Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Microarray Analysis of Gene Expression in Human Donor Corneas

Microarray Analysis of Gene Expression in Human Donor Corneas ObjectivesTo use microarray analysis to identify genes expressed in human donor corneas and to create a preliminary, comprehensive database of human corneal gene expression.MethodsA complementary DNA (cDNA) library was constructed from transplant-quality, human donor corneas. Biotin-labeled RNA was transcribed from the cDNA library and hybridized in duplicate to microarrays containing approximately 5600 human genes. Results were analyzed using a gene database of the National Institutes of Health, Bethesda, Md. Reverse transcriptase polymerase chain reaction analysis was performed to confirm corneal expression of genes identified by microarray analysis.ResultsDuplicate microarrays identified the expression of 1200 genes in human donor corneas. Chromosomal loci had been assigned to 1025 (85%) of these genes. A preliminary database of human corneal gene expression was compiled. A Web site containing these genes was created. Six collagen genes were identified that had not previously been localized within the cornea. Five apoptosis-related genes were identified, 4 of which had not previously been localized within the cornea. Three genes previously shown to cause corneal diseases were identified. Reverse transcriptase polymerase chain reaction analysis of genes identified by microarray analysis confirmed the corneal expression of 2 apoptosis-related genes and 1 collagen gene.ConclusionsMicroarray analysis of healthy human donor corneas has produced a preliminary, comprehensive database of corneal gene expression. Large-scale analysis of gene expression has the potential to generate large amounts of data, which should be made readily accessible to the scientific community. The Internet offers many potential advantages as a medium for the maintenance of these large data sets.Clinical RelevanceIdentification of structural, apoptosis-related, and disease-causing genes within the cornea by microarrays may increase the understanding of normal and abnormal corneal function with likely relevance to corneal diseases and transplants.FUNDAMENTAL to understanding normal tissue function is a global knowledge of the thousands of genes expressed in the various cell types that comprise that tissue. This baseline knowledge should facilitate the identification of alterations from normal gene expression that play important roles in disease pathogenesis. Efforts to comprehensively study gene expression patterns in normal tissues and altered gene expression patterns in diseased tissues will require a complete knowledge of the human genetic sequence and methods to accurately and simultaneously analyze large amounts of genetic information. Both of these requirements have recently become available through the ongoing progress of the Human Genome Project and breakthroughs in high-efficiency genetic analysis techniques such as DNA microarrays.DNA microarrays are a new and powerful technique to study the expression of thousands of genes in a single experiment.A microarray is a solid substrate such as a glass slide or nylon membrane to which known, single-stranded DNA molecules are attached at distinct locations. The density of these locations on a microarray can reach upwards of 250 000/cm2, as demonstrated by van Hal et al.Experimental messenger RNA (mRNA) is labeled as a complex mixture and exposed to the microarray. Labeled mRNA molecules will bind to complementary sequences on the microarray and can be detected in a semiquantitative manner using automated techniques. Advantages of DNA microarrays include simultaneous screening for the expression of large numbers of genes, the ability to use small amounts of starting material, and mass production, which enables standardized, comparative analysis between samples.The acceptance of this technology is growing rapidly as microarrays are being used in an increasing number of experimental applications. These include analysis of gene expression in normal embryonic developmentand pathologic states such as breast cancerand myocardial infarction.Microarrays also have been used to identify novel genes expressed in brain tissueand for positional cloning of a disease gene in Niemann-Pick disease, type C.Given the successful use of microarray analysis in other biological systems, we sought to apply this technique to study gene expression in human donor corneal tissue. Such analysis may be useful for understanding the genetic basis of normal corneal function as well as corneal disease processes such as graft failure, inflammation, degenerations, and dystrophies. Knowledge of what genes are or are not expressed in a given corneal disorder could lead to new and definitive treatment strategies, including interventional drugs and gene therapies. These strategies may be particularly relevant and feasible for the cornea because the tissue is relatively less complex, can be manipulated ex vivo, and can be easily assessed visually.Microarray analysis is a feasible method to begin compiling a comprehensive database of genes expressed in human corneas. Such a database of genes may have broad applications for corneal genetics research. Any comprehensive database of corneal genes would be expected to be relatively large and to grow as more genes are identified in this tissue and as the assembly phase of the Human Genome Project defines novel genes from currently available sequence information. Such a large database would be most useful if it could be readily updated, freely accessible to the global research community, and effectively interfaced with preexisting gene databases. Given these desirable features, an Internet Web site could be an ideal format for a comprehensive corneal gene database. Such a Web site could potentially enhance the progress of corneal genetics research by increasing the accessibility of relevant genetic information and facilitating discussion among corneal genetics researchers. As the proposed comprehensive corneal gene database grows and becomes more clinically relevant, it also could serve as a model for similar efforts in other clinical disciplines.MATERIALS AND METHODSTwenty corneal-scleral rims were obtained at the time of penetrating keratoplasty, and peripheral corneal tissue was carefully dissected, placed in microcentrifuge tubes, and immediately stored at −80°C. Two entire transplant-quality donor corneal buttons were placed in microcentrifuge tubes and immediately stored at −80°C. The death to preservation time of all tissues used in this study was less than 12 hours. All tissues used in this study were obtained from donors younger than age 65 years. A complementary DNA (cDNA) library was constructed using standard methods from the pooled corneal tissues described.The number of clones contained in the primary cDNA library was estimated to be 1.0 × 106.Standard methods were used to recover phagemids by mass excision protocol (pBluescript; Stratagene, La Jolla, Calif). The number of plasmids excised was 1.6 × 106. The ratio of clones excised to the number of independent clones in the library was 1.6:1. Excised clones were used to transfect a large-volume cell culture (SOLR; Stratagene, and plasmid "maxi-preps" were performed with a standard kit and protocol (Qiagen, Valencia, Calif). Plasmids were digested using restriction endonuclease (NotI; Life Technologies, Rockville, Md), phenol-chloroform extracted, and ethanol precipitated.Biotin-labeled cRNA molecules were produced by in vitro transcription (Enzo Diagnostics, Farmingdale, NY), digested