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Profile of the Genes Expressed in the Human Peripheral Retina, Macula, and Retinal Pigment Epithelium Determined Through Serial Analysis of Gene Expression (SAGE)

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Profile of the Genes Expressed in the Human Peripheral Retina, Macula, and Retinal Pigment Epithelium Determined Through Serial Analysis of Gene Expression (SAGE) Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE) Dror Sharon*†, Seth Blackshaw†‡, Constance L. Cepko‡, and Thaddeus P. Dryja*§ *Ocular Molecular Genetics Institute, Massachusetts Eye and Ear Infirmary, and ‡Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02114 Contributed by Thaddeus P. Dryja, October 31, 2001 We used the serial analysis of gene expression (SAGE) technique to a short piece of each transcript (a SAGE tag that is 9–14 bases in catalogue and measure the relative levels of expression of the genes length) is linked in series with 20–50 other such tags in a cloned expressed in the human peripheral retina, macula, and retinal pig- DNA fragment. Sequencing of such a clone provides the sequence ment epithelium (RPE) from one or both of two humans, aged 88 and of 20–50 tags, and a set of a few thousand such clones represents 44 years. The cone photoreceptor contribution to all transcription in a library of SAGE tags. Most individual SAGE tags can be assigned the retina was found to be similar in the macula versus the retinal to specific genes by inspecting available NCBI sequences periphery, whereas the rod contribution was greater in the periphery (SAGEmap). The relative abundance of each tag reliably reflects versus the macula. Genes encoding structural proteins for axons were the level of gene expression based on a comparison with Northern found to be expressed at higher levels in the macula versus the retinal blot analysis (9). The SAGE technique has been used to compare periphery, probably reflecting the large proportion of ganglion cells the profile of mRNA expression in different types of cancer cells in the central retina. In comparison with the younger eye, the (10, 11) and to identify genes that are up- or down-regulated in peripheral retina of the older eye had a substantially higher propor- response to exposure to drugs or other stimuli (12, 13). tion of mRNAs from genes encoding proteins involved in iron me- tabolism or protection against oxidative damage and a substantially Here we describe SAGE libraries constructed from the human lower proportion of mRNAs from genes encoding proteins involved peripheral retina, macula, and RPE. We compared the expression in rod phototransduction. These differences may reflect the differ- profile of the retinal periphery versus the macula of the same eye ence in age between the two donors or merely interindividual and the expression profile of the peripheral retina from two variation. The RPE library had numerous previously unencountered individuals (ages 44 and 88 years). Finally, we evaluated the tags, suggesting that this cell type has a large, idiosyncratic repertoire potential value of the library as a resource for identifying candidate of expressed genes. Comparison of these libraries with 100 reported genes for hereditary retinal diseases. nonocular SAGE libraries revealed 89 retina-specific or enriched genes expressed at substantial levels, of which 14 are known to cause a Methods retinal disease and 53 are RPE-specific genes. We expect that these Procurement of Tissue. The patients who donated eyes for this study libraries will serve as a resource for understanding the relative had periocular malignancies that required exenteration of an oth- GENETICS expression levels of genes in the retina and the RPE and for identi- erwise normal eye as part of clinically indicated surgical therapy. fying additional disease genes. Before surgery, the patients were contacted and gave their consent to donate parts of their to-be-enucleated eye for this research. The he human retina is a highly specialized tissue that converts harvesting of the intraocular tissue fragments for this study did not Tphotons into neural signals that are communicated to the brain. interfere with the pathological evaluation of the extraocular ma- This process involves different types of retinal cells, mainly neuronal lignancies. Each exenteration specimen was placed in saline after it cells (e.g., photoreceptors, bipolar cells, and others) and the non- was removed from the patient and promptly transported to the neuronal retinal pigment epithelium (RPE). The set of genes pathology laboratory. Each eye was cut open with a virgin razor expressed by the human neural retina has been elucidated partially, blade, and retinal fragments were excised. The RPE was harvested mainly through reported expressed sequence tag (EST) libraries by gentle scraping with a Pasteur pipette, and fragments of RPE [nonnormalized (1–3) or enriched (4, 5)] that contain over 16,000 were collected by aspiration in sterile 0.9% saline. Tissue fragments EST sequences derived from less than 5,000 genes (6). Our knowl- were collected and frozen within 