|Molecular Vision 2004;
Received 3 October 2003 | Accepted 16 March 2004 | Published 22 March 2004
Microarray analysis of gene expression in human donor sclera
Terri L. Young,1,2
Genaro S. Scavello,1,2 Prasuna
C. Paluru,1,2 Jonathan D. Choi,2,3 Eric F. Rappaport,2
Jody A. Rada4
Divisions of 1Ophthalmology and 2Genetics, Children's Hospital of Philadelphia and the University of Pennsylvania, Philadelphia, PA; 3Department of Medical and Molecular Genetics, GKT School of Medicine, London; 4Department of Cell Biology, University of Oklahoma Health Science Center, Oklahoma City, OK
Correspondence to: Terri L. Young, M.D., Division of Ophthalmology, Children's Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, PA, 19104; Phone: (215) 590-9950; FAX: (215) 590-3850; email: firstname.lastname@example.org
Purpose: To develop gene expression profiles of human sclera to allow for the identification of novel, uncharacterized genes in this tissue-type, and to identify candidate genes for scleral disorders.
Methods: Total RNA was isolated from 6 donor sources of human sclerae, and reverse transcribed into cDNA using a T7-(dT) 24 primer. The resulting cDNA was in vitro transcribed to produce biotin-labeled cRNA, fragmented, and mixed with hybridization controls before a 16 h hybridization step with oligonucleotide probes on 6 Affymetrix U95A chips. The chips were scanned twice at 570 nM and the data collected using GeneChip software. Array analyses were carried out with Microarray Suite, version 5.0 (Affymetrix), using the expression analysis algorithm to run an absolute analysis after cell intensities were computed. All arrays were scaled to the same target intensity using all probe sets. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to validate the microarray results.
Results: There were 3,751 genes with "present" calls assigned independently to all six human scleral samples. These genes could be clustered into 4 major categories; transcription (10%), metabolism (8.8%), cell growth and proliferation (5.4%), and extracellular matrix (2%). Many extracellular matrix proteins, such as collagens 6A3 and 10A1, thrombospondins 2 and 4, and dystroglycan have not previously been shown to be expressed in sclera. RT-PCR results confirmed scleral expression in 7 extracellular matrix genes examined.
Conclusions: This study demonstrated the utility of gene microarray technology in identifying global patterns of scleral gene expression, and provides an extended list of genes expressed in human sclera. Identification of genes expressed in sclera contributes to our understanding of scleral biology, and potentially provides positional candidate genes for scleral disorders such as high myopia.
The sclera, the tough outer wall of the eye, is a specialized connective tissue that provides the structural framework that defines the shape (such as the axial length) of the eye. It consists largely of collagenous lamellae in close association with proteoglycans and glycoproteins [1-5]. Changes in the extracellular matrix components of the sclera or in molecules required for the synthesis and degradation of the scleral matrix may lead to significant changes in scleral biomechanical properties, leading to changes in scleral shape, ocular size and therefore the refractive state of the eye [6-10].
The profiling of gene expression in specific tissues provides useful information to characterize gene function and tissue physiology [11-13]. This baseline knowledge should facilitate the identification of alterations from normal gene expression that play important roles in disease pathogenesis. Prior research on the human sclera has focused on the detection of individual proteins of interest using techniques such as immunohistochemistry and in situ hybridization. Due to the rapid progress of the Human Genome Project and the development of high-throughput techniques such as cDNA microarray analysis [14-16], messenger RNA (mRNA) expression can be determined on a global scale with parallel assessment of gene expression for hundreds or thousands of genes in a single experiment.
Microarray expression analysis has features that make it the most widely used method for profiling mRNA expression. DNA segments representing the collection of genes to be assayed are amplified by polymerase chain reaction (PCR) and mechanically spotted at high density on glass microscope slides or nylon membranes using robotic systems. Experimental mRNA is labeled as a complex mixture and exposed to the microarray. Labeled mRNA will bind to complementary sequences on the microarray and can be detected in a semi-quantitative manner using automated techniques. The microrarrays are queried in a co-hybridization assay using fluorescently labeled probes prepared from mRNA from the cellular phenotypes of interest. The kinetics of hybridization allows relative expression levels to be determined based on the ratio with which each probe hybridizes to an individual array element. Hybridization is assayed using a confocal laser scanner to measure fluorescence intensities, which allows the simultaneous determination of the relative expression levels of all the genes represented in the array [16-19].
Because of the success of microarray analysis use in other biological systems and other eye tissue types [16-23], we sought to apply this technology to study gene expression in human donor scleral tissue. This analysis provides baseline expression information regarding the genetic basis of normal scleral function, as well as for scleral disease processes such as pathologic myopia with excess axial elongation, microphthalmia, scleral ectasia, focal staphylomatous formation, and inflammation. Knowledge of genes expressed or not expressed in a particular scleral disorder could lead to novel and definitive treatment strategies, such as interventional or gene therapies. These strategies may be particularly relevant for the sclera because the tissue is relatively less complex, can be manipulated ex vivo, and can be readily assessed visually.
