Profile of the expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of 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 (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 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. represented in SAGE libraries. After the program identifies a tag, Examples are the genes encoding red and green cone opsins it searches for a nearby polyadenylation signal sequence (OPN1LW and OPN1MW), which appear to have the same, newly (AATAAA or AATTAA). This software, written by John Keene identified tag AGGTCTGCCT. The program also found a putative of D. H. Keene Associates, runs on a Microsoft WINDOWS platform rhodopsin tag, CTCACCCGCC, expressed at a low level and and will be available upon request at eyegene.meei.harvard.edu. possibly derived from a rare alternative transcript not yet cata- The ␹2 statistic was calculated for all comparisons of tag fre- logued in EST sequence databases. quency, taking into account the frequency of each tag and the total Even after this search for new tag assignments, there remained number of tags per library. To identify tissue-specific tags, the most a few genes we expected to be represented but for which no common tags were compared with tags from 100 nonocular librar- matching tag was encountered (e.g., CRX, USH2A, RHOK in the ies (www.ncbi.nlm.nih.gov͞SAGE͞). The assignment of a ’s retina, and MERTK in the RPE). These tags may be absent because function was, for the most part, based on the database LocusLink the corresponding genes are expressed at a very low level (for (www.ncbi.nlm.nih.gov͞LocusLink͞). example, no ESTs have been reported from RHOK in the retina and MERTK in the RPE), because they produce unstable mRNA Results and Discussion molecules that were degraded in the 45 min between enucleation SAGE Libraries. We harvested mRNA from one eye from each of and the harvesting of tissues, or because of an erroneous prediction two humans without retinal disease, an 88-year-old female (patient of the expected tag through inaccurate knowledge of the 3Ј end of 1) and a 44-year-old male (patient 2). SAGE libraries were pre- the mRNA sequence. pared from the peripheral retina (one from each eye), the macula (from eye 2), and the RPE (from eye 1). SAGE tags from 53,666 Validity of the Libraries. We assessed the validity of the SAGE to 105,312 mRNA molecules were sequenced from each library for libraries by evaluating tags from genes previously known to be a total of 320,998 tags from the four SAGE libraries (Table 1; also expressed specifically in the retina or RPE. For example, the genes deposited in NCBI database). Approximately 15% of the retinal for rhodopsin (RHO), the rod subunits (GNB3, GNAT1, tags and 52% of the RPE tags were observed only once. Some of and GNGT1), the rod cGMP phosphodiesterase subunits (PDE6A, these singleton tags may be due to sequencing errors with the PDE6B, and PDE6G), the subunits of the rod cGMP-gated cation remainder corresponding to mRNAs expressed at very low levels. channel (CNGA1 and CNGB1), other proteins involved in rod The small proportion of spurious tags caused by sequencing errors phototransduction (RCV1, SAG, GUCA1A, and SLC24A1), and is likely to be approximately the same in the different libraries we other rod photoreceptor proteins (HRG4, ABCR, RDS, and ROM1) compiled. Thus, it seems likely that the large number of singleton are known to be expressed in the retina. As expected, tags corre- tags in the RPE reflects an abundance of rare transcripts in that cell sponding to all these genes were present in the retinal SAGE type. We have confined the analyses presented in this paper to tags libraries derived from the peripheral retina and the macula. Sim- encountered at least twice. ilarly, the genes RPE65, 11-cis-retinol dehydrogenase (RDH5),

316 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.012582799 Sharon et al. Downloaded by guest on September 26, 2021 transthyretin (TTR), bestrophin (VMD2), cathepsin D (CTSD), and (Table 6, which is published as supporting information on the PNAS cystatin C (CST3) are all known to be expressed by the RPE; all web site) in the retinal libraries with the levels of those genes in five were detected in the RPE library. nonocular SAGE libraries from the following normal human We also evaluated the purity of the RPE library by examining tissues: cerebellum, thalamus, kidney, prostate, and lung. In com- whether tags corresponding to genes known to be expressed in the parison with these