Towards a comprehensive description of the human retinal transcriptome: identification and characterization of differentially expressed genes (2003)

The human retina is a multilayered neuroectodermal tissue specialized in the transformation of light energy into electric impulses which can be transmitted to the brain where they are perceived as vision. Since the retina is easily accessible and functional aspects are directly recordable, the study of this tissue has been at the forefront of neuroscience research for over a century. Studies have revealed that the distinct functions of the retina require a large degree of differentiation which is achieved by the coordinated function of approximately 55 different cell types. The highly structured anatomy and the functional differentiation of the retina is a result of its distinctive transcriptome and proteome. Due to the complexity of the retina it has been difficult to estimate the number of genes actively transcribed in this tissue. Great efforts in the elucidation of retinal disease genes have led to the identification of 139 retina disease loci with 90 of the corresponding genes cloned thus far . In contrast to the success in the hereditary disorders, efforts to identify the genetic factors conferring manifestations known as age-related macular degeneration (AMD) have revealed sparse results. AMD is a retinal disease affecting a significant percentage of the older population. This disorder is likely due to exogenic as well as genetic factors. To further our understanding of retinal physiology and facilitate the identification of genes underlying retinal degenerations, particularly AMD, our efforts concentrated on the systematic analysis of the retinal transcriptome. Since approximately half of all retinal degeneration-associated genes identified to date are preferentially expressed in retina, it is plausible that the investigation of gene expression profiles and the identification of retina-expressed transcripts could be an important starting point for characterizing candidate genes for the retinal diseases. The expressed sequence tags approach included the assessment of all retinal expressed sequence tags (EST) clusters indexed in the UniGene database and of 1080 single-pass ESTs derived from an in-house generated human retina suppression subtracted hybridization (SSH) cDNA library. In total, 6603 EST clusters were evaluated during this thesis and detailed in-silico analysis was performed on 750 EST clusters. The expression of the genes was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR), followed by confirmation using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), as well as conventional and virtual Northern blot analysis. The expression profiling of 337 selected EST clusters led to the identification of 111 transcripts, of which 60 are specific or abundant to the retina, 3 are expressed at high levels in the retinal pigment epithelium (RPE), and 48 are expressed in brain as well as in retina. The EST approach used to select candidate transcripts allowed us to assess the effectiveness of the two available resources, the UniGene database and the retinal SSH (retSSH) cDNA library. From the results obtained, it is evident that the generation of suppression subtracted libraries to identify cell-specific transcripts constitutes the most straight-forward and efficient strategy. In addition to the high percentage of candidate genes that are identified from an SSH cDNA library, it has the added benefit that genes expressed at low levels can be identified. Furthermore, comparison of our retina-enriched gene set with previously published studies demonstrated only limited overlap of the identified genes further confirming the valuable source of retinal genes from our retinal SSH cDNA library. The effort of our and other groups has resulted in the establishment of the full-length coding sequence of 55 of the 111 genes uniquely or preferentially expressed in the retina. Using various methods such as bioinformatical analysis, EST assembly, cDNA library screening, and rapid amplification of cDNA ends (RACE) a number of genes were cloned in the scope of this thesis including C1orf32, C4orf11, C7orf9, C12orf7, C14orf29, DAPL1, and GRM7. Bioinformatic analyses and cDNA library screening were used to isolate the full-length cDNA sequence and determine the genomic organization of C7orf9, also identified as RFRP. This 1190 bp retina-specific transcript from chromosome 7p15.3 encodes a precursor protein for at least two small neuropeptides, referred to as RFRP-1 and RFRP-3. Since C7orf9 is localized in the critical region for dominant cystoid macular dystrophy (CYMD) its role in the pathology was investigated. Southern blot analysis and sequencing of samples from two affected individuals of the original pedigree used to localize the disease gene excluded the gene from involvement in this disease. Multiple isoforms of the C12orf7 gene were assembled from a number of clones identified from library screenings, PCR amplifications, and RACE experiments. The gene variants, transcribed from chromosome 12q13.13, have been found to be expressed exclusively in retina. Because of the multiple alternative splicing of the gene, we can only speculate about the nature of the protein it encodes. The longest transcript, which includes all six exons plus the last intervening sequence, encodes a 471 aa protein which contains a nuclear localization signal and five ankyrin repeats. The existence of many isoforms is also observed in mouse suggesting that they may have a relevant role in cellular physiology. Five novel splice variants of the glutamate metabotropic receptor 7 (GRM7) resulting from the use of alternative 3’-end exons were identified and characterized. One of the novel variants, GRM7_v3, encodes a 924 aa protein and is therefore the longest putative GRM7 protein reported to date. Even though they are not retina-specific, the isoforms are preferentially expressed in the nervous system. Although the functional properties of the specific carboxyl-termini are still unclear, it is known that axon targeting of GRM7_v1 is mediated by the last 60 aa of the protein. Hence the novel isoforms may direct the protein to specific subcellular localizations. The C1orf32 gene, preferentially expressed in retina, is organized in 10 exons and is transcribed from chromosome 1q24.1. Bioinformatic analyses of the 639 aa putative protein not only identified the mouse and rat orthologous genes but also the LISCH7 gene as a potential member of the same family. Since the LISCH7 protein has been shown to function as a low density lipoprotein receptor, the C1orf32 protein may be involved in retinal lipid homeostasis. Disturbances in lipid metabolism have been proposed as one of the pathways involved in AMD etiology. Thus, the role of C1orf32 in this complex disease should be investigated. Expression analyses of the death-associated protein-like 1 (DAPL1) gene revealed that it is expressed in both the retina and the RPE at high levels. The 552 bp transcript encodes a 107 aa putative protein and is transcribed from chromosome 2q24.1. In-silico analyses identified an additional 12 related proteins from various species which share high similarity constituting a novel protein family. The similarity to the death-associated-protein (DAP) is particularly interesting since this protein has been found to be indispensable for programmed cell death. Therefore, DAPL1 is an excellent candidate for retinal disease as apoptosis is generally the ultimate cause in retinal degeneration. The retina-specific C4orf11 and C14orf29 genes localized on chromosome 4q21.22 and 14q22.1, respectively, are both transcribed in more than one isoform. The encoded proteins do not contain any known domains but because of their retina-specific expression they may be important for proper retinal physiology. As part of the long-term goals of the project, several of the cloned genes are being genotyped to construct single nucleotide polymorphism (SNP) maps. Projects to investigate haplotype frequencies of candidate genes in large cohorts of controls and AMD patients are ongoing. Thus, by establishing a collection of 111 genes expressed exclusively or preferentially in the retina, the present work has laid the foundation for future research in retinal diseases.