45 min of the surgical removal of edge of the set of genes expressed by the RPE is more rudimentary, each specimen. The exenteration specimens, including the remain- because only 624 ESTs have been sequenced (refs. 4 and 7, and GenBank Library 6359). Little is known about the variation in gene expression between different regions of the retina (e.g., the retinal Abbreviations: RPE, retinal pigment epithelium; EST, expressed sequence tag; SAGE, serial periphery versus the macula); only a few genes with a preferential analysis of gene expression. expression in the fovea have been identified (1). There is no Data deposition: The tag sequences from the four SAGE libraries reported in this paper have been deposited in the Gene Expression Omnibus database (accession nos. GSM571– counterpart to these different retinal regions in mouse retinas. GSM574). Knowledge of the set of genes expressed in these regions is valuable, †D.S. and S.B. contributed equally to this work. because some common diseases of the retina can affect one region §To whom reprint requests should be addressed at: Massachusetts Eye and Ear Infirmary, preferentially (e.g., age-related macular degeneration). 243 Charles Street, Boston, MA 02114. E-mail: [email protected]. The serial analysis of gene expression (SAGE) procedure, first The publication costs of this article were defrayed in part by page charge payment. This described by Velculescu et al. (8), allows the compilation of article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. thousands of transcripts from a tissue sample. With this technique, §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.012582799 PNAS ͉ January 8, 2002 ͉ vol. 99 ͉ no. 1 ͉ 315–320 Downloaded by guest on September 26, 2021 Table 1. SAGE tags in the human retinal and RPE libraries Peripheral retina Eye 1* Eye 2* Combined Macula, Eye 2* Retina totals RPE, Eye 1* Total no. of tags 59,661 105,312 164,973 102,359 267,332 53,666 Tags for analysis†, (%) 50,193 (84) 90,444 (86) 140,637 (85) 87,555 (86) 228,192 (85) 25,712 (48) Different transcripts 13,344 19,199 23,112 18,660 26,355 10,404 UniGenes‡, (%) 10,942 (82) 15,373 (80) 18,119 (78) 15,023 (81) 20,251 (77) 6,401 (62) *Eye 1 was enucleated from an 88-year-old female, and eye 2 was enucleated from a 44-year-old male. †The number of tags represented at least twice in the current study. The percentage of nonsingleton tags out of the total number of tags is presented in parentheses. ‡Transcripts that correspond to UniGene entries. In parentheses is the percentage of UniGene transcripts out of all different transcripts. ing ocular tissues, were subsequently placed in fixative and pro- After excluding singletons and combining tags derived from cessed as required for diagnostic pathology. the same transcript, the total number of different transcripts The peripheral retinal fragments were derived from regions was 26,355 in the combined retinal libraries and 10,404 in the outside the central vascular arcades. The macular fragment was Ϸ6 RPE library. Approximately 77% of the retinal tags could be mm in diameter and centered at the foveola. The RPE fragments assigned to UniGene entries, but only 62% of the RPE tags could were from the posterior pole and the periphery. be assigned. The high proportion of unassigned RPE tags (38%), together with the high proportion of singleton RPE tags men- SAGE Library Construction and Analysis. We used a modification of tioned in the previous paragraph, suggest that a large proportion the microSAGE protocol (14) to generate the four human SAGE of genes expressed by the RPE are tissue-specific. Many RPE- libraries. The sequences were analyzed by using the SAGE2000 specific tags are from unknown genes. This may be due in part software (courtesy of Victor Velculescu and Ken Kinzler, Johns to the small number of ESTs that have been sequenced from Hopkins University School of Medicine). Tags with ambiguous RPE libraries (4, 7). bases, duplicate ditags, and abnormally short tags (Ͻ14 bases) were eliminated automatically. The identity of the mRNAs correspond- New Tag Assignments. Most of the genes that were known previously ing to the SAGE tags was determined through inspection and to be expressed in the retina or the RPE had corresponding tags in comparison with the SAGEmap (www.ncbi.nlm.nih.gov͞SAGE͞ our SAGE libraries. However, we came across a number of genes SAGEtag.cgi) and UniGene (www.ncbi.nlm.nih.gov͞UniGene͞) known to be expressed in the retina but for which there initially databases. appeared to be no representative tag in our libraries. We considered To identify tags in genomic sequences, we developed a computer the possibility that these genes may produce mRNAs with 3Ј ends program that we have named TAGSEARCH. This program searches different than those reported previously. By using the TAGSEARCH the genomic sequence at the 3Ј end of a specified gene for each software, we tentatively assigned some of the tags in our libraries 14-bp tag, including an NlaIII recognition sequence (CATG), to known genes for which no tags had otherwise been found.
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