other tissues, the retina devotes the highest retina and not the RPE were present only in the retinal libraries and proportion of mRNA synthesis to these genes. The total number of not in the RPE library. Genes encoding photoreceptor proteins these tags in the retina (21,610 per 106)is2.7–4.2 times greater than (e.g., RHO, GNAT1, GNB1, GNGT1, PDE6A, PDE6B, PDE6G, in the cerebellum (7,376), thalamus (8,086), kidney (5,347), prostate ROM1, RDS, RCV1, SAG, CNGA1, CNGB1, GUCA1A, and (5,180), or lung (6,666). The high expression level of genes encoding SLC24A1) were ideal for this evaluation, because they are ex- proteins involved in energy metabolism is consistent with the high pressed by photoreceptor cells immediately adjacent to the RPE but metabolic rate of the retina (in particular of the photoreceptor cells) not by the RPE itself. In total, these genes accounted for 1.7% of and is consistent with data from a retinal EST library (3). the tags in the retinal libraries overall, although they only accounted for 0.05% of the tags in the RPE library. The small percentage of Gene Expression in the RPE. The RPE exhibits an expression profile photoreceptor-specific tags in the RPE library probably is the result very different from the neural retina. We scanned the 1,000 most of minor contamination of the RPE with retina during dissection. highly expressed tags (each represented three or more times) to Evaluating the amount of RPE that contaminated the retinal obtain a list of the 798 most highly expressed tags for which an fragments used to construct SAGE libraries was more difficult unambiguous gene assignment could be made. These 798 tags because the few RPE-specific genes of which we are aware (e.g., correspond to 445 distinct, known genes and 333 unknown tags in RPE65, RDH5, and VMD2) are not expressed at a high level in the the UniGene database. Of the 445 known genes, no function could RPE and therefore could not serve as sensitive markers of con- be assigned to 49% (accounting for 50.2% of the tags from the 445 tamination. Our evaluation of the tags corresponding to these genes known genes). Of the remaining 227 genes with a function assigned suggested some contamination (these tags represented 0.05% of the to the corresponding gene product, Ϸ15% (15.4% of tags from the tags in the RPE versus 0.005% of the tags in the combined retinal 227 known genes) encode proteins involved in translation, and libraries). However, it is possible that one or more of these genes 11.5% (11.2% of tags) encode transcription factors (Fig. 1B). is actually expressed by the neural retina at low levels. Other Approximately 5.3% (9.5% of tags) encode proteins involved in evidence that the amount of RPE contamination in the retinal protein degradation. One of the major functions of the RPE is the libraries is low came from our observation of seven tags (tag phagocytosis and digestion of the photoreceptor outer segments. sequences ACAAAACCAA, ATAACACATA, GGGTT- This task requires metabolic pathways to catabolize the phagocy- GGGGA, GCTCCAGCTG, CTCCCGCTGG, ATACTGACAC, tosed proteins and other components of the outer segment disks and TGGGTGCTGG) that were abundant in the RPE library and plasma membrane. Thus, the relatively high percentage of tags (each was at a frequency Ͼ100 tags per 105) but were absent or very from genes encoding proteins involved in protein degradation rare (less than 2 tags per 105) in the peripheral retinal library of the might be expected. Indeed, although the RPE devotes more than same eye. 9.5% of its transcripts to produce proteins involved in protein degradation, the neural retina devotes less than 3%. Gene Expression in the Retina. Of the 1,000 most frequent tags in the Of the 50 most common tags in the RPE library, 29 do not combined peripheral retina and macular libraries, 216 were derived correspond to any known gene. Of these 29 orphan tags, 22 appear from mitochondrial DNA or were ambiguous tags that could not be to be expressed exclusively by the RPE, because they could not be assigned to a single gene. The remaining 784 tags correspond to 690 found in any of the 100 available SAGE libraries derived from other distinct, known genes and 47 unknown tags in the UniGene cell types. Some of these orphan tags are highly expressed: 5 of the database. A provisional function could be assigned to the proteins 7 most abundant tags are orphans, and 3 of the 4 most abundant tags expressed by 65.2% of the known genes, which together accounted are RPE-specific