Affymetrix prefabricated chips
Each probe set on an Affymetrix chip is represented by multiple features or probes consisting of synthesized oligonucleotides. Half of the features are exact match representations at different positions along the length of the expressed regions of the gene. The other half contains the same oligonucleotide probes, but with a single mismatch in the middle. The range of 22-40 cells on the HG-U95A chip includes some controls for which there is a smaller or larger number than the 32 used as the probe sets for experimental genes. On the Affymetrix HG-U95A chip, for any given experimental gene there are 16 positions at which hybridization can be assessed, and the comparison between the perfect match and mismatch probes provides a control for non-specific hybridization. The Affymetrix HG-U95A chip contains 12,626 probe sets with some redundancy for certain genes or splice variants.
Target preparation, hybridization, and washing
Total RNA was isolated from the sclera of 6 human donor eyes. The eyes were from both male and female donors, ages 35-68 years. The human eyes were obtained from the Lions Eye Bank of Minnesota under an approved Institutional Review Board protocol at the University of Minnesota. The procurement and use of human tissues was in compliance with the tenets of the Declaration of Helsinki. The donor eyes were obtained as either whole globes or posterior poles with the cornea removed. The eyes were treated by submersion in RNALater solution (Ambion Inc., Austin, TX) within 2-6 h post mortem. The entire sclera, minus the lamina cribosa was used for extraction of total RNA. The scleral tissue was snap frozen, and stored in a -80 °C freezer until RNA extraction. Frozen scleral tissue was cryogenically ground into a powder form using a freezer mill (6750 SPEX, CertiPrep Inc., Metuchen, NJ). Total RNA was extracted from pulverized samples using TRIZOL reagent (Invitrogen Inc., Carlsbad, CA) . A high salt (0.8 M sodium citrate and 1.2 M NaCl) precipitation step was added to increase yield and improve the quality of the total RNA. The quantity and quality of RNA was assessed by obtaining the ratio of absorbance values at 260 and 280 nm using a Beckman spectrophotometer, and by visualization of intact 28S and 18S ribosomal RNA bands on denaturing formaldehyde agarose gels after electrophoresis. Individual RNA samples were assessed with electrophoresis to document RNA integrity using the Agilent Caliper system (Agilent Technologies Inc., Palo Alto, CA) . The yield of total RNA per scleral sample ranged from 89-201 μg/eye. There was no correlation of quality with donor age.
Target was prepared using 5-20 μg of total RNA for each donor sclera. First strand cDNA was synthesized using Superscript II Reverse Transcriptase (Invitrogen Corporation, Carlsbad, CA) and a T7-(dT)24 primer to incorporate the T7 priming site into the cDNA. Following RNA degradation with RNase H and second strand cDNA synthesis with DNA polymerase I, the double-stranded cDNA was extracted with phenol:chlorform:isoamyl alcohol (25:24:1). Approximately 1 μg of cDNA was used as template in an in vitro transcription assay reaction (Enzo Life Sciences, Inc., Farmingdale, NY) that incorporates biotin into the resulting cRNA. The cRNA was fragmented to a size range of 35-200 bases prior to use in hybridization by incubation at 94 °C for 35 min in fragmentation buffer (40 mM Tris acetate, pH 8.1, 125 mM KOAc, 30 mM MgOAc). Fifteen μg of fragmented probe was mixed with hybridization controls, herring sperm DNA (final concentration 100 μg/ml), and acetylated BSA (final concentration 100 μg/ml) in hybridization buffer (100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween 20). The hybridization mixture was heated at 99 °C for five min, incubated at 45 °C for five min, and centrifuged at 13,000x g for 5 min. Test 3 chips were pre-hybridized with 80 μl of 1X hybridization buffer for 10 min at 45 °C and 60 RPM in the hybridization oven. Following removal of the pre-hybridization buffer, the 6 Affymetrix chips were filled with 200 μl of the hybridization mixture and incubated at 45 °C and 60 RPM for 16 h [26,27].
Hybridization mixture was removed and saved, and each chip was filled with 250 μl of non-stringent wash buffer (6X SSPE, 0.01% Tween 20). Further washing and staining of the chips was conducted on the fluidics station with non-stringent washing buffer, stringent washing buffer (100 mM MES, 0.1 M [Na+], 0.01% Tween 20), and stain buffer (100 mM MES, 1 M [Na+], 0.05% Tween 20) containing 10 μg /ml of streptavidin phycoerythrin (SAPE, Molecular Probes, Inc., Eugene, OR). The signal was amplified by an additional treatment with goat IgG (0.1 mg/ml), biotinylated antibody (3 μg/ml) and a second staining with SAPE. The chips were then scanned and the data analyzed using the Affymetrix Microarray Analysis Suite software as described below.
Scanning, data collection, and analysis
Following staining and washing, each chip was scanned twice at 570 nm with a confocal scanner (Agilent Technologies, Inc., Palo Alto, CA). The output fluorescence was collected using the Affymetrix Microarray Analysis Suite 5.0 software and the average for the two scans yielded an image file used for further data analysis.
