The klotho-related protein KLPH (lctl) has preferred expression in lens and is essential for expression of clic5 and normal lens suture formation.

KLPH/lctl belongs to the Klotho family of proteins. Expressed sequence tag analyses unexpectedly revealed that KLPH is highly expressed in the eye lens while northern blots showed that expression is much higher in the eye than in other tissues. In situ hybridization in mouse localized mRNA to the lens, particularly in the equatorial epithelium. Immunofluorescence detected KLPH in lens epithelial cells with highest levels in the germinative/differentiation zone. The gene for KLPH in mouse was deleted by homologous recombination. Littermate knockout (KO) and wild type (WT) mice were compared in a wide panel of pathology examinations and were all grossly normal, showing no systemic effects of the deletion. However, the lens, while superficially normal at young ages, had focusing defects and exhibited age-related cortical cataract by slit lamp examination. Whole-lens imaging showed that KO mice had disorganized lens sutures, forming a loose double-y or x instead of the tight y formation of WT. RNA-seq profiles for KO and WT littermates confirmed the absence of KLPH mRNA in KO lens and also showed complete absence of transcripts for Clic5, a protein associated with cilium/basal body related auditory defects in a mouse model. Immunofluorescence of lens epithelial flat mounts showed that Clic5 localized to cilia/centrosomes. Mice mutant for Clic5 (jitterbug) also had defective sutures. These results suggest that KLPH is required for lens-specific expression of Clic5 and that Clic5 has an important role in the machinery that controls lens fiber cell extension and organization.

Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

PURPOSE: The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. METHODS: A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. RESULTS: Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. CONCLUSIONS: The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

Expressed sequence tag analysis of human retina for the NEIBank Project: retbindin, an abundant, novel retinal cDNA and alternative splicing of other retina-preferred gene transcripts.

PURPOSE: Expressed sequence tag (EST) analysis was performed on un-normalized, unamplified cDNA libraries constructed from adult human retina to examine the expression profile of the tissue and to contribute resources for functional genomics studies. METHODS: Two size fractionated cDNA libraries (designated hd and he) were constructed from human retina RNA. Clones were randomly selected for sequencing and analyzed using the bioinformatics program GRIST (GRouping and Identification of Sequence Tags). PCR, Northern blotting and other techniques have been used to examine selected novel transcripts. RESULTS: After informatics analysis, 2200 retina cDNAs yield 1254 unique clusters, potentially representing individual genes. Opsin is the most abundant transcript and other retina transcripts are prominently represented. One abundant cluster of cDNAs encodes retbindin, a novel, retina preferred transcript which has sequence similarity to riboflavin binding proteins and whose gene is on chromosome 19. Variant transcripts of known retina genes are also observed, including an alternative exon in the coding sequence of the transcription factor NRL and a skipped coding sequence exon in the phosphodiesterase gammasubunit (PDE6G). CONCLUSIONS: The new retina cDNA libraries compare favorably in quality with those already represented in public databases. They are rich in retina specific sequences and include abundant cDNAs for a novel protein, retbindin. The function of retbindin remains to be determined, but it is a candidate for flavinoid or carotenoid binding. Analysis of multiple clones for highly expressed retina genes reveals several alternative splice variants in both coding and noncoding sequences which may have functional significance. The validated set of retina cDNAs will contribute to a nonredundant set for microarray construction.

Expressed sequence tag analysis of adult human iris for the NEIBank Project: steroid-response factors and similarities with retinal pigment epithelium.

PURPOSE: The iris is a specialized tissue with important roles in the development and function of the eye. It is involved in diseases, including glaucoma and ocular melanoma, and its pigmented cells share an origin with the retinal pigment epithelium (RPE). Expressed sequence tag (EST) analysis of human iris has been performed to explore the repertoire of genes expressed in this tissue. METHODS: An unamplified, un-normalized cDNA library (designated bx) was constructed from pooled (4-80 years old) human iris tissue. Over 2000 clones were picked and sequenced. Sequences were analyzed and clustered using GRIST (GRouping and Identification of Sequence Tags). The library was then normalized (and designated fg) and a further 2200 clones were sequenced for deeper examination of rarer sequence. Some sequences of interest were investigated further by standard methods. RESULTS: From bx and fg libraries respectively, 1263 and 1604 clusters of expressed genes have been identified, giving a combined total of almost 2700 potentially unique genes. The most abundant novel transcript in bx is oculoglycan/opticin. Others include glucocorticoid induced leucine zipper protein (GILZ), Ris, a novel member of the Ras family, Iris Ring Finger (IRF), a member of the midline family, melastatin 2 (MLSN2), a member of the transient receptor potential calcium channel family, and iris expressed growth factor (IEGF), a member of the VEGF/PDGF family. Several factors involved in steroid responses are also represented. CONCLUSIONS: The iris libraries are a rich source of novel as well as known genes, including molecular markers for pigmented cells that are also shared with RPE. A number of transcripts code for proteins involved in steroid response, with interesting implications for control of intraocular pressure. These sequence verified clones provide a nonredundant set for micro-array construction.

Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.

PURPOSE: To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses. METHODS: A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. RESULTS: The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. CONCLUSIONS: The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.

Grouping and identification of sequence tags (GRIST): bioinformatics tools for the NEIBank database.

NEIBank is a project to develop and organize genomics and bioinformatics resources for the eye. As part of this effort, tools have been developed for bioinformatics analysis and web based display of data from expressed sequence tag (EST) analyses. EST sequences are identified and formed into groups or clusters representing related transcripts from the same gene. This is carried out by a rules-based procedure called GRIST (GRouping and Identification of Sequence Tags) that uses sequence match parameters derived from BLAST programs. Linked procedures are used to eliminate non-mRNA contaminants. All data are assembled in a relational database and assembled for display as web pages with annotations and links to other informatics resources. Genome projects generate huge amounts of data that need to be classified and organized to become easily accessible to the research community. GRIST provides a useful tool for assembling and displaying the results of EST analyses. The NEIBank web site contains a growing set of pages cataloging the known transcriptional repertoire of eye tissues, derived from new NEIBank cDNA libraries and from eye-related data deposited in the dbEST section of GenBank.

Interaction of complement factor h and fibulin3 in age-related macular degeneration.

Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch's membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for "dry" AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.