orphans (Table 5, which is published as supporting for 77.5% of the tags from the 690 known genes. The largest information on the PNAS web site). Of the 21 most frequent tags GENETICS functional categories are proteins involved in protein translation, for which a gene assignment could be made, some were from genes accounting for 17.6% of the genes (23.1% of the tags), and proteins previously known to be expressed in the RPE, including transthy- involved in energy metabolism, accounting for 14.2% of the genes retin [a protein involved in vitamin A transport (7, 15)], cathepsin (19.4% of the tags). Among the genes involved in energy metab- D [a lysosomal enzyme important in the proteolysis of opsin and in olism are those encoding 10 of the 28 enzyme subunits in the the digestion of phagocytosed photoreceptor outer segments (16)], glycolytic pathway. Approximately 4.2% of the genes (8.5% of and ␤-amyloid binding protein [a protein implicated in Alzheimer’s the tags) encode proteins that participate in phototransduction disease (17)]. (Fig. 1A). We separately tabulated the 50 most highly expressed tags in the The Retinal Periphery Versus the Macula. The posterior pole of the combined retinal libraries, including tags from genes with unknown human retina has a specialized region, called the macula, that has function (Table 4, which is published as supporting information on the highest density of photoreceptor cells and provides the best the PNAS web site, www.pnas.org). Six tags in this set have visual acuity. The anatomic boundary of the macula is not stan- as-yet-unknown gene assignments including the most common tag dardized. Here it is considered to be a 6-mm-diameter disk centered sequence representing 1.59% of all tags in the retinal libraries. In at the foveola; this area processes visual information from the this list are many tags from genes expected to be expressed at high central 20° of visual field diameter. The macula occupies less than levels based on previously reported EST libraries (2, 3) such as those 1.4% of the surface of the retina, yet it contains Ϸ8.4% of the encoding proteins involved in phototransduction and other proteins cone photoreceptors, 3.4% of the rod photoreceptors, and including HRG4, ABCR, and ROM1. Genes encoding two proteins Ϸ60% of the ganglion cells of the retina (18). Furthermore, the involved in iron metabolism, transferrin and ferritin, are repre- ratio of rods to cones is lower in the macula (Ϸ8:1) than in the sented among the 10 most highly expressed tags; these genes will be peripheral retina [Ϸ20:1 (19)]. discussed below. Many genes encoding proteins involved in protein We compared the set of 87,555 nonsingleton tags in the macula translation and enzymes involved in energy metabolism are also in library with the set of 90,444 nonsingleton tags in the peripheral the top-50 list. retinal library derived from the same eye (donor 2). For this We further compared the expression levels of all 28 genes comparison, the number of tags in the libraries first was adjusted encoding the subunits of the 10 enzymes of the glycolytic pathway mathematically (i.e., normalized) to account for the fact that we had

Sharon et al. PNAS ͉ January 8, 2002 ͉ vol. 99 ͉ no. 1 ͉ 317 Downloaded by guest on September 26, 2021 Fig. 1. (A) A pie chart depicting the percentage of distinct genes encoding proteins in various functional categories out of the total number of genes encoding proteins with known function. Data from the macula library and the two peripheral retinal libraries were combined. (B) A pie chart similar to that shown in A but based on data from the RPE library. (C) A comparison of the expression levels of the genes that are expressed at higher levels in the macula than in the retinal periphery (left of the vertical red line) or higher in the periphery than in the macula (right of the vertical red line). (D) A comparison of the expression levels of the genes that are expressed at higher levels in the peripheral retina of eye 1 than in the peripheral retina of eye 2 (left of the vertical red line) or higher in the peripheral retina of eye 2 than in the peripheral retina of eye 1 (right of the vertical red line). In both C and D, the number above each pair of bars is the ratio of tags from that gene in the two SAGE libraries unless there are zero tags in one library, in which case the actual tag frequencies (tags per 105) are shown. Tags that do not correspond to any known genes are represented by their 10-base sequence.