with DNase I (Life Technologies), and purified using commercially available spin columns (RNeasy; Qiagen). Analysis of biotin-labeled cRNA using the HuGeneFL microarray (Affymetrix, Santa Clara, Calif) was performed in duplicate (Research Genetics, Huntsville, Ala) by hybridizing the same labeled cRNA sample to 2 identical microarrays within a 6-week time period.Microarray analysis results were analyzed using GenBankand LocusLink,online genetic databases sponsored by the National Library of Medicine of the National Institutes of Health, Bethesda, Md. A corneal genetics web site was created using the y-Base Informatics Engine (y-DNA Inc, Palo Alto, Calif).Total RNA was extracted using TRIZOL reagent (Life Technologies) from 2 pooled, whole, transplant-quality donor corneas. Reverse transcriptase polymerase chain reaction (RTPCR) was performed using standard methods (Applied Biosystems, Foster City, Calif). One microgram of total RNA was used as template for first-strand cDNA synthesis. Polymerase chain reaction (PCR) was performed using 5 µL of each cDNA sample in a final reaction volume of 100 µL. A final concentration of 2.5 µM was used for each PCR primer. The PCR cycling conditions included an initial denaturation for 105 seconds at 95°C, followed by 35 cycles of denaturation for 15 seconds at 95°C, annealing for 30 seconds at 60°C, and extension for 7 minutes at 72°C. The PCR primers for α1 type IV collagen included (sense) 5′-CAAGTTCAGCACAATGCCCTTC-3′ and (antisense) 5′-AATGGTCTGGCTGTGCACGGC-3′. The predicted PCR fragment corresponds to nucleotide positions +141 to +351 for an overall length of 211 base pairs (bp).The PCR primers for caspase 7 included (sense) 5′-ATGGCAGATGATCACGGCTGTATTG-3′ and (antisense) 5′-TATAGACAATCACGTCAAAACCCA-3′. The predicted PCR fragment corresponds to nucleotide positions +44 to +377 for an overall length of 334 bp.The PCR primers for TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) included (sense) 5′-GAAGGAAGGGCTTCAGTGACCGG-3′ and (antisense) 5′-CTAACGAGCTGACGGAGTTGC-3′. The predicted PCR fragment corresponds to nucleotide positions +33 to +361 for an overall length of 329 bp.The RTPCR products were visualized after dilution (caspase 7, 1:3; TRAIL, 1:10; and α1 collagen IV, 1:5), electrophoresis in 1.2% agarose gels, and staining with 1-µg/mL ethidium bromide.RESULTSMicroarray analysis of a cDNA library constructed from transplant-quality human donor corneas was performed in duplicate. The first microarray identified the expression of 1794 human genes. The second microarray identified the expression of 1406 human genes. A total of 1200 shared genes were identified on both microarrays. The concordance rate between microarrays was 67%, calculated as the number of shared genes identified on both microarrays (1200) divided by the larger number of genes identified on a single microarray (1794). Only the 1200 genes confirmed by both microarrays as expressed in the cornea were used in subsequent analyses in this study. As the microarrays used in both experiments contained approximately 5600 human genes, the 1200 genes with confirmed corneal expression represent approximately 22% of the total genes contained on the microarrays.The 1200 genes with confirmed corneal expression were analyzed using GenBankand LocusLink.Of the 1200 confirmed corneal genes, 1025 (85%) had assigned chromosomal loci in GenBank or LocusLink (Table 1).Thus, 175 confirmed corneal genes (15%) had unassigned chromosomal loci in GenBank or LocusLink (Table 1).Table 1. Chromosome Loci of Genes Identified in Human Donor Corneas by Microarray Analysis*ChromosomeNo. of Corneal Genes Identified11182703454375426677538359291044116712691323144015331628174818171948202321172229X42Y1Unmapped†175*Chromosome loci of confirmed corneal genes were determined using GenBankand LocusLink.†Chromosome loci not assigned in GenBankor LocusLink.The 1200 genes with confirmed corneal expression were used as the basis of a corneal genetics Web site named CorneaNet.CorneaNet includes the 1200 genes identified in the present study in addition to 53 genes identified from the literature as being expressed in human corneas or cultured human corneal cell lines. Each entry in CorneaNet includes a gene name, symbol, chromosome locus, and GenBank accession number with an active link to the GenBank entry for the specified gene. CorneaNet is open to contributions from the research community and is updated regularly with genes newly reported in the literature as being expressed in human corneal tissues.Six types of collagen subunits were included among the confirmed corneal genes identified by microarray analysis (Table 2). These included α1 type IV, α1 type XI, α1 type XVI, α2 type V, α3 type IV, and α3 type VI collagens (Table 2). Mutations in α1 type XI collagen cause Stickler syndrome with a "beaded" type 2 vitreous phenotype.Mutations in α2 type V collagen cause Ehlers-Danlos syndrome type II.Mutations in α3 type IV collagen cause autosomal recessive Alport syndrome.Mutations in α3 type VI collagen cause Bethlem myopathy.None of these 6 collagen subunits has previously been identified in human corneas.Table 2. Detection of Collagen Gene Expression by MIcroarray Analysis of Human Donor Corneas*Collagen TypeChromosome LocusGenBankAccession No.Associated DiseaseReferenceCOL4A113q34M26576. . .14COL11A11p21J04177Stickler syndrome15COL16A11p34-p35M92642. . .16COL5A22q14-q32M11718Ehlers-Danlos syndrome type II17COL4A32q36-q37M81379Alport syndrome18COL6A32q37X52022Bethlem myopathy19*Ellipses indicate no associated disease reported.Five apoptosis-related genes were included among the confirmed corneal genes identified by microarray analysis (Table 3).These included caspase-like apoptosis regulatory protein 2, TRAIL, Bcl-xL, Bcl2/p53-binding protein (BBP/53BP2), and caspase 7 (Table 3). Caspase-like apoptosis regulatory protein 2 is a protein with a homologous sequence to caspase 8 and caspase 10 that may stimulate apoptosis through regulatory effects on caspase 8.The TRAIL is a member of the tumor necrosis factor family that can induce apoptosis of activated T lymphocytes.Bcl-xL is an inhibitor of apoptosis that is often overexpressed in solid tumors and shares sequence homology with Bcl-2, another apoptosis inhibitor.BBP/53BP2 is a proapoptotic protein that interacts with p53, Bcl-2, and the p65 subunit of NF-kappaBand is overexpressed in lung cancer cell lines and in vitro cell lines exposed to UV stress.Caspase 7 is a proapoptotic cysteine protease that induces massive apoptosis when overexpressed in human prostate cell lines.Selective inhibition of caspase 7 prevents apoptosis and maintains cell functionality.Of these 5 genes, only Bcl-xL has previously been identified in human corneal cells.Table 3. Detection of Apoptosis-Related Gene Expression in Human Donor Corneas by Microarray AnalysisGene Name*Chromosome LocusGenBankAccession No.ReferenceCLARP2q33-q34AF00577521TRAIL3q26U3751822, 23Bcl-xL20Z2311524, 25Bcl2/p53 binding protein (BBP/53BP2)1q42.1U5833426-29Caspase 710q25NM00122730, 31*CLARP indicates caspase-like apoptosis regulatory protein 2; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.Three corneal disease–causing genes were included among the confirmed corneal genes identified by microarray analysis (Table 4).These included keratoepithelin (BIGH3), keratin 12 (KRT12), and PAX6(Table 4). Numerous mutations in the BIGH3gene produce an abnormal protein that accumulates in the cornea and produces granular dystrophy I, lattice dystrophies I and IIIA, Avellino dystrophy, Reis-Bucklers dystrophy, and Thiel-Behnke dystrophy.Six mutations