The Microarray Analysis Suite software performs absolute analysis of the data from each chip, including background calculation, detection call (present, absent, or marginal) and signal, which serves as a measure of relative expression level for the various genes. Several metrics are used in these calculations, including the intensities of each probe pair (perfect match and mismatch) in a probe set (the 11-20 pairs representing each gene) and a decision matrix is formed from which the absolute call is made. The software can also perform comparative analysis between any two chips, normalizing signals, calculating more metrics and using them to make a difference call (increased, decreased, marginally increased, marginally decreased, no change), to generate an average difference change between the same genes (i.e., probe sets) on the different chips, and to provide a fold change calculation about the magnitude of any expression difference measured.
The identified expressed genes with present calls for all 6 samples were compared with the following NCBI databases: GenBank, On-Line Mendelian Inheritance in Man (OMIM), and PubMed.
Reverse transcription-polymerase chain reaction
Total RNA was extracted from 4 pooled, human donor sclera using TRIZOL reagent as described above. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed with oligo-dT oligonucleotide primer using standard methods to synthesize cDNA with SuperScript II (Invitrogen Corporation, Carlsbad, CA). Total RNA from sclera (1 μg) or eye tissues such as optic nerve, retina, and cornea, as well as commercially prepared poly-A RNA from various human organs (Clontech Inc., Pal Alto, CA) were used as a template for first-strand cDNA synthesis. PCR was performed using Platinum Taq polymerase using 2 μl of each cDNA sample in a final reaction volume of 50 μl. A final concentration of 2.5 μM was used for each PCR primer. The PCR cycling conditions included an initial denaturation for 120 s at 95 °C, followed by 34 cycles of denaturation for 15 s at 95 °C, annealing for 30 s at 54 °C, extension for 45 s at 68 °C, and a final extension for 4 min at 68 °C. The 5' sense and 3' antisense PCR primer pairs designed for collagens 6A3 and 10A1, thrombospondins 2 and 4, dystroglycan, biglycan, and decorin are listed in Table 1, along with their corresponding amplicon size. The RT-PCR products, along with the amplicon products of the housekeeping gene β-actin as a control were visualized on 2% agarose gels after electrophoresis and staining with ethidium bromide.
There were 3,751 genes with "present" calls assigned independently to all six human scleral samples. Only the 3,751 genes confirmed by all 6 microarrays as expressed in sclera were used in subsequent analyses in this study. These genes could be clustered into 4 major categories; transcription (10%), metabolism (8.8%), cell growth and proliferation (5.4%), and extracellular matrix (2%). The 3,751 genes with confirmed scleral expression were analyzed using GenBank and LocusLink at NCBI. Of the 3,751 genes, 3,096 (82.5%) had assigned chromosomal loci. Six hundred fifty-five confirmed scleral genes (17.5%) had unassigned chromosomal loci.
The 3,751 genes detected with this microarray analysis form the basis of a scleral genetics sub-web site named ScleraNet at our research laboratory web site. The raw data for this study can also be found in Appendix 1. ScleraNet includes the 3,751 genes identified in the present study in addition to genes identified from the literature (sclera or scleral fibroblast cell lines), and non-overlapping genes identified from a human scleral cDNA library developed previously in our laboratory .
Table 2 displays all extracellular matrix genes identified by microarray analysis, along with their chromosomal locus and GenBank accession numbers. The primary structural proteins which sclera is comprised of are the collagens. As expected, a variety of collagen genes were expressed in all samples. These include COL1A2, COL2A1, COL3A1, COL4A1-4,6, COL5A1-3, COL6A1-3, COL7A1, COL8A1-2, COL9A1-3, COL10A1, COL11A1-2, COL12A1, COL13A1, COL14A1, COL15A1, COL18A1, and COL19A1. Many of these extracellular matrix proteins, such as collagens 6A3 and 10A1, have not been shown to be expressed in sclera previously. Interestingly, there was minimal overlap with collagens expressed in the cornea, where only 6 collagens (COL4A1, 11A1, 16A1, 5A2, 4A3, and 6A3) were detected by similar microarray analysis .
Even though the scleral samples were from adult human eyes presumptively no longer in a growth phase, evidence of continued remodeling is supported by the detection of expressed regulators of collagen metabolism. Secreted proteases are known to play a major role in remodeling the stromal extracellular matrix [29,30]. Multiple human metalloproteinase proteins (MMP) were detected (MMP 1-3, 7-17, 20, and 24). These are proteins originally identified in macrophages . MMP 12, for example, has multiple extracellular matrix substrates and disrupts basement membranes. Manganese super-oxide dismutase and leukotriene C4 synthase were detected, possibly indicating regulated scleral degradation by MMPs based on other scleral studies [32,33].
Secreted proteases are in turn regulated by various inhibitors. These have been shown to help control the degradation of the extracellular matrix [34,35]. Three tissue inhibitor metalloproteinases were detected (TIMPs 1, 2, and 3). The tissue inhibitors of metalloproteinases (TIMPs) block matrix metalloproteinase (MMP)-mediated increases in cell proliferation, migration, and invasion that