sequenced slightly fewer tags from the macula than from the test yielded a P value Ͻ 3.2 ϫ 10Ϫ5. This value would roughly peripheral retina. We considered that the abundance of a tag to be correspond to a P value threshold of 0.01 after a Bonferroni statistically significantly different between the two libraries if a ␹2 correction for the 15,625 comparisons that were performed.

318 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.012582799 Sharon et al. Downloaded by guest on September 26, 2021 Table 2. Transcripts specific to or enriched in the retina and RPE Table 3. Candidate genes for retinal disease Retina RPE Total Chromosomal Gene (symbol) localization Specific tags 68 45 113 Enriched tags 21 8 29 Hs. 43586 1p31–32 Total 89 53 142 Hs. 310689 1q25 Tags corresponding to known 37 2 39 Frizzled-related protein (FRZB) 2q24–31 retinal or RPE genes Palmitoylated 4 2q33.2 Tags corresponding to known 14 1 15 Hs. 66803 3 retinal disease genes HT017 protein 3p14.3 Transferrin (TF) 3q21 The 1,000 most common tags in the three retinal libraries combined and the Hermansky–Pudlak syndrome-3 (HPS3) 3q24 100 most common tags in the RPE library were compared to tag expression Hs. 300880 4p16 levels in 100 SAGE libraries (SAGEmap) from nonocular tissues. Specific tags , (rdgC homolog) (PPEF2) 4q13–21 are those with 0–1 tags in any nonocular library, whereas enriched tags have Hs. 151710 5 an abundance ratio of at least 10 between the retina or RPE and any nonocular Glutathione peroxidase 3 (GPX3) 5q23 libraries. Hs. 246420 6p21 Lysosomal apyrase-like 1 (LYSAL1) 8 Clusterin (CLU) 8p21–12 Twenty-four genes were statistically significantly more frequent Hs. 154131 9p24 in the macula than in the periphery (Fig. 1C and Table 7, which is MT-protocadherin (KIAA1775) 10q22 published as supporting information on the PNAS web site). None Cathepsin D (CTSD) 11p15 of these genes are expressed exclusively by the retina. Five of the Hs. 239444 11q14 genes that showed a preferential expression in the macula encode Immunoglobulin superfamily (IGSF4) 11q23.2 Hs. 13768 12p13 cell-structural proteins found in neuronal axons [neurofilament 3 ͞ ␣ Sentrin SUMO-specific protease (SENP1) 12q12–13 (NEF3), light neurofilament (NEFL), brain-specific -tubulin Hs. 33792 12q15 (TUBA3), ␣-tubulin (K-␣-1), and ␤-tubulin (FKBP1A)]. Their rel- Hs. 3821 13q13 ative abundance in the macula is likely to reflect the high density Hs. 21299 14q21 of ganglion cells in the retina. Ganglion cells are the only cells in the Creatine kinase B (CKB) 14q32 retina with axons more than a few millimeters in length; their axons Hs. 98927 (FLJ13993) 15q22.3–23 extend many centimeters and end in the central nervous system. Cellular retinoic acid-binding protein 1 (CRABP1) 15q24 Hs. 261526 17 Also, the axons of photoreceptor cells in the macula (which form Retinal clusterin 1 (CLUL1) 18p11.3 Henle’s nerve fiber layer) are much longer than elsewhere in the Hs. 122245 18p11.3 retina (20). mRNAs from genes encoding cell-trafficking and Transthyretin (TTR) 18q12.1 signal-transduction proteins also account for a higher percentage of Calcium-binding protein 5 (CABP5) 19q13.3 transcription in the macula versus the retinal periphery. Hs. 98881 20q11 Although the cone-specific genes encoding cone ␣-transducin Hs. 35493 Multiple (GNAT2), the ␣Ј and ␥ subunits of cone cGMP-phosphodiesterase (PDE6C and PDE6H), and the cone opsins (OPN1SW and OPN1LW͞OPN1MW) as a group were more abundant in the in the two libraries first was normalized. The same ␹2 