in the keratin 12 gene have been demonstrated to cause Meesmann dystrophy.Mutations in the PAX6gene cause anterior segment abnormalities such as aniridia and Peter anomaly, as well as autosomal-dominant keratitis.Table 4. Detection of Genes Causing Corneal Diseases by Microarray Analysis of Human Donor CorneasGene NameChromosome LocusGenBankAccession No.Corneal Disease(s)ReferenceTransforming growth factor β–induced gene product (BIGH3,keratoepithelin)5q31M77349Granular dystrophy I, lattice dystrophies I and IIIA, Avellino dystrophy, Reis-Bucklers dystrophy, Thiel-Behnke dystrophy33, 34Keratin 12 (KRT12)17q12D78367Meesmann dystrophy34-36PAX611p13M93650Peters anomaly, autosomal dominant keratitis37-39Three genes, caspase 7, TRAIL, and α1 collagen IV, were selected at random for RTPCR analysis to confirm corneal expression as determined by microarray analysis. Specific amplification products of the expected sizes were detected for all 3 genes (Figure 1).Reverse transcriptase polymerase chain reaction analysis of human corneal total RNA. Lane 1, DNA size ladder; lane 2, caspase 7 (334 base pairs [bp]); lane 3, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) (329 bp); lane 4, α1 collagen IV (211 bp).COMMENTThe recent completion of the sequencing phase of the Human Genome Project provides a wealth of genetic information that should facilitate clinically relevant studies of normal and abnormal cellular processes. One potentially useful application of this information is the creation of comprehensive databases of genes expressed in a given normal or abnormal tissue or cell type. An initial attempt to investigate quantitative and qualitative aspects of gene expression in the corneal epithelium was performed using the conventional technique of sequencing 1069 randomly selected cDNA clones.A similar study reported the sequencing of 1060 cDNA clones from a human trabecular meshwork cDNA library.DNA microarrays represent a powerful technique to screen large amounts of genetic material for known sequences. This method has been used in ophthalmology to study alterations in gene expression caused by the photoreceptor homeobox gene CRXand elevations in intraocular pressure.In the present study, this technique was used to identify the expression of 1200 known genes in transplant-quality human donor corneas. Only those genes positively identified on 2 identical microarrays were included in this study with a concordance rate of 67%. This conservative approach was followed to minimize the possibility of false-positive genes. Further studies are in progress to confirm corneal expression for the genes identified by only a single microarray in these experiments.As this study exemplifies, current techniques of genetic analysis can generate extremely large amounts of information in a single experiment. Disseminating this information via the Internet offers the advantages of being easily accessible and modifiable. Furthermore, the Internet allows such gene databases to interface with preexisting, high-quality, and authoritative online genetic Web sites such as GenBankand Online Mendelian Inheritance in Man.This approach was used in the creation of CorneaNet,which may become a useful resource for the cornea research community by improving the dissemination of genetic information. If successful, CorneaNet may serve as a model for online databases of gene expression in other tissues.One limitation of microarray analysis is the inability to identify previously unreported genes. However, the ongoing assembly phase of the Human Genome Project should identify all of the estimated 30 000 genes in the human genome. This information, combined with continuing advances in microarray construction, should yield full-genome microarrays in the near future. Such tools should greatly facilitate the development of truly comprehensive gene expression databases.Microarray analysis is an efficient way to investigate the genetic basis of normal and abnormal biological processes.In this study, microarray analysis identified corneal expression of 6 collagen genes, none of which had previously been localized to this tissue. These results may lead to a more sophisticated understanding of the contributions of various collagens to the structural integrity of the corneal stroma. Similarly, the expression of 5 apoptosis-related genes were identified in the donor-quality corneas used in this study. Four of these genes had not been previously localized to the cornea. These results may provide insights into the possible role of apoptosis in the ultimate success or failure of corneal grafts. Caspase 7 is a powerful initiator of apoptosis,and potent inhibitors of its activity have been shown to block caspase 7–mediated apoptosis.Such inhibitors could ultimately be useful additives to corneal storage solutions to improve the viability of donor corneas.Much progress has been made recently in identifying genes causing corneal dystrophies.Several corneal dystrophies such as central crystalline dystrophy, posterior polymorphous dystrophy, congenital hereditary endothelial dystrophies I and II, keratoconus, and X-linked megalocornea have been mapped to chromosome loci but await identification of causative genes (Table 5).The search for these disease-causing genes might be facilitated by knowledge of which genes present at specific chromosome loci are expressed in the cornea. Thus, the database of corneal genes identified by microarray analysis includes chromosome loci when available.Table 5. Genes Expressed in Human Donor Corneas That Map to Chromosome Loci Associated With Corneal DystrophiesCorneal DystrophyInheritance*Chromosome LocusNo. of Corneal Genes at LocusReferenceCentral crystallineAD1p361245Posterior polymorphousAD20q11246Congenital hereditary endothelial IAD20p747Congenital hereditary endothelial IIAR20 tel248KeratoconusAD21q21.1-q21.1249X-linked megalocorneaXRXq12-q261650*AD indicates autosomal dominant; AR, autosomal recessive; and XR, X-linked recessive.The present study is the first to our knowledge to apply microarray analysis to study corneal gene expression. The results were used to create a preliminary, online database of genes expressed in normal donor corneas. Microarray analyses of corneal ulcers, dystrophies, graft rejection, and others may provide insights into the genetic bases of these pathologic processes that may, in turn, lead to better treatments for corneal diseases.ABrazmaJViloGene expression data analysis.FEBS Lett.2000;480:17-24.NLWvan HalOVorstAMMLvan HouwelingenThe application of DNA microarrays in gene expression analysis.J Biotechnol.2000;78:271-280.TSTanakaSAJaradatMKLimGenome-wide expression profiling of mid-gestation placenta and embryo using a 15 000 mouse developmental cDNA microarray.Proc Natl Acad Sci.2000;97:9127-9132.CMPerouTSorlieMBElsenMolecular portraits of human breast tumors.Nature.2000;406:747-752.LWStantonLJGarrardDDammAltered patterns of gene expression in response to myocardial infarction.Circ Res.2000;86:939-945.TYoshikawaYNagasugiTAzumaIsolation of novel mouse genes differentially expressed in brain using cDNA microarray.Biochem Biophys Res Commun.2000;275:532-537.DAStephanYChenYJiangPositional cloning utilizing genomic DNA microarrays: the Niemann-Pick type C gene as a model system.Mol Genet Metab.2000;70:10-18.JDGottschWJStarkSHLiuCloning and sequence analysis of human and bovine corneal antigen (CO-Ag) cDNA: identification of host-parasite protein calgranulin C.Trans Am Ophthalmol Soc.1997;95:111-125.Not AvailableGenBank resources page.National Center for Biotechnology Information Web site. Available at: http://www.ncbi.nlm.nih.gov/Genbank/. Accessibility verified July 19, 2001.Not AvailableLocusLink resources page.National Center for Biotechnology Information Web site. Available at: http://www.ncbi.nlm.nih.gov/locuslink. Accessibility verified July 19, 2001.TPihlajaniemiKTryggvasonJCMyerscDNA clones coding for the pro-α1(IV) chain of human type IV procollagen reveal and unusual homology of amino acid sequences in two halves of the carboxyl-terminal domain.J Biol Chem.1985;260:7681-7687.MMarcelliGRCunninghamSJHaidacherCaspase-7 is activated during lovastatin-induced apoptosis of the prostate cancer cell line LnCaP.Cancer Res.1998;58:76-83.QWangYJiXWangBMEversIsolation and molecular characterization of the 5′-upstream region of the human TRAIL gene.Biochem Biophys Res Commun.2000;276:466-471.YSadoMKagawaINaitoOrganization and expression of basement membrane collagen IV genes and their roles in human disorders.J Biochem (Tokyo).1998;123:767-776.SMartinAJRichardsJRYatesJDScottMPopeMPSneadStickler syndrome: further mutations in COL11A1 and evidence for additional locus heterogeneity.Eur J Hum Genet.1999;7:807-814.NYamaguchiSKimuraOWMcBrideMolecular cloning and partial characterization of a novel collagen chain, alpha 1 (XVI), consisting of repetitive collagenous domains and cysteine-containing non-collagenous segments.J Biochem (Tokyo).1992;112:856-863.AJRichardsSMartinACNichollsJBHarrisonFMPopeNPBurrowsA single base mutation in COL5A2 causes Ehlers-Danlos syndrome type II.J Med Genet.1998;35:846-848.RTorraCBadenasFCofanLCallisLPerez-OllerADarnellAutosomal recessive Alport syndrome: linkage analysis and clinical features in two families.Nephrol Dial Transplant.1999;14:627-630.GJJobsisHKeizersJPVreijlingType VI collagen mutations in Bethlem myopathy, and autosomal dominant myopathy with contractures.Nat Genet.1996;14:113-115.ASJunWJStarkJDGottschThe Cornea Information Network.CorneaNet Web site, The Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, Md. Available at: http://www.corneanet.net. Accessibility verified July 17, 2001.NInoharaTKosekiYHuSChenGNunezCLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis.Proc Natl Acad Sci U S A.1997;94:10717-10722.SRWileyKSchooleyPJSmolakIdentification and characterization of a new member of the TNF family that induces apoptosis.Immunity.1995;3:673-682.QWangYJiXWangBMEversIsolation and molecular characterization of the 5′-upstream region of the human TRAIL gene.Biochem Biophys Res Commun.2000;276:466-471.LHBoiseMGonzalez-GarciaCEPostemabcl-x, A bcl-2-related gene that functions as a dominant regulator of apoptotic cell death.Cell.1993;74:597-608.UZangemeister-WittkeSHLeechRAOlieA novel bispecific antisense oligonucleotide inhibiting both bcl-2 and bcl-xL expression efficiently induces apoptosis in tumor cells.Clin Cancer Res.2000;6:2547-2555.LNaumovskiMLClearyThe p53-binding protein 53BP2 also interacts with bcl2 and impedes cell cycle progression at G2/M.Mol Cell Biol.1996;16:3884-3892.JPYangMHoriNTakahashiTKawabeHKatoTOkamotoNF-kappaB subunit p65 binds to 53BP2 and inhibits cell death induced by 53BP2.Oncogene.1999;18:5177-5186.TMoriHOkamotoNTakahashiRUedaTOkamotoAberrant overexpression of 53BP2 mRNA in lung cancer cell lines.FEBS Lett.2000;465:124-128.CDLopezYAoLHRohdeProapoptotic p53-interacting protein 53BP2 is induced by UV irradiation but suppressed by p53.Mol Cell Biol.2000;20:8018-8025.MMarcelliTCShaoXLiInduction of apoptosis in BPH stromal cells by adenoviral-mediated overexpression of caspase-7.J Urol.2000;164:518-525.DLeeSALongSLAdamsPotent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.J Biol Chem.2000;275:16007-16014.SEWilsonQLiJWengThe Fas-Fas ligand system and other modulators of apoptosis in the cornea.Invest Ophthalmol Vis Sci.1996;37:1582-1592.EKorvatskaFLMunierPChaubertOn the role of kerato-epithelin in the pathogenesis of 5q31-linked corneal dystrophies.Invest Ophthalmol Vis Sci.1999;40:2213-2219.ABronGenetics of the corneal dystrophies: what we have learned in the past twenty-five years.Cornea.2000;19:699-711.ADIrvineLDCordenOSwenssonMutations in cornea-specific keratin K3 or K12 genes cause Meesmann corneal dystrophy.Nat Genet.1997;16:184-187.KNishidaYHonmaADotaIsolation and chromosomal localization of a cornea-specific human keratin 12 gene and detection of four mutations in Meesmann corneal epithelial dystrophy.Am J Hum Genet.1997;61:1268-1275.TGlaserDSWaltonRLMaasGenomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene.Nat Genet.1992;2:232-239.IMHansonJMFletcherTJordanMutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly.Nat Genet.1994;6:168-173.FMirzayansWGPearceIMMacDonaldMAWalterMutation of the PAX6 gene in patients with autosomal dominant keratitis.Am J Hum Genet.1995;57:539-548.KNishidaWAdachiAShimizu-MatsumotoA gene expression profile of corneal epithelium and the isolation of human keratin 12 cDNA.Invest Ophthalmol Vis Sci.1996;37:1800-1809.PGonzalezDLEpsteinTBorrasCharacterization of gene expression in human trabecular meshwork using single-pass sequencing of 1060 clones.Invest Ophthalmol Vis Sci.2000;41:3678-8693.FJLiveseyTFurukawaMASteffenGMChurchCLCepkoMicroarray analysis of the transcriptional network controlled by the photoreceptor homeobox gene Crx.Curr Biol.2000;10:301-310.PGonzalezDLEpsteinTBorrasGenes up-regulated in the human trabecular meshwork in response to elevated intraocular pressure.Invest Ophthalmol Vis Sci.2000;41:352-361.Not AvailableOnline Mendelian Inheritance in Man resource page.National Center for Biotechnology Information Web site. Available at: http://www.ncbi.nlm.nih.gov/omim. Accessibility verified July 19, 2001.AMShearmanTJHudsonJMAndresenThe gene for Schnyder's crystalline corneal dystrophy maps to human chromosome 1p34-p36.Hum Mol Genet.1996;5:1667-1672.EHeonWDMathersWLAlwardLinkage of posterior polymorphous dystrophy to 20q11.Hum Mol Genet.1995;4:485-488.NMTomaNDEbenezerCFInglehearnCPlantLAFickerSSBhattacharyaLinkage of congenital hereditary endothelial dystrophy to chromosome 20.Hum Mol Genet.1995;4:2395-2398.CKHandDLHarmonSMKennedyJSFitzsimonLMCollumNAParfreyLocalization of the gene for autosomal recessive congenital hereditary endothelial dystrophy (CHED2) to chromosome 20 by homozygosity mapping.Genomics.1999;61:1-4.YSRabinowitzLZuHYangYWangJRotterSPulstKeratoconus: further gene linkage studies on chromosome 21.Invest Ophthalmol Vis Sci.2000;41(suppl):S539.DAMackeyRGButteryGMWiseMJDentonDescription of X-linked megalocornea with identification of the gene locus.Arch Ophthalmol.1991;109:829-833.Accepted for publication June 8, 2001.This work was supported by unrestricted grants from Research to Prevent Blindness Inc, New York, NY; Tissue Banks International, Baltimore, Md (Dr Gottsch); the Irvin and Ginger Gomprecht Research Fund (Dr Gottsch); the Deborah Black Research Fund (Dr Stark); and the Raymond Kwok Research Fund (Dr Stark).The authors are thankful to Morton F. Goldberg, MD, for continued support of this work and Elizabeth Bell for research assistance.Corresponding author and reprints: John D. Gottsch, MD, Cornea and External Disease Division, Wilmer Ophthalmological Institute, The Johns Hopkins Medical Institutions, Maumenee 317, 600 N Wolfe St, Baltimore, MD 21287 (e-mail: jgottsch@jhmi.edu.). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JAMA Ophthalmology American Medical Association

Microarray Analysis of Gene Expression in Human Donor Corneas

Loading next page...
 
/lp/american-medical-association/microarray-analysis-of-gene-expression-in-human-donor-corneas-wJzpqLCFTV

References (52)

Publisher
American Medical Association
Copyright
Copyright 2001 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.
