminals synapse on second-order neurons and, to a lesser extent, to the inner segments, where polarized protein translocation occurs. ERG analyses in young tulp1-/- mice indicated a decreased b-wave at ages when the retina retained a full complement of photoreceptor cells. CONCLUSIONS: These data indicated that TULP1 interacts with Dynamin-1 and suggested that TULP1 is involved in the vesicular trafficking of photoreceptor proteins, both at the nerve terminal during synaptic transmission and at the inner segment during protein translocation to the outer segment. These results also raised the possibility that normal synaptic function requires TULP1, and they motivate a closer look at synaptic architecture in the developing tulp1-/- retina.

Mutation screen of the cone-specific gene, CLUL1, in 376 patients with age-related macular degeneration.

Clusterin is a secreted glycoprotein expressed ubiquitously in many tissues that appears to function as a molecular chaperone capable of protecting stressed proteins. It is upregulated in many different forms of neurodegeneration and is thought to represent a defense response against neuronal damage. Clusterin has been found to be a common protein identified in drusen preparations isolated from the retina of donor eyes of patients with age-related macular degeneration (AMD), the leading cause of blindness in the elderly population of developed countries. A retina-specific clusterin-like protein (CLUL1) showing nearly 25% identity to clusterin at the protein level was recently cloned and shown to be expressed specifically in cone photoreceptor cells. For these reasons, we investigated CLUL1 as a candidate gene for AMD. A mutation screen of the entire coding region of the CLUL1 gene in 376 unrelated patients with AMD uncovered three sequence variations, one isocoding change and two intronic changes. One intronic change appears significantly less frequent in patients with the more severe forms of AMD than in control subjects, suggesting that this variant may reduce the risk for AMD or may be linked to a nearby variant that may reduce AMD risk. Variant alleles of the CLUL1 gene were found; however, none are considered pathogenic. None of the variants identified are predicted to create or destroy splice donor or acceptor sites based on splice-site prediction software.

Mutation screen of the TUB gene in patients with retinitis pigmentosa and Leber congenital amaurosis.

TUB is the first identified member of the TULP family of four proteins with unknown function. A spontaneous mutation in murine tub causes retinal degeneration, obesity, and deafness. Mutations in another member of the TULP family, TULP1, are a cause of autosomal recessive retinitis pigmentosa (RP). These findings prompted us to investigate TUB as a candidate gene for RP and Leber congenital amaurosis (LCA). A mutation screen of the entire coding region of the TUB gene in 159 unrelated patients with autosomal recessive RP, 114 unrelated patients with simplex RP, and 21 unrelated patients with LCA uncovered 18 sequence variations. Of these, seven were missense mutations, six were isocoding changes, and five were intronic polymorphisms. All seven missense mutations were identified as heterozygous changes and no defect could be found in the other allele. None of the isocoding variants or intronic polymorphisms are predicted to create or destroy splice donor or acceptor sites based on splice-site prediction software. Although variant alleles of the TUB gene were found, none could be definitively associated with a specific retinal disease.