test and the macula than the periphery, the difference was not statistically same threshold for statistical significance were used. significant (P ϭ 0.06). The lack of a substantially higher number of The 36 genes and 20 orphan tags with unknown gene assignment cone-specific tags in the macula versus the periphery simply may that were expressed at significantly different levels in the two indicate that the fraction of all cells that are cones may not be very donors are presented in Fig. 1D and Tables 9 and 10, which are GENETICS different in the two regions. In other words, not only cones but also published as supporting information on the PNAS web site. This set other retinal cell types such as ganglion cells are at their highest of genes does not appear to be distributed randomly among the two densities in the macula, and the tags from these other cell types donors: 24 genes and 12 orphan tags are expressed at higher levels dilute the contribution from cones. in donor 1, whereas 12 genes and 8 orphan tags were expressed at Twenty-nine genes were significantly more abundant in the higher levels in donor 2. Some genes have exceptionally different frequencies between the peripheral retinal library than the macula library (Fig. 1C and Table two donors. For example, donor 1 has six tags from genes with 8, which is published as supporting information on the PNAS web unknown function that are each found at a frequency of 25–111 per site). Approximately a third of these tags corresponds to genes 105 yet are absent from donor 2; donor 2 has one such high- encoding proteins in the rod phototransduction cascade including ␤ ␥ frequency tag that is absent from donor 1. Other remarkably rhodopsin (RHO), the and subunits of transducin (GNB1 and discrepant genes are those encoding RNA helicase (tag frequency GNGT1), the ␤ and ␥ subunits of rod cGMP-phosphodiesterase 5 5 ␣ of 70 per 10 in donor 1 but only 2 per 10 in donor 2) and the (PDE6B and PDE6G), the subunit of the cGMP-gated cation unassigned tag TCTCCATAAC (484 per 105 in donor 1 but only 2 channel (CNGA1), and recoverin (RCV1). The relative abundance per 105 in donor 2). It seems unlikely that the striking differences of rod-specific tags in the periphery is likely a reflection of the in the expression of these genes in the two donors is simply due to higher proportion of rod photoreceptors there compared with the polymorphisms in the 3Ј untranslated regions of the genes causing macula. However, tags from some genes that are not specific to rod alternative polyadenylation signal sequences to be used. If that were photoreceptors also were more abundant in the periphery [e.g., the case, one would expect some symmetry in the occurrence of transferrin (TF) and dopa decarboxylase (DDC), among others]. such tags in the two donors. That is, for every highly discrepant tag that is overrepresented in one donor, there should be, on average, Comparison of the Peripheral Retina in Two Individuals. We com- another discrepant tag that is overrepresented in the other donor. pared the mRNA expression profile in the peripheral retinal library This is not the case: the majority of the strikingly discrepant genes derived from the 88-year-old donor 1 with the peripheral retinal are overrepresented in donor 1. The discrepancies in the expression library derived from the 44-year-old donor 2. As with the compar- of these genes may be caused by the difference in age between the ison of the retinal periphery versus the macula, the number of tags two donors.

Sharon et al. PNAS ͉ January 8, 2002 ͉ vol. 99 ͉ no. 1 ͉ 319 Downloaded by guest on September 26, 2021 Most of the genes that are expressed at statistically significantly 185430). A related gene that encodes retinal clusterin (clusterin-like different levels in the two donors are not as strikingly discrepant as protein, or CLUL1) is expressed at a significantly higher level in the those discussed in the previous paragraph but instead are only 2–20 younger donor. times more abundant in one donor versus the other. Three of the A number of genes specifically expressed by rod photoreceptors genes that are expressed at a higher level in the older donor 1 versus are expressed at significantly higher levels in the younger donor donor 2 encode proteins involved in iron metabolism (ferritin light (e.g., rhodopsin and the ␣ subunit of the rod cGMP-gated channel). polypeptide, ferritin heavy polypeptide, and transferrin). The heavy The number of rod photoreceptors in the human retina decreases and light polypeptides of ferritin are expressed at high levels in many with age, with Ϸ32% loss from the second to the ninth decade (25), tissues including the retina. Transferrin is expressed also in many and there is a concomitant fall in average electroretinogram (ERG) tissues but achieve the highest known expression level in the retina amplitudes during this interval (26). It is reasonable to suppose that (Ͼ5 times higher than any of the 100 SAGE libraries in SAGEmap). the reduction of tags from rod-specific genes in the older retina Iron’s role in the retina is important, as evidenced by the observa- reflects this phenomenon. tion that null mutations in the ceruloplasmin gene, encoding a protein important for iron homeostasis, produce a phenotype that SAGE as a Tool to Identify Candidate Genes. Aiming to identify retinal includes retinal degeneration (21). The expression of iron metab- and RPE-specific transcripts, we compared the expression level of olism genes can increase synchronously in response to the increas- the 1,000 most common retinal tags to 100 nonocular SAGE ing iron concentrations (22). The higher expression of these three libraries (SAGEmap), and we separately compared the 100 most iron metabolism genes in the older donor 1 could be related to the common RPE tags to the same nonocular libraries. The analysis increase in iron stores with age. Iron in the body generally increases revealed 89 retinal tags and 53 RPE tags that were either specific Ϸ2-fold in the 44–88-year age interval (23). However, adult males to those tissues or highly enriched in them (Table 2 and Tables 11 generally have much greater iron stores than adult females, and one and 12, which are published as supporting information on the PNAS might expect the iron stores in donor 1, an 88-year-old female, to web site). Fourteen of the 89 retinal tags correspond to genes be comparable to those in donor 2, a 44-year-old male. Thus, we are already known to cause a retinal disease. All but 6 of the 53 RPE uncertain as to the actual basis for the greatly increased expression tags are unassigned tags, and 1 of these 6 corresponds to a gene of these iron metabolism genes in the retina of the older donor. (VMD2) known to cause a retinal disease. Most of the remaining A few other genes that show a preferential expression in the high frequency retina- or RPE-enriched genes have not been retina from the older donor are of particular interest. Glutathione evaluated for pathogenic mutations among patients with hereditary peroxidase 3 (GPX3) catalyzes the reduction of hydrogen peroxide, retinal diseases. Many are within chromosomal regions that are organic hydroperoxide, and lipid peroxides by reduced glutathione known to harbor unidentified retinal disease genes (Table 3 and and functions in the protection of cells against oxidative damage Table 13, which is published as supporting information on the (Online Mendelian Inheritance in Man database no. 138321). The PNAS web site), and it is reasonable to consider them as candidates high level of expression of this gene in the retina compared with for these or other retinal diseases. We expect that the candidate nonocular SAGE libraries and the higher expression levels in the genes presented here, in combination with candidate genes based older eye makes this gene an attractive candidate for age-related on mouse SAGE retinal libraries (9), will be useful for identifying retinal diseases. The gene is localized to 5q32–33 and was suggested retinal disease genes. previously as a candidate for age-related macular degeneration (24). Another gene showing a higher expression level in the older This work was supported by National Institutes of Health Grants donor encodes clusterin (also known as apolipoprotein J), which is EY08683 and EY09676, Massachusetts Lions Eye Research Fund, Inc., speculated to be involved in , lipid transport, and neuro- the Foundation for Retinal Research, the Ruth and Milton Steinbach degeneration (Online Mendelian Inheritance in Man database no. Fund, Inc., and the Howard Hughes Medical Institute (to S.B.).

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