ISSN
2168-6165
eISSN
2168-6173
DOI
10.1001/archopht.119.11.1629
Publisher site
See Article on Publisher Site

Abstract

ObjectivesTo use microarray analysis to identify genes expressed in human donor corneas and to create a preliminary, comprehensive database of human corneal gene expression.MethodsA complementary DNA (cDNA) library was constructed from transplant-quality, human donor corneas. Biotin-labeled RNA was transcribed from the cDNA library and hybridized in duplicate to microarrays containing approximately 5600 human genes. Results were analyzed using a gene database of the National Institutes of Health, Bethesda, Md. Reverse transcriptase polymerase chain reaction analysis was performed to confirm corneal expression of genes identified by microarray analysis.ResultsDuplicate microarrays identified the expression of 1200 genes in human donor corneas. Chromosomal loci had been assigned to 1025 (85%) of these genes. A preliminary database of human corneal gene expression was compiled. A Web site containing these genes was created. Six collagen genes were identified that had not previously been localized within the cornea. Five apoptosis-related genes were identified, 4 of which had not previously been localized within the cornea. Three genes previously shown to cause corneal diseases were identified. Reverse transcriptase polymerase chain reaction analysis of genes identified by microarray analysis confirmed the corneal expression of 2 apoptosis-related genes and 1 collagen gene.ConclusionsMicroarray analysis of healthy human donor corneas has produced a preliminary, comprehensive database of corneal gene expression. Large-scale analysis of gene expression has the potential to generate large amounts of data, which should be made readily accessible to the scientific community. The Internet offers many potential advantages as a medium for the maintenance of these large data sets.Clinical RelevanceIdentification of structural, apoptosis-related, and disease-causing genes within the cornea by microarrays may increase the understanding of normal and abnormal corneal function with likely relevance to corneal diseases and transplants.FUNDAMENTAL to understanding normal tissue function is a global knowledge of the thousands of genes expressed in the various cell types that comprise that tissue. This baseline knowledge should facilitate the identification of alterations from normal gene expression that play important roles in disease pathogenesis. Efforts to comprehensively study gene expression patterns in normal tissues and altered gene expression patterns in diseased tissues will require a complete knowledge of the human genetic sequence and methods to accurately and simultaneously analyze large amounts of genetic information. Both of these requirements have recently become available through the ongoing progress of the Human Genome Project and breakthroughs in high-efficiency genetic analysis techniques such as DNA microarrays.DNA microarrays are a new and powerful technique to study the expression of thousands of genes in a single experiment.A microarray is a solid substrate such as a glass slide or nylon membrane to which known, single-stranded DNA molecules are attached at distinct locations. The density of these locations on a microarray can reach upwards of 250 000/cm2, as demonstrated by van Hal et al.Experimental messenger RNA (mRNA) is labeled as a complex mixture and exposed to the microarray. Labeled mRNA molecules will bind to complementary sequences on the microarray and can be detected in a semiquantitative manner using automated techniques. Advantages of DNA microarrays include simultaneous screening for the expression of large numbers of genes, the ability to use small amounts of starting material, and mass production, which enables standardized, comparative analysis between samples.The acceptance of this technology is growing rapidly as microarrays are being used in an increasing number of experimental applications. These include analysis of gene expression in normal embryonic developmentand pathologic states such as breast cancerand myocardial infarction.Microarrays also have been used to identify novel genes expressed in brain tissueand for positional cloning of a disease gene in Niemann-Pick disease, type C.Given the successful use of microarray analysis in other biological systems, we sought to apply this technique to study gene expression in human donor corneal tissue. Such analysis may be useful for understanding the genetic basis of normal corneal function as well as corneal disease processes such as graft failure, inflammation, degenerations, and dystrophies. Knowledge of what genes are or are not expressed in a given corneal disorder could lead to new and definitive treatment strategies, including interventional drugs and gene therapies. These strategies may be particularly relevant and feasible for the cornea because the tissue is relatively less complex, can be manipulated ex vivo, and can be easily assessed visually.Microarray analysis is a feasible method to begin compiling a comprehensive database of genes expressed in human corneas. Such a database of genes may have broad applications for corneal genetics research. Any comprehensive database of corneal genes would be expected to be relatively large and to grow as more genes are identified in this tissue and as the assembly phase of the Human Genome Project defines novel genes from currently available sequence information. Such a large database would be most useful if it could be readily updated, freely accessible to the global research community, and effectively interfaced with preexisting gene databases. Given these desirable features, an Internet Web site could be an ideal format for a comprehensive corneal gene database. Such a Web site could potentially enhance the progress of corneal genetics research by increasing the accessibility of relevant genetic information and facilitating discussion among corneal genetics researchers. As the proposed comprehensive corneal gene database grows and becomes more clinically relevant, it also could serve as a model for similar efforts in other clinical disciplines.MATERIALS AND METHODSTwenty corneal-scleral rims were obtained at the time of penetrating keratoplasty, and peripheral corneal tissue was carefully dissected, placed in microcentrifuge tubes, and immediately stored at −80°C. Two entire transplant-quality donor corneal buttons were placed in microcentrifuge tubes and immediately stored at −80°C. The death to preservation time of all tissues used in this study was less than 12 hours. All tissues used in this study were obtained from donors younger than age 65 years. A complementary DNA (cDNA) library was constructed using standard methods from the pooled corneal tissues described.The number of clones contained in the primary cDNA library was estimated to be 1.0 × 106.Standard methods were used to recover phagemids by mass excision protocol (pBluescript; Stratagene, La Jolla, Calif). The number of plasmids excised was 1.6 × 106. The ratio of clones excised to the number of independent clones in the library was 1.6:1. Excised clones were used to transfect a large-volume cell culture (SOLR; Stratagene, and plasmid "maxi-preps" were performed with a standard kit and protocol (Qiagen, Valencia, Calif). Plasmids were digested using restriction endonuclease (NotI; Life Technologies, Rockville, Md), phenol-chloroform extracted, and ethanol precipitated.Biotin-labeled cRNA molecules were produced by in vitro transcription (Enzo Diagnostics, Farmingdale, NY), digested with DNase I (Life Technologies), and purified using commercially available spin columns (RNeasy; Qiagen). Analysis of biotin-labeled cRNA using the HuGeneFL microarray (Affymetrix, Santa Clara, Calif) was performed in duplicate (Research Genetics, Huntsville, Ala) by hybridizing the same labeled cRNA sample to 2 identical microarrays within a 6-week time period.Microarray analysis results were analyzed using GenBankand LocusLink,online genetic databases sponsored by the National Library of Medicine of the National Institutes of Health, Bethesda, Md. A corneal genetics web site was created using the y-Base Informatics Engine (y-DNA Inc, Palo Alto, Calif).Total RNA was extracted using TRIZOL reagent (Life Technologies) from 2 pooled, whole, transplant-quality donor corneas. Reverse transcriptase polymerase chain reaction (RTPCR) was performed using standard methods (Applied Biosystems, Foster City, Calif). One microgram of total RNA was used as template for first-strand cDNA synthesis. Polymerase chain reaction (PCR) was performed using 5 µL of each cDNA sample in a final reaction volume of 100 µL. A final concentration of 2.5 µM was used for each PCR primer. The PCR cycling conditions included an initial denaturation for 105 seconds at 95°C, followed by 35 cycles of denaturation for 15 seconds at 95°C, annealing for 30 seconds at 60°C, and extension for 7 minutes at 72°C. The PCR primers for α1 type IV collagen included (sense) 5′-CAAGTTCAGCACAATGCCCTTC-3′ and (antisense) 5′-AATGGTCTGGCTGTGCACGGC-3′. The predicted PCR fragment corresponds to nucleotide positions +141 to +351 for an overall length of 211 base pairs (bp).The PCR primers for caspase 7 included (sense) 5′-ATGGCAGATGATCACGGCTGTATTG-3′ and (antisense) 5′-TATAGACAATCACGTCAAAACCCA-3′. The predicted PCR fragment corresponds to nucleotide positions +44 to +377 for an overall length of 334 bp.The PCR primers for TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) included (sense) 5′-GAAGGAAGGGCTTCAGTGACCGG-3′ and (antisense) 5′-CTAACGAGCTGACGGAGTTGC-3′. The predicted PCR fragment corresponds to nucleotide positions +33 to +361 for an overall length of 329 bp.The RTPCR products were visualized after dilution (caspase 7, 1:3; TRAIL, 1:10; and α1 collagen IV, 1:5), electrophoresis in 1.2% agarose gels, and staining with 1-µg/mL ethidium bromide.RESULTSMicroarray analysis of a cDNA library constructed from transplant-quality human donor corneas was performed in duplicate. The first microarray identified the expression of 1794 human genes. The second microarray identified the expression of 1406 human genes. A total of 1200 shared genes were identified on both microarrays. The concordance rate between microarrays was 67%, calculated as the number of shared genes identified on both microarrays (1200) divided by the larger number of genes identified on a single microarray (1794). Only the 1200 genes confirmed by both microarrays as expressed in the cornea were used in subsequent analyses in this study. As the microarrays used in both experiments contained approximately 5600 human genes, the 1200 genes with confirmed corneal expression represent approximately 22% of the total genes contained on the microarrays.The 1200 genes with confirmed corneal expression were analyzed using GenBankand LocusLink.Of the 1200 confirmed corneal genes, 1025 (85%) had assigned chromosomal loci in GenBank or LocusLink (Table 1).Thus, 175 confirmed corneal genes (15%) had unassigned chromosomal loci in GenBank or LocusLink (Table 1).Table 1. Chromosome Loci of Genes Identified in Human Donor Corneas by Microarray Analysis*ChromosomeNo. of Corneal Genes Identified11182703454375426677538359291044116712691323144015331628174818171948202321172229X42Y1Unmapped†175*Chromosome loci of confirmed corneal genes were determined using GenBankand LocusLink.†Chromosome loci not assigned in GenBankor LocusLink.The 1200 genes with confirmed corneal expression were used as the basis of a corneal genetics Web site named CorneaNet.CorneaNet includes the 1200 genes identified in the present study in addition to 53 genes identified from the literature as being expressed in human corneas or cultured human corneal cell lines. Each entry in CorneaNet includes a gene name, symbol, chromosome locus, and GenBank accession number with an active link to the GenBank entry for the specified gene. CorneaNet is open to contributions from the research community and is updated regularly with genes newly reported in the literature as being expressed in human corneal tissues.Six types of collagen subunits were included among the confirmed corneal genes identified by microarray analysis (Table 2). These included α1 type IV, α1 type XI, α1 type XVI, α2 type V, α3 type IV, and α3 type VI collagens (Table 2). Mutations in α1 type XI collagen cause Stickler syndrome with a "beaded" type 2 vitreous phenotype.Mutations in α2 type V collagen cause Ehlers-Danlos syndrome type II.Mutations in α3 type IV collagen cause autosomal recessive Alport syndrome.Mutations in α3 type VI collagen cause Bethlem myopathy.None of these 6 collagen subunits has previously been identified in human corneas.Table 2. Detection of Collagen Gene Expression by MIcroarray Analysis of Human Donor Corneas*Collagen TypeChromosome LocusGenBankAccession No.Associated DiseaseReferenceCOL4A113q34M26576. . .14COL11A11p21J04177Stickler syndrome15COL16A11p34-p35M92642. . .16COL5A22q14-q32M11718Ehlers-Danlos syndrome type II17COL4A32q36-q37M81379Alport syndrome18COL6A32q37X52022Bethlem myopathy19*Ellipses indicate no associated disease reported.Five apoptosis-related genes were included among the confirmed corneal genes identified by microarray analysis (Table 3).These included caspase-like apoptosis regulatory protein 2, TRAIL, Bcl-xL, Bcl2/p53-binding protein (BBP/53BP2), and caspase 7 (Table 3). Caspase-like apoptosis regulatory protein 2 is a protein with a homologous sequence to caspase 8 and caspase 10 that may stimulate apoptosis through regulatory effects on caspase 8.The TRAIL is a member of the tumor necrosis factor family that can induce apoptosis of activated T lymphocytes.Bcl-xL is an inhibitor of apoptosis that is often overexpressed in solid tumors and shares sequence homology with Bcl-2, another apoptosis inhibitor.BBP/53BP2 is a proapoptotic protein that interacts with p53, Bcl-2, and the p65 subunit of NF-kappaBand is overexpressed in lung cancer cell lines and in vitro cell lines exposed to UV stress.Caspase 7 is a proapoptotic cysteine protease that induces massive apoptosis when overexpressed in human prostate cell lines.Selective inhibition of caspase 7 prevents apoptosis and maintains cell functionality.Of these 5 genes, only Bcl-xL has previously been identified in human corneal cells.Table 3. Detection of Apoptosis-Related Gene Expression in Human Donor Corneas by Microarray AnalysisGene Name*Chromosome LocusGenBankAccession No.ReferenceCLARP2q33-q34AF00577521TRAIL3q26U3751822, 23Bcl-xL20Z2311524, 25Bcl2/p53 binding protein (BBP/53BP2)1q42.1U5833426-29Caspase 710q25NM00122730, 31*CLARP indicates caspase-like apoptosis regulatory protein 2; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.Three corneal disease–causing genes were included among the confirmed corneal genes identified by microarray analysis (Table 4).These included keratoepithelin (BIGH3), keratin 12 (KRT12), and PAX6(Table 4). Numerous mutations in the BIGH3gene produce an abnormal protein that accumulates in the cornea and produces granular