Isolation and Mapping of Novel Candidate Genes for Retinal Disorders Using Suppression Subtractive Hybridization

Genomics 58, 240 –249 (1999) Article ID geno.1999.5823, available online at http://www.idealibrary.com on

Isolation and Mapping of Novel Candidate Genes for Retinal Disorders Using Suppression Subtractive Hybridization Anneke I. den Hollander,* ,1 Marc A. van Driel,* Yvette J. M. de Kok,* Dorien J. R. van de Pol,* Carel B. Hoyng,† Han G. Brunner,* August F. Deutman,† and Frans P. M. Cremers* *Department of Human Genetics and †Department of Ophthalmology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Received December 28, 1998; revised March 7, 1999

We have constructed human cDNA libraries enriched for retina- and retinal pigment epithelium (RPE)/choroid-specific cDNAs through suppression subtractive hybridization. The sequence of 314 cDNAs from the retina enriched library and 126 cDNAs from the RPE/choroid enriched library was analyzed. Based on the absence of a database match, 25% of the retina cDNA clones and 16% of the RPE/choroid cDNA clones are novel cDNAs. The expression profiles of 86 retina and 21 RPE/choroid cDNAs were determined by a semiquantitative reverse transcription polymerase chain reaction technique. Thirty-three cDNAs were expressed exclusively or most prominently in retina or RPE/choroid. These cDNAs were mapped in the human genome by radiation hybrid mapping. Eleven cDNAs colocalized with loci involved in retinal disorders. One cDNA mapped in a 1.5-megabase critical region for autosomal recessive retinitis pigmentosa (RP12). Another cDNA was assigned to the 7.7-cM RP17 linkage interval. Seven cDNAs colocalized with four loci involved in Bardet–Biedl syndrome. © 1999 Academic Press

Key Words: suppression subtractive hybridization; retina; retinal pigment epithelium; RP12; RP17.

INTRODUCTION

The highly specialized function of the retina requires a large number of specifically expressed genes. Isolation of such genes has contributed to the understanding of the function of the retina and the pathogenesis of retinal disease. Over the last few years the molecular causes of several retinal disorders have been elucidated using positional cloning and candidate gene approaches. As most nonsyndromic retinal disease genes Sequence data from this article have been deposited with the GenBank/dbEST Data Libraries under Accession Nos. AI444803AI444839, AI461440, and AI461441. 1 To whom correspondence should be addressed. Fax: 131.24.3540488. E-mail: [email protected]. 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

identified thus far are expressed exclusively or predominantly in the retina or in the retinal pigment epithelium (RPE), the candidate gene approach is becoming more and more popular (Dryja, 1990, 1997; Sullivan and Daiger, 1996). Thirteen retinal disease genes encode proteins that are part of the phototransduction cascade, for example, rhodopsin, the a subunit of transducin, and the a and b subunits of cyclic guanosine monophosphate phosphodiesterase (cGMP-PDE) (Stryer, 1991; Dryja and Li, 1995; Sullivan and Daiger, 1996; MacDonald et al., 1998). Two genes encode proteins essential for the structure of the photoreceptor disks (RDS/peripherin and ROM-1) (Dryja and Li, 1995). Other retina-specific genes involved in retinal disorders include the retinaspecific ATP-binding cassette transporter (ABCR) gene (Allikmets et al., 1997), a retina-specific transcription factor (CRX) (Freund et al., 1997), the Tubby-like protein (TULP-1) gene (Banerjee et al., 1998; Hagstrom et al., 1998), the X-linked retinoschisis gene (Sauer et al., 1997), and a retina-specific calcium channel (CACNA1F) gene (Bech-Hansen et al., 1998; Strom et al., 1998). Visual processing and survival of the retina depend on a number of highly specialized functions carried out by the RPE. The molecular mechanisms underlying these processes are likely to require a broad spectrum of proteins, some of which may be unique and specific to the RPE. Defects in genes expressed specifically in the RPE may disrupt this supportive function and can lead to disorders of the retina, as exemplified by RPE65 (Gu et al., 1997; Marlhens et al., 1997), cellular retinaldehyde-binding protein (CRALBP) (Maw et al., 1997) and bestrophin (Petrukhin et al., 1998). RPE65 and CRALBP have a role in the transport and metabolism of vitamin A from RPE cells to photoreceptor cells in the retina (Saari et al., 1994; Redmond et al., 1998). Bestrophin may have a role in the metabolism and transport of polyunsaturated fatty acids (Petrukhin et al., 1998). Although a number of genes involved in retinal dis-

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orders do not show expression that is restricted to the eye, e.g., gyrate atrophy (OAT) (Valle and Simell, 1989), choroideremia (REP1) (Cremers et al., 1990), Sorsby’s fundus dystrophy (TIMP-3) (Weber et al., 1994), RP3 (RPGR) (Meindl et al., 1996; Roepman et al., 1996), and RP2 (Schwahn et al., 1998), the isolation of a large number of novel genes expressed exclusively or predominantly in the eye will contribute greatly to the understanding of the function of the retina and the pathogenesis of retinal disease. Several methods have been used to isolate retina- and RPE-specific genes, including differential hybridization (Bascom et al., 1992; Bernstein et al., 1995; Swanson et al., 1997), subtractive hybridization (Swaroop et al., 1991, 1992; Gieser and Swaroop, 1992; Murakami et al., 1993; Agarwal et al., 1995; Higashide et al., 1996; Imamura et al., 1997), and large-scale sequencing of retinal cDNA libraries (Bernstein et al., 1996; Shimizu-Matsumoto et al., 1997). To isolate novel retina- and RPE-specific genes, we constructed cDNA libraries enriched for retina- and RPE/choroid-specific cDNAs using a polymerase chain reaction (PCR)-based suppression subtractive hybridization technique, which normalizes sequence abundance and achieves high enrichment for differentially expressed cDNAs by a single round of subtractive hybridization (Diatchenko et al., 1996). Subtraction was performed against a mixture of cDNAs from several nonocular tissues. The sequence of 440 cDNAs was analyzed and compared with sequences deposited in the GenBank and EMBL databases. The expression profiles of 107 (predominantly novel) cDNAs were determined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Thirty-three cDNAs were expressed specifically or at highest levels in the retina or RPE/choroid compared with other tissues. These were mapped in the human genome by radiation hybrid mapping. Several cDNAs map near loci involved in retinal disorders. MATERIALS AND METHODS Suppression subtractive hybridization. For suppression subtractive hybridization, poly(A) 1 RNA from five human tissues (brain, kidney, liver, heart, skeletal muscle) and total placental RNA were purchased from Clontech. Total RNA from retina, RPE/choroid, psoas muscle, and stomach (mucosal layer) was isolated with RNAzol B (Campro Scientific). Retina and RPE/choroid poly(A) 1 RNA was isolated with the Oligotex mRNA Kit (Qiagen). Complementary DNA (cDNA) was synthesized using the SMART PCR cDNA Synthesis Kit (Clontech). First-strand synthesis was performed on 0.4 mg poly(A) 1 RNA (brain, kidney, liver, heart, skeletal muscle, retina, RPE/choroid) or 1 mg total RNA (placenta, stomach, psoas muscle). Singlestranded cDNA was amplified by long-distance PCR with the Advantage cDNA PCR Kit (Clontech). Prior to subtraction, cDNA was size fractionated with Chroma Spin-1000 columns (Clontech) and digested by RsaI. Digested cDNA was purified with the Advantage PCR-Pure Kit (Clontech). Subtraction of retina and RPE/choroid cDNA was performed with the PCR-Select cDNA Subtraction Kit (Clontech) according to the method described by Diatchenko et al. (1996). Tester cDNA consisted either of retina or of RPE/choroid cDNA. Driver cDNA consisted of a

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mixture of cDNAs from brain, kidney, liver, heart, skeletal muscle, placenta, stomach (mucosal layer), and psoas muscle. Tester cDNA was divided into two portions and ligated to two different adaptors. In a first hybridization step, an excess of denatured driver (450 ng) was added to each sample of denatured tester (12 ng). In the second hybridization step, the two primary hybridization samples were mixed without denaturing, and freshly denatured driver cDNA (300 ng) was added. Differentially expressed cDNAs were amplified by two suppression PCR amplifications with the Advantage cDNA PCR Kit (Clontech). Cloning and analysis of the subtracted cDNA. Subtracted PCR products were ligated into plasmid vector pCRII using a T/A cloning kit (Invitrogen). The resulting constructs were transformed to TOP10F9 competent cells (Invitrogen) and selected by blue/white screening. Approximately 2000 colonies from the retina enriched library and 400 colonies from the RPE/choroid enriched library were picked manually and grown for 16 h in 100 ml LB containing ampicillin (50 mg/ml) in 96-well tissue culture plates. After the addition of 100 ml 30% (w/v) glycerol, the plates were stored at 280°C. Inserts from cDNA clones were recovered by direct PCR amplification of the culture using the vector primers T7 and M13 Reverse. PCR products were electrophoresed with a Centipede horizontal electrophoresis system (200 samples/gel; Owl Scientific). Gels were blotted on Qiabrane Nylon Plus membranes (Qiagen). To determine which cDNA clones had a poly(A) tail, blots were hybridized with a (T) 24A/C/G oligonucleotide. To screen for abundant cDNA clones, the blots were also hybridized with oligonucleotides designed from cDNA sequences encoding rhodopsin (RHO: 59-CTTCTTGCTATTTACAAAGTGC-39), the b subunit of cGMP-PDE (PDE6B: 59-TGATTTCAGAGTTCATGGACC-39), MEK kinase 1 (MEKK1: 59-CAGTATTACTTTGTAGATCACC-39), and an abundant retina-specific cDNA (RET1: 59-AATCCAAAGGAACAGGGAGG-39). Oligonucleotides (20 pmol) were radioactively labeled with 2 pmol [g- 32P]ATP using 10 U T4 polynucleotide kinase (New England Biolabs) and the kinase buffer provided by the manufacturer. Oligonucleotide hybridizations were performed in 53 SSPE, 0.3% SDS at 50°C for 16 h. Blots were washed with 53 SSPE, 0.3% SDS at 45°C. Insert PCR products were isolated from an agarose gel with the Qiaquick Gel Extraction Kit (Qiagen). DNA sequencing was performed with the Thermo Sequenase Dye Terminator Cycle Sequencing Pre-mix Kit (Amersham) using an automated DNA sequencer (Applied Biosystems, Inc, Model 370A). Clones containing a poly(A) tail were sequenced with a (T) 24A/C/G oligonucleotide, the remaining cDNA clones were sequenced with the vector primers. Nucleic acid homology searches were performed using the FASTA program (Pearson and Lipman, 1988) against the GenBank and EMBL databases. Expression profile analysis by semiquantitative RT-PCR. For semiquantitive RT-PCR, total RNA from eight human tissues (brain, liver, lung, skeletal muscle, placenta, heart, spleen, kidney) was purchased from Clontech. Total RNA from retina, RPE/choroid, and two RPE cell lines, D407 (Davis et al., 1995) and ARPE-19 (Dunn et al., 1995), was isolated by RNAzol B (Campro Scientific) and treated with DNase I (Gibco/BRL). Randomly primed cDNA was synthesized in a volume of 250 ml from 3.1 mg total RNA using 4 pmol random hexanucleotides [pd(N) 6; Pharmacia], 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 0.01% (w/v) gelatin, 5 mM MgCl 2, 1 mM DTT, 95 U RNAguard (Pharmacia), and 2500 U MMLV reverse transcriptase (Gibco/BRL). Primer sets were chosen with the PRIMER program (Version 0.5, Whitehead Institute for Biomedical Research) from the nucleotide sequence of 107 retina and RPE/choroid cDNA clones, amplifying products of 100 –250 bp. The primer sets were first tested on genomic DNA to test the optimal annealing temperature and MgCl 2 concentration of the PCR. Semiquantitative RT-PCRs were performed in a volume of 35 ml using 87 ng of randomly primed cDNA, 11.2 pmol of each primer, 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 0.01% gelatin, 0.5 mM MgCl 2 (for most primer sets), 160 mM of each dNTP, and 1.75 units Taq DNA polymerase (Gibco/BRL). Amplification was performed at 94°C for 1 min, 60°C for 1 min (for most primer sets), and 72°C for 1 min in a microtiter PCR machine (MG Research). After 25 cycles a 10-ml sample was taken from the PCRs,

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and an additional 5 cycles (for a total of 30 cycles) were performed. Again, a 10-ml sample was taken from the PCRs, and the PCR was continued for an additional 5 cycles for a total of 35 cycles. Northern blot analysis. RNA from the tissues used for the RTPCR were also used for the Northern blot analysis. Ten micrograms of total RNA of each tissue was loaded on a 1% agarose gel containing 13 MOPS and 18% formaldehyde. Gels were blotted on GeneScreen Plus membranes (NEN Life Science Products) with 103 SSC. Hybridizations were performed in 53 SSPE, 50% deionized formamide, 53 Denhardt’s, 1% SDS at 60°C. Blots were washed in 23 SSPE, 2% SDS at 60°C. Radiation hybrid mapping. Radiation hybrid mapping was performed with the Stanford G3 Radiation Hybrid Panel (Research Genetics). PCRs were performed in a volume of 10 ml using 25 ng DNA of each hamster/human hybrid cell line, 4 pmol of each primer, 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 (for most primer sets), 5 mM DTT, 200 mM of each dNTP, and 0.4 unit Taq DNA polymerase (Gibco/BRL). Amplification was performed for 35 cycles at 94°C for 1 min, 60°C for 1 min (for most primer sets), and 72°C for 1 min in a microtiter PCR machine (MG Research). The localization of the cDNA fragments in the Stanford G3 Radiation Hybrid Map was determined at the Stanford Human Genome Center website (http://www-shgc.stanford.edu).

RESULTS

Subtraction Efficiency An efficient suppression subtractive hybridization decreases the concentration of ubiquitously expressed (housekeeping) genes, and increases the concentration of tissue-specific genes (Diatchenko et al., 1996). Equal amounts of subtracted and unsubtracted cDNA from the retina and RPE/choroid were size-separated and blotted. These blots were hybridized with two housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and b-actin (ACTB). The retina cDNA blot was hybridized with three retina-specific

FIG. 1. Southern blot analysis of retina and RPE/choroid cDNA before (lane B) and after (lane A) subtraction. The concentrations of two housekeeping genes, GAPDH and ACTB, are considerably lower after subtraction. The concentrations of three retina-specific genes, RHO, PDE6B, and RET10, and two RPE-specific genes, RPE65 and CRALBP, are increased considerably after subtraction.

TABLE 1 Database Match of cDNA Clones from the Retina and RPE/Choroid Enriched cDNA Libraries a Database match 1. Known genes a. Not retina specific b. Retina specific 2. ESTs/cDNAs a. Not retina specific b. Retina specific c. Pineal gland specific 3. No database match Total

Retina cDNA library

RPE/choroid cDNA library

39 (14) b 27 (10) b

24 (19) 7 (5)

109 (39) 27 (10) b 4 (2) 69 (25) 275 b

71 (56) 2 (2) 2 (2) 20 (16) 126

a

The number (and percentage) of clones that show more than 95% identity to known genes (1) or ESTs and cDNAs (2) or that show no database match (3) are given. b Thirty-nine repetitively encountered clones were excluded from sequence analysis and were not included in this table.

genes: rhodopsin (RHO), the b subunit of cGMP-PDE (PDE6B), and a retina-specific cDNA isolated in this study (RET10), which is expressed at a low level in retina. The RPE/choroid cDNA blot was hybridized with two RPE-specific genes, RPE65 and CRALBP. In both libraries the concentration of the two housekeeping genes was considerably decreased and the concentration of the tissue-specific genes was increased considerably after subtraction (Fig. 1). Sequence Analysis of cDNAs from the Retina and RPE/Choroid Enriched Libraries Prior to sequencing, cDNA clones were analyzed for the presence of a poly(A) sequence by Southern blot analysis with a (T) 24A/C/G oligonucleotide. From the retina enriched library, 121 of 314 cDNA cDNA clones (39%) were positive; from the RPE/choroid enriched library 65 of 126 (52%) were positive. Positive clones in most cases represent 39-end cDNA fragments carrying a poly(A) tail, but may also be cDNA fragments with an internal poly(A) or poly(T) stretch. Initially, 118 cDNAs from the retina enriched library were partially sequenced and compared with the GenBank and EMBL databases. Four cDNAs were found more than once, including PDE6B (13/118), RHO (3/ 118), MEK kinase 1 (MEKK1) (4/118), and RET1 (4/ 118) (Table 2). The sequencing of additional cDNA clones was preceded by Southern blot analysis of 196 retina cDNAs with radioactively labeled oligonucleotides specific for PDE6B, RHO, MEKK1, and RET1. Thirty-nine cDNA clones were found to be positive, and were excluded from sequence analysis. The remaining 157 cDNAs were sequenced. In conclusion, we analyzed a total of 314 cDNAs from the retina enriched library, 275 of which were sequenced. From the RPE/choroid enriched library, 126 cDNA clones were sequenced and compared with sequences deposited in the GenBank and EMBL databases. Re-

NOVEL CANDIDATE GENES FOR RETINAL DISORDERS

TABLE 2 Known Genes Encountered in the Retina Enriched Library Gene cGMP phosphodiesterase b MEK kinase 1 (mouse) Nucleosome assembly protein Rhodopsin cGMP gated channel a Hexokinase II pseudogene Phospholipase A2 Prothymosin a a-Tropomyosin Capping protein a1 Chromosomal protein Cytochrome c oxidase Dystrobrevin isoform DTN-2 HS1 protein Insulin growth factor-binding protein 5 KIAA0168 gene KIAA0240 gene Myosin VIIA Mouse tob family Na-dependent neurotransmitter transporter (rat) Protein phospatase 1 subunit b scg (mouse) Stanniocalcin precursor Transcription factor ETR103 Voltage-dependent Ca 21 channel b-2c U2 snRNA associated B antigen X16 gene (mouse)

No. of clones .20 .11 4 .3 3 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

petitively encountered clones in this library included insulin-like growth factor-binding protein 5 (IGFBP5) (12/126), RHO (3/126), and MEKK1 (3/126). Comparison of the cDNA sequences with cDNAs and ESTs deposited in databases resulted in “expression profiles” based on their representation in other cDNA libraries (Table 1). In the retina and RPE/choroid enriched libraries, 58 (22%) and 11 (9%) of the cDNA clones respectively show homology to known retina- or pineal gland-specific cDNAs or known genes; 69 (25%) and 20 (16%) of the cDNA clones respectively represent novel cDNAs; and 148 (53%) and 95 (75%) respectively show homology to cDNAs and known genes expressed in other tissues as well. However, when the 39 frequently encountered cDNAs from the retina enriched library that were not sequenced are also considered, approximately 30% of the retina enriched cDNAs show homology to known retina- or pineal gland-specific cDNAs or genes, 50% show homology to cDNAs expressed in other tissues as well, and 20% are novel. Several genes already known to be involved in retinal disorders were isolated from the retina and RPE/ choroid enriched libraries, including PDE6B, RHO, the a subunit of cGMP gated channel (CNGC), myosin VIIA (MYO7A), and tissue inhibitor of metalloproteinases-3 (TIMP-3). The 314 analyzed retina cDNA clones represent 199 different cDNA fragments; 161 were found only once, 20 were found twice, 6 were found 3 times, and 12 were

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found 4 times or more. The 126 analyzed RPE/choroid cDNA clones represent 101 different cDNA fragments; 87 were found only once, 9 were found twice, 3 were found 3 times, and only 2 were found 4 times or more. Seventeen cDNAs were encountered both in the retina and in the RPE/choroid enriched library. Expression Profile Analysis of Novel Retina and RPE/Choroid cDNAs Eighty-six cDNA clones from the retina enriched library and 21 cDNA clones from the RPE/choroid enriched library were selected for expression profile analysis by RT-PCR. Based on their comparison with DNA sequences in the GenBank and EMBL databases, 60 of these clones were novel, 11 were identical to cDNAs represented specifically in other retina cDNA libraries, and 18 were identical to cDNAs represented in retina and/or pineal gland, brain, neuroepithelium, or cochlea cDNA libraries. Furthermore, 5 were identical to cDNAs listed in the Bodymap database (http://cookie. imcb.osaka-u.ac.jp/bodymap/) and 13 cDNA clones were identical to cDNAs represented in other libraries that were of interest because of their homology to known genes or their high abundance in retina, pineal gland, or brain cDNA libraries. We performed a semiquantitative RT-PCR (25, 30, and 35 cycles) using RNA from 12 tissues, including liver, lung, skeletal muscle, placenta, heart, brain, spleen, kidney, retina, two RPE cell lines (ARPE-19 and D407), and RPE/choroid. To test the usefulness of our semiquantitive RTPCR, we determined the expression profile of several known retina- or RPE-specific genes, e.g., ABCR (Allikmets et al., 1997), RDS (Travis et al., 1991), ROM-1 (Bascom et al., 1992), phosducin (PDC) (Abe et al., 1990), and CRALBP (Bunt-Milam and Saari, 1983) (Table 3). All genes were expressed considerably higher in retina or RPE/choroid than in all other tissues, but for some, lower levels of expression were also detected in other tissues (e.g., Fig. 2). Based on the expression profiles of the 86 cDNA clones selected from the retina enriched library, 30 were considered candidate genes for retinal disorders. Of these 30 cDNA clones, 9 were expressed only in retina or RPE/choroid, 12 were expressed considerably higher in retina or RPE/choroid than in all other tissues tested, and 9 were expressed in retina or RPE/ choroid and in one or two other tissues after the same number of PCR cycles. Of the 21 cDNA clones selected from the RPE/choroid enriched library, 2 were expressed only in retina or RPE/choroid, and 1 was expressed considerably higher in the retina and RPE/ choroid than in the other tissues. Thus, after expression profiling, 33 of 107 cDNAs were considered good candidates for retinal disorders. Of these 33 cDNA clones, 15 showed no database match; 8 clones were identical to cDNAs represented specifically in other retina cDNA libraries; 4 were identical to cDNAs represented in retina and/or pineal

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TABLE 3 Expression Profile Analysis of Five Known Retina- or RPE-Specific Genes by Semiquantitative RT-PCR a Gene

Liver

Lung

Skeletal muscle

Placenta

Heart

Brain

Spleen

Kidney

Retina

ARPE-19

D407

RPE 1 choroid

ABCR RDS ROM-1 PDC CRALBP

222 222 221 222 222

222 222 221 222 222

222 222 221 222 222

221 221 221 222 222

222 221 221 222 222

221 221 211 222 211

222 221 211 222 222

221 221 211 222 222

211 111 111 221 111

222 222 211 222 221

222 222 221 222 222

211 211 111 222 111

RT-PCR products detected in agarose gel electrophoresis after 25, 30, and 35 cycles are indicated by a 1 at the first, second, or third position, respectively, for each tissue analyzed. a

gland, brain, neuroepithelium, or cochlea cDNA libraries; 3 were identical to cDNAs from the Bodymap database; and 3 were identical to cDNAs represented in other libraries that were considered interesting by homology to known genes or high abundance in retina, pineal gland, or brain cDNA libraries. The nucleotide sequences of the 33 retina- and RPE/choroid-specific cDNAs were submitted to the GenBank/dbEST databases; GenBank accession numbers are listed in Table 4. Clones that show no database match to other ESTs (,90% homology) in boldface. Radiation Hybrid Mapping of Retina- and RPE/Choroid-Specific cDNAs Thirty of the thirty-three selected cDNA clones were mapped in the human genome by radiation hybrid mapping (Table 4). One cDNA, RET2E2, was mapped by its homology to a genomic clone of which the complete sequence was deposited in the database. Two cDNAs (RET1D1 and RET4B7) could not be mapped. The genetic intervals of the mapped cDNA clones were determined relative to framework markers. These genetic intervals were compared with linkage intervals of nonsyndromic and syndromic retinal disorders using the RetNet database (http://www.sph.uth.tmc.edu/ Retnet/) and reviews listing retinal disease loci (Rosenfeld et al., 1994; Sullivan and Daiger, 1996; MacDonald et al., 1998; Simunovic and Moore, 1998). Eleven cDNA

clones localized near loci involved in retinal diseases (Table 5). RET3C11 is a positional candidate for autosomal recessive RP (RP12) on 1q31– q32.1 (Van Soest et al., 1996). The RP12 critical region has been refined to a 1.3-cM interval (Van Soest et al., in press). RET3C11 was mapped on a YAC contig spanning the RP12 critical region (Van Soest et al., in press). Recently, a locus for age-related macular degeneration (ARMD1) was mapped to 1q25–1q31 (Klein et al., 1998). The involvement of RET3C11 in RP and ARMD is currently under investigation. RET1G11 is a positional candidate for autosomal dominant RP (RP17), the gene defect of which was localized on 17q22. The RP17 locus was first described in two South African families (Bardien et al., 1995, 1997). Recently, we found linkage to the RP17 locus in a large Dutch family and refined the critical region to a 7.7-cM interval between markers D17S1607 and D17S948 (den Hollander et al., 1999). By radiation hybrid mapping, RET1G11 was localized near marker D17S1607 and is currently being analyzed. RET1G11 may also be a candidate gene for cone–rod dystrophy (CORD4) on 17q (Kylstra and Aylsworth, 1993). Seven cDNA clones are positional candidate genes for Bardet–Biedl syndrome (BBS), which is characterized by pigmentary retinopathy, mental retardation, obesity, and polydactyly. Five BBS loci have been iden-

FIG. 2. Expression profile analysis of the ABCR gene and two clones isolated from the retina enriched library (RET1 and RET1D2) by semiquantitative RT-PCR (25, 30, and 35 cycles). GAPDH serves as a positive control for the RNA. (1) Liver, (2) lung, (3) skeletal muscle, (4) placenta, (5) heart, (6) brain, (7) spleen, (8) kidney, (9) retina, (10) ARPE-19, (11) D407, (12) RPE/choroid.

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TABLE 4 Chromosomal Localization and Expression Profiles of 33 Retina (RET) and RPE/Choroid cDNA Clones That Are Expressed Specifically in the Retina or RPE/Choroid, or Considerably Higher in the Retina or RPE/Choroid than in Most Other Tissues Tested a

Chrom. 1 3 5 6

7 8 11 12 14 15 16

17 19

21 X

?

Chromosomal region (cM)

Clone name

GenBank Accession No.

Liver

Lung

Skeletal muscle

Placenta

Heart

Brain

Spleen

Kidney

Retina

ARPE-19

D407

RPE 1 choroid

213–216 117 117 74–79 141 13–17 13–17 60–65 60–65 60–66 65–66 73–74 74 82–93 62–65 110 90–96 46–58 22–26 70–71 75–77 75–77 75–77 75–82 50–58 50–58 69–81 44–50 122–150 122–150 122–150 ? ?

RET3C11 RPE63 RPE1H6 RET2E2 RET1A6 RET1F6 RET1F12 RET5 RET1B10 b RET1A4 RET1 RET3 b RET3E4 b RET1B3 b RET2G9 RET1D2 RET2F8 b RPE1G2 b RET3F3 b RET1B8 RET1H7 b RET3B3 RET3E3 b RET1G11 RET29 RET3D11 b RET1F4 b RET2C9 b RET10 b RET1A12 b RET2A6 b RET1D1 RET4B7

AI444813/14 AI444822 AI444824 AI444808 AI444832 AI444803 AI444804 AI444827 AI444837 AI444831 AI444825 AI444826 AI444817/18 AI444835 AI444810 AI444838 AI444809 AI444823 AI444819 AI444836 AI461441 AI444811/12 AI444816 AI444805 AI444830 AI444815 AI444839 AI444807 AI444828/29 AI444834/35 AI444806 AI461440 AI444821

222 222 222 222 222 222 221 221 221 221 221 222 211 222 222 222 222 222 222 211 222 222 222 222 221 222 222 221 222 222 222 221 221

222 222 222 222 222 222 211 221 211 211 222 222 221 221 221 222 222 222 221 211 222 222 222 222 211 221 222 221 222 222 222 221 221

222 222 222 222 222 222 221 221 211 211 221 222 221 222 222 222 222 222 222 221 222 222 222 222 211 221 222 221 222 222 222 221 221

222 222 222 222 222 222 211 211 211 211 221 222 222 222 211 222 222 222 222 211 222 222 222 221 221 221 222 221 222 221 221 221 222

222 222 222 222 222 222 211 222 211 221 211 222 221 222 221 222 222 222 222 211 222 222 222 222 211 221 222 221 222 222 222 221 221

222 222 222 221 222 222 211 221 211 211 211 222 221 211 221 222 222 221 211 211 222 221 222 2221 211 221 222 211 222 222 222 211 211

222 222 222 222 222 222 211 211 211 221 221 222 221 221 221 222 222 222 222 211 222 222 222 222 221 222 222 221 222 221 222 221 211

222 222 222 222 222 222 211 221 211 221 221 221 211 211 221 222 222 222 222 211 222 222 222 222 211 221 222 211 222 222 222 221 221

221 221 211 221 221 211 111 211 111 111 111 221 211 211 211 111 221 211 211 111 221 211 221 211 111 211 111 211 221 211 211 211 111

222 222 222 222 222 222 221 221 221 221 221 222 222 222 221 222 222 221 222 221 222 222 222 222 221 222 222 221 222 222 222 211 211

222 222 222 222 222 222 221 221 221 221 222 222 222 222 222 222 222 222 222 221 222 222 222 222 221 222 222 221 222 222 222 211 222

222 222 222 222 221 222 211 222 221 211 222 222 221 221 222 222 222 211 221 211 222 222 222 222 211 222 211 222 222 221 222 221 211

a Chromosomal localizations were determined by radiation hybrid mapping. RT-PCR products detected in agarose gel electrophoresis after 25, 30, or 35 cycles are indicated by a 1 at the first, second, or third position, respectively, for each tissue analyzed. b Clones that show no database match to other ESTs (,90% homology) are in boldface.

tified until now; BBS1 on chromosome 11q13 (Leppert et al., 1994), BBS2 on 16q21 (Kwitek-Black et al., 1993), BBS3 on 3p13–p12 (Sheffield et al., 1994), BBS4 on 15q22.3– q23 (Carmi et al., 1995), and BBS5 on 2q31 (Young et al., 1999). Positional candidates are RET2G9 for BBS1; RET1H7, RET3B3, and RET3E3 for BBS2; RPE63 and RPE1H6 for BBS3; and RET1B8 for BBS4. RPE1G2 may be a positional candidate for total col-

orblindness (ACHM1) on chromosome 14 (Pentao et al., 1992). RET1F4 maps near a locus for optic atrophy with ataxia and 3-methylglutaconic aciduria (OPA3) on chromosome 19q13.2– q13.3 (Nystuen et al., 1997). Some cDNAs mapped in this study were localized in the same chromosomal region, and may represent different RsaI fragments from the same gene. For example, RPE63 and RPE1H6 map to the same region on

TABLE 5 Comparison of Chromosomal Map Locations between 11 Chorioretinal cDNAs and Inherited Retinal Disease Loci a Chromosome

Clone name

Chromosomal region (cM)

1 3 11 14 15 16

RET3C11 RPE63/RPE1H6 RET2G9 RPE1G2 RET1B8 RET1H7/RET3B3/RET3E3

213–216 117 62–65 46–58 70–71 75–77

17 19

RET1G11 RET1F4

a

75–82 69–81

Disease locus RP12 BBS3 BBS1 ACHM1 BBS4 BBS2 RP17 CORD4 OPA3

Cytogenetic location

Chromosomal region (cM)

1q31–q32.1 3p13–p12 11q13 14 15q22.3–q23 16q21 17q22 17q 19q13.2–q13.3

213–216 114–125 63–72 ? 63–71 72–81 76–84 ? 68–69

The genetic mapping intervals of these clones were determined relative to framework markers.

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Subtraction Efficiency and Evaluation of the Libraries

FIG. 3. Northern blot analysis of RET1. Lanes contain 10 mg of total RNA from the following human tissues: (1) liver, (2) lung, (3) skeletal muscle, (4) placenta, (5) heart, (6) brain, (7) spleen, (8) kidney, (9) retina, (10) ARPE-19, (11) D407, (12) RPE/choroid.

chromosome 3 and have similar expression profiles (Table 4). A cluster of four cDNAs (RET5, RET1B10, RET1A4, RET1) maps to 6p21 (60 – 66 cM), and the expression profiles of these cDNAs are very similar (Table 4). We have isolated a cDNA clone from a full-length retina cDNA library, and demonstrated that RET1 and RET1A4 belong to the same gene. RET1A4 colocalizes with the RDS gene in the radiation hybrid map, and the expression profile of RDS is similar to that of all four cDNAs (Table 3). This result suggests that these cDNAs may belong to the RDS gene. Northern blot analysis shows that the RDS gene is represented by transcripts of approximately 3 and 6.5 kb (Travis et al., 1991). The 3-kb transcript has been sequenced and deposited in the database. The four cDNAs isolated in this study could represent RsaI fragments of the 6.5-kb transcript. RET1 was analyzed by Northern blot analysis and hybridizes to a transcript of approximately 6 kb (Fig. 3). Other cDNA fragments that may belong to the same gene are RET1F6 and RET1F12 on chromosome 6 (13–17 cM); RET3 and RET3E4 on chromosome 7 (73–74 cM); RET1H7, RET3B3, and RET3E3 on chromosome 16 (75–77 cM); RET29 and RET3D11 on chromosome 19 (50 –58 cM); and RET10, RET1A12, and RET2A6 on the X chromosome (122–150 cM). DISCUSSION

This study describes the isolation and mapping of 33 retina- and RPE-specific cDNAs. Libraries enriched for retina- and RPE/choroid-specific cDNAs were constructed through suppression subtractive hybridization. The sequence of 314 cDNAs from the retina enriched library and 126 cDNAs from the RPE/choroid enriched library was analyzed and compared with the GenBank and EMBL databases. The expression profiles of 86 retina and 21 RPE/choroid (predominantly novel) cDNAs were determined by a semiquantitative RT-PCR. Thirty-three cDNAs were expressed specifically or considerably higher in retina or RPE/choroid than in most other tissues analyzed, and were mapped in the human genome by radiation hybrid mapping.

The subtraction of the retina and RPE/choroid cDNA was very efficient, as the concentration of two housekeeping genes (GAPDH and ACTB) is considerably decreased after subtraction, and the concentration of several tissue-specific genes (RHO, PDE6B, and RET10 for the retina and RPE65 and CRALBP for the RPE) is considerably increased after subtraction. Based on homology with retina specific genes/cDNAs and on expression profiling by RT-PCR, approximately 30% of the retina enriched cDNA library is estimated to be retina specific. In an unsubtracted, nonnormalized retina cDNA library, approximately 2% of the cDNAs are estimated to be retina specific (S. Bernstein, personal communication), suggesting a considerable enrichment of retina-specific transcripts in the retina enriched cDNA library. The RPE/choroid enriched library was constructed from RPE that was attached to the choroid. The attached choroidal tissue hampers the isolation of RPEspecific genes from this library. Nevertheless, this library is the first human RPE subtracted library reported in the literature. One subtracted RPE library has been constructed previously from an RPE cell line (Gieser and Swaroop, 1992). Gene expression in this cell line differs from that in RPE tissue in vivo, as several of the known genes isolated from this library are genes involved in cell growth and proliferation (Gieser and Swaroop, 1992). In contrast to conventional subtraction methods and differential hybridization techniques previously used to isolate retina-specific genes, suppression subtractive hybridization allows the isolation of tissue-specific transcripts expressed at moderate or low levels in the tissue of interest. Such transcripts are underrepresented in the collection of retina ESTs deposited in the public databases. Consequently, the suppression subtractive hybridization technique enables the isolation of novel retinal and RPE/choroidal cDNAs. Twenty-five percent of the retina cDNA clones and 16% of the RPE/choroid cDNA clones were novel. Thirty-nine percent of the cDNA clones from the retina enriched library and 52% of the cDNA clones from the RPE/choroid enriched library contain a poly(A) stretch, most of which represent cDNA fragments containing a poly(A) tail. As most ESTs deposited in the public databases represent 39 ends of genes, most cDNAs encountered in these libraries that show no database match are truly novel. The representation of RsaI fragments for several known genes in the retina enriched cDNA library was analyzed (data not shown). Most RsaI fragments encountered are small RsaI fragments (,500 bp) from the 39 region of the genes. As suppression subtractive hybridization is a PCR-based technique, small RsaI fragments will preferentially be amplified over large RsaI fragments. A disadvantage of this method is that

NOVEL CANDIDATE GENES FOR RETINAL DISORDERS

genes that lack small RsaI fragments are underrepresented in the libraries. To bypass this problem, other restriction enzymes might be used instead of RsaI. The cDNA used for the subtraction was synthesized by oligo(dT) priming with the SMART PCR cDNA Synthesis Kit (Clontech). With this kit high yields of cDNA can be amplified from nanogram amounts of poly(A) 1 or total RNA. As the cDNA is amplified by PCR and not all transcripts will be amplified equally efficiently, the cDNA will not be a complete representation of the mRNA in vivo. Moreover, we encountered cloning artifacts in two genes (PDE6B and RHO), which are both probably caused by priming to an internal poly(T) stretch during the PCR amplification of the cDNA. This may have led to the preferential amplification of the 39 end of the PDE6B gene during the cDNA synthesis, as one of the RsaI fragments was encountered 13 times in the first 118 cDNA clones analyzed from the retina enriched library (Table 2). Expression Profiling Another important aspect of our strategy in isolating novel retina- and RPE-specific genes is the expression profiling of a large number of cDNAs from the retinaand RPE/choroid-enriched libraries by a semiquantitative RT-PCR method. For most ESTs deposited in the public databases, no expression data have been determined. The different cDNA libraries in which an EST is encountered may give an idea of its expression, but will depend largely on how many cDNAs from each tissue type are deposited in the public databases. From 314 retina cDNA clones that were analyzed, 86 were selected for expression profile analysis by semiquantitative RT-PCR. Thirty cDNAs are expressed specifically in the retina or considerably higher in the retina than in most other tissues tested. From 126 analyzed RPE/choroid cDNA clones, 21 were selected for expression profile analysis. Three cDNAs are expressed specifically in the retina or RPE/choroid. No cDNAs were isolated that are expressed exclusively in the RPE. This may have two reasons. First, retina and RPE cannot be separated completely. Therefore, retina RNA will contain approximately 1% RPE RNA and vice versa. Consequently, RPE-specific cDNAs will also show expression in the retina on RT-PCR. Second, the RNA used for the semiquantitative RT-PCR was isolated from RPE that was attached to the choroid. As the amount of RNA from the RPE is only a fraction of the isolated RNA, truly RPE-specific cDNAs will be detected only after a larger number of cycles. It is therefore possible that the expression detected in the RPE/choroid by RT-PCR would actually be higher in pure RPE tissue. To compensate for this problem, we included two RPE cell lines in the RT-PCR panel. However, the expression of genes in these cell lines does not represent the in vivo situation.

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Mapped Retina- and RPE-Specific cDNAs The 33 cDNAs expressed specifically or at a high level in the retina or RPE/choroid represent good candidate genes for retinal disorders. These cDNAs were mapped in the human genome by radiation hybrid mapping. Eleven of these cDNAs localized near loci involved in retinal disorders. Candidate cDNAs were isolated for retinitis pigmentosa, age-related macular degeneration, Bardet–Biedl syndrome, total colorblindness, cone–rod dystrophy, and optic atrophy. We are currently investigating two cDNAs (RET3C11 and RET1G11) for their involvement in two forms of RP (RP12 and RP17). Several cDNAs mapped in this study localize to the same chromosomal region. These cDNAs may represent different RsaI fragments of the same gene. A cluster of four cDNAs on 6p21 (60 – 66 cM) may represent RsaI fragments of a 6.5-kb transcript of the RDS gene. In the near future we will scale up the analysis of the retina and RPE/choroid enriched cDNA libraries. The expression profiling of cDNAs provides important information that is highly complementary to the ongoing human genome sequencing project. We will concentrate future efforts on the structural characterization of retina- or RPE-specific genes which should enable us to identify several novel genes underlying chorioretinal diseases. ACKNOWLEDGMENTS We thank Dr. L. Pels of The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands, for the collection of retina and RPE/choroid tissues. We thank Dr. J. Janssen, Dr. A. Davis, and Dr. L. Hjelmeland for kindly providing the RPE cell lines. This work was supported by The Foundation Fighting Blindness, Inc., USA, and The British Retinitis Pigmentosa Society.

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