dystrophy I, lattice dystrophies I and IIIA, Avellino dystrophy, Reis-Bucklers dystrophy, and Thiel-Behnke dystrophy.Six mutations in the keratin 12 gene have been demonstrated to cause Meesmann dystrophy.Mutations in the PAX6gene cause anterior segment abnormalities such as aniridia and Peter anomaly, as well as autosomal-dominant keratitis.Table 4. Detection of Genes Causing Corneal Diseases by Microarray Analysis of Human Donor CorneasGene NameChromosome LocusGenBankAccession No.Corneal Disease(s)ReferenceTransforming growth factor β–induced gene product (BIGH3,keratoepithelin)5q31M77349Granular dystrophy I, lattice dystrophies I and IIIA, Avellino dystrophy, Reis-Bucklers dystrophy, Thiel-Behnke dystrophy33, 34Keratin 12 (KRT12)17q12D78367Meesmann dystrophy34-36PAX611p13M93650Peters anomaly, autosomal dominant keratitis37-39Three genes, caspase 7, TRAIL, and α1 collagen IV, were selected at random for RTPCR analysis to confirm corneal expression as determined by microarray analysis. Specific amplification products of the expected sizes were detected for all 3 genes (Figure 1).Reverse transcriptase polymerase chain reaction analysis of human corneal total RNA. Lane 1, DNA size ladder; lane 2, caspase 7 (334 base pairs [bp]); lane 3, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) (329 bp); lane 4, α1 collagen IV (211 bp).COMMENTThe recent completion of the sequencing phase of the Human Genome Project provides a wealth of genetic information that should facilitate clinically relevant studies of normal and abnormal cellular processes. One potentially useful application of this information is the creation of comprehensive databases of genes expressed in a given normal or abnormal tissue or cell type. An initial attempt to investigate quantitative and qualitative aspects of gene expression in the corneal epithelium was performed using the conventional technique of sequencing 1069 randomly selected cDNA clones.A similar study reported the sequencing of 1060 cDNA clones from a human trabecular meshwork cDNA library.DNA microarrays represent a powerful technique to screen large amounts of genetic material for known sequences. This method has been used in ophthalmology to study alterations in gene expression caused by the photoreceptor homeobox gene CRXand elevations in intraocular pressure.In the present study, this technique was used to identify the expression of 1200 known genes in transplant-quality human donor corneas. Only those genes positively identified on 2 identical microarrays were included in this study with a concordance rate of 67%. This conservative approach was followed to minimize the possibility of false-positive genes. Further studies are in progress to confirm corneal expression for the genes identified by only a single microarray in these experiments.As this study exemplifies, current techniques of genetic analysis can generate extremely large amounts of information in a single experiment. Disseminating this information via the Internet offers the advantages of being easily accessible and modifiable. Furthermore, the Internet allows such gene databases to interface with preexisting, high-quality, and authoritative online genetic Web sites such as GenBankand Online Mendelian Inheritance in Man.This approach was used in the creation of CorneaNet,which may become a useful resource for the cornea research community by improving the dissemination of genetic information. If successful, CorneaNet may serve as a model for online databases of gene expression in other tissues.One limitation of microarray analysis is the inability to identify previously unreported genes. However, the ongoing assembly phase of the Human Genome Project should identify all of the estimated 30 000 genes in the human genome. This information, combined with continuing advances in microarray construction, should yield full-genome microarrays in the near future. Such tools should greatly facilitate the development of truly comprehensive gene expression databases.Microarray analysis is an efficient way to investigate the genetic basis of normal and abnormal biological processes.In this study, microarray analysis identified corneal expression of 6 collagen genes, none of which had previously been localized to this tissue. These results may lead to a more sophisticated understanding of the contributions of various collagens to the structural integrity of the corneal stroma. Similarly, the expression of 5 apoptosis-related genes were identified in the donor-quality corneas used in this study. Four of these genes had not been previously localized to the cornea. These results may provide insights into the possible role of apoptosis in the ultimate success or failure of corneal grafts. Caspase 7 is a powerful initiator of apoptosis,and potent inhibitors of its activity have been shown to block caspase 7–mediated apoptosis.Such inhibitors could ultimately be useful additives to corneal storage solutions to improve the viability of donor corneas.Much progress has been made recently in identifying genes causing corneal dystrophies.Several corneal dystrophies such as central crystalline dystrophy, posterior polymorphous dystrophy, congenital hereditary endothelial dystrophies I and II, keratoconus, and X-linked megalocornea have been mapped to chromosome loci but await identification of causative genes (Table 5).The search for these disease-causing genes might be facilitated by knowledge of which genes present at specific chromosome loci are expressed in the cornea. Thus, the database of corneal genes identified by microarray analysis includes chromosome loci when available.Table 5. Genes Expressed in Human Donor Corneas That Map to Chromosome Loci Associated With Corneal DystrophiesCorneal DystrophyInheritance*Chromosome LocusNo. of Corneal Genes at LocusReferenceCentral crystallineAD1p361245Posterior polymorphousAD20q11246Congenital hereditary endothelial IAD20p747Congenital hereditary endothelial IIAR20 tel248KeratoconusAD21q21.1-q21.1249X-linked megalocorneaXRXq12-q261650*AD indicates autosomal dominant; AR, autosomal recessive; and XR, X-linked recessive.The present study is the first to our knowledge to apply microarray analysis to study corneal gene expression. The results were used to create a preliminary, online database of genes expressed in normal donor corneas. Microarray analyses of corneal ulcers, dystrophies, graft rejection, and others may provide insights into the genetic bases of these pathologic processes that may, in turn, lead to better treatments for corneal diseases.ABrazmaJViloGene expression data analysis.FEBS Lett.2000;480:17-24.NLWvan HalOVorstAMMLvan HouwelingenThe application of DNA microarrays in gene expression analysis.J Biotechnol.2000;78:271-280.TSTanakaSAJaradatMKLimGenome-wide expression profiling of mid-gestation placenta and embryo using a 15 000 mouse developmental cDNA microarray.Proc Natl Acad Sci.2000;97:9127-9132.CMPerouTSorlieMBElsenMolecular portraits of human breast tumors.Nature.2000;406:747-752.LWStantonLJGarrardDDammAltered patterns of gene expression in response to myocardial infarction.Circ Res.2000;86:939-945.TYoshikawaYNagasugiTAzumaIsolation of novel mouse genes differentially expressed in brain using cDNA microarray.Biochem Biophys Res Commun.2000;275:532-537.DAStephanYChenYJiangPositional cloning utilizing genomic DNA microarrays: the Niemann-Pick type C gene as a model system.Mol Genet Metab.2000;70:10-18.JDGottschWJStarkSHLiuCloning and sequence analysis of human and bovine corneal antigen (CO-Ag) cDNA: identification of host-parasite protein calgranulin C.Trans Am Ophthalmol Soc.1997;95:111-125.Not AvailableGenBank resources page.National Center for Biotechnology Information Web site. Available at: http://www.ncbi.nlm.nih.gov/Genbank/. Accessibility verified July 19, 2001.Not AvailableLocusLink resources page.National Center for Biotechnology Information Web site. Available at: http://www.ncbi.nlm.nih.gov/locuslink. Accessibility verified July 19, 2001.TPihlajaniemiKTryggvasonJCMyerscDNA clones coding for the pro-α1(IV) chain of human type IV procollagen reveal and unusual homology of amino acid sequences in two halves of the carboxyl-terminal domain.J Biol Chem.1985;260:7681-7687.MMarcelliGRCunninghamSJHaidacherCaspase-7 is activated during lovastatin-induced apoptosis of the prostate cancer cell line LnCaP.Cancer Res.1998;58:76-83.QWangYJiXWangBMEversIsolation and molecular characterization of the 5′-upstream region of the human TRAIL gene.Biochem Biophys Res Commun.2000;276:466-471.YSadoMKagawaINaitoOrganization and expression of basement membrane collagen IV genes and their roles in human disorders.J Biochem (Tokyo).1998;123:767-776.SMartinAJRichardsJRYatesJDScottMPopeMPSneadStickler syndrome: further mutations in COL11A1 and evidence for additional locus heterogeneity.Eur J Hum Genet.1999;7:807-814.NYamaguchiSKimuraOWMcBrideMolecular cloning and partial characterization of a novel collagen chain, alpha 1 (XVI), consisting of repetitive collagenous domains and cysteine-containing non-collagenous segments.J Biochem (Tokyo).1992;112:856-863.AJRichardsSMartinACNichollsJBHarrisonFMPopeNPBurrowsA single base mutation in COL5A2 causes Ehlers-Danlos syndrome type II.J Med Genet.1998;35:846-848.RTorraCBadenasFCofanLCallisLPerez-OllerADarnellAutosomal recessive Alport syndrome: linkage analysis and clinical features in two families.Nephrol Dial Transplant.1999;14:627-630.GJJobsisHKeizersJPVreijlingType VI collagen mutations in Bethlem myopathy, and autosomal dominant myopathy with contractures.Nat Genet.1996;14:113-115.ASJunWJStarkJDGottschThe Cornea Information Network.CorneaNet Web site, The Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, Md. Available at: http://www.corneanet.net. Accessibility verified July 17, 2001.NInoharaTKosekiYHuSChenGNunezCLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis.Proc Natl Acad Sci U S A.1997;94:10717-10722.SRWileyKSchooleyPJSmolakIdentification and characterization of a new member of the TNF family that induces apoptosis.Immunity.1995;3:673-682.QWangYJiXWangBMEversIsolation and molecular characterization of the 5′-upstream region of the human TRAIL gene.Biochem Biophys Res Commun.2000;276:466-471.LHBoiseMGonzalez-GarciaCEPostemabcl-x, A bcl-2-related gene that functions as a dominant regulator of apoptotic cell death.Cell.1993;74:597-608.UZangemeister-WittkeSHLeechRAOlieA novel bispecific antisense oligonucleotide inhibiting both bcl-2 and bcl-xL expression efficiently induces apoptosis in tumor cells.Clin Cancer Res.2000;6:2547-2555.LNaumovskiMLClearyThe p53-binding protein 53BP2 also interacts with bcl2 and impedes cell cycle progression at G2/M.Mol Cell Biol.1996;16:3884-3892.JPYangMHoriNTakahashiTKawabeHKatoTOkamotoNF-kappaB subunit p65 binds to 53BP2 and inhibits cell death induced by 53BP2.Oncogene.1999;18:5177-5186.TMoriHOkamotoNTakahashiRUedaTOkamotoAberrant overexpression of 53BP2 mRNA in lung cancer cell lines.FEBS Lett.2000;465:124-128.CDLopezYAoLHRohdeProapoptotic p53-interacting protein 53BP2 is induced by UV irradiation but suppressed by p53.Mol Cell Biol.2000;20:8018-8025.MMarcelliTCShaoXLiInduction of apoptosis in BPH stromal cells by adenoviral-mediated overexpression of caspase-7.J Urol.2000;164:518-525.DLeeSALongSLAdamsPotent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.J Biol Chem.2000;275:16007-16014.SEWilsonQLiJWengThe Fas-Fas ligand system and other modulators of apoptosis in the cornea.Invest Ophthalmol Vis Sci.1996;37:1582-1592.EKorvatskaFLMunierPChaubertOn the role of kerato-epithelin in the pathogenesis of 5q31-linked corneal dystrophies.Invest Ophthalmol Vis Sci.1999;40:2213-2219.ABronGenetics of the corneal dystrophies: what we have learned in the past twenty-five years.Cornea.2000;19:699-711.ADIrvineLDCordenOSwenssonMutations in cornea-specific keratin K3 or K12 genes cause Meesmann corneal dystrophy.Nat Genet.1997;16:184-187.KNishidaYHonmaADotaIsolation and chromosomal localization of a cornea-specific human keratin 12 gene and detection of four mutations in Meesmann corneal epithelial dystrophy.Am J Hum Genet.1997;61:1268-1275.TGlaserDSWaltonRLMaasGenomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene.Nat Genet.1992;2:232-239.IMHansonJMFletcherTJordanMutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly.Nat Genet.1994;6:168-173.FMirzayansWGPearceIMMacDonaldMAWalterMutation of the PAX6 gene in patients with autosomal dominant keratitis.Am J Hum Genet.1995;57:539-548.KNishidaWAdachiAShimizu-MatsumotoA gene expression profile of corneal epithelium and the isolation of human keratin 12 cDNA.Invest Ophthalmol Vis Sci.1996;37:1800-1809.PGonzalezDLEpsteinTBorrasCharacterization of gene expression in human trabecular meshwork using single-pass sequencing of 1060 clones.Invest Ophthalmol Vis Sci.2000;41:3678-8693.FJLiveseyTFurukawaMASteffenGMChurchCLCepkoMicroarray analysis of the transcriptional network controlled by the photoreceptor homeobox gene Crx.Curr Biol.2000;10:301-310.PGonzalezDLEpsteinTBorrasGenes up-regulated in the human trabecular meshwork in response to elevated intraocular pressure.Invest Ophthalmol Vis Sci.2000;41:352-361.Not AvailableOnline Mendelian Inheritance in Man resource page.National Center for Biotechnology Information Web site. Available at: http://www.ncbi.nlm.nih.gov/omim. Accessibility verified July 19, 2001.AMShearmanTJHudsonJMAndresenThe gene for Schnyder's crystalline corneal dystrophy maps to human chromosome 1p34-p36.Hum Mol Genet.1996;5:1667-1672.EHeonWDMathersWLAlwardLinkage of posterior polymorphous dystrophy to 20q11.Hum Mol Genet.1995;4:485-488.NMTomaNDEbenezerCFInglehearnCPlantLAFickerSSBhattacharyaLinkage of congenital hereditary endothelial dystrophy to chromosome 20.Hum Mol Genet.1995;4:2395-2398.CKHandDLHarmonSMKennedyJSFitzsimonLMCollumNAParfreyLocalization of the gene for autosomal recessive congenital hereditary endothelial dystrophy (CHED2) to chromosome 20 by homozygosity mapping.Genomics.1999;61:1-4.YSRabinowitzLZuHYangYWangJRotterSPulstKeratoconus: further gene linkage studies on chromosome 21.Invest Ophthalmol Vis Sci.2000;41(suppl):S539.DAMackeyRGButteryGMWiseMJDentonDescription of X-linked megalocornea with identification of the gene locus.Arch Ophthalmol.1991;109:829-833.Accepted for publication June 8, 2001.This work was supported by unrestricted grants from Research to Prevent Blindness Inc, New York, NY; Tissue Banks International, Baltimore, Md (Dr Gottsch); the Irvin and Ginger Gomprecht Research Fund (Dr Gottsch); the Deborah Black Research Fund (Dr Stark); and the Raymond Kwok Research Fund (Dr Stark).The authors are thankful to Morton F. Goldberg, MD, for continued support of this work and Elizabeth Bell for research assistance.Corresponding author and reprints: John D. Gottsch, MD, Cornea and External Disease Division, Wilmer Ophthalmological Institute, The Johns Hopkins Medical Institutions, Maumenee 317, 600 N Wolfe St, Baltimore, MD 21287 (e-mail: jgottsch@jhmi.edu.).

Journal

JAMA OphthalmologyAmerican Medical Association

Published: Nov 1, 2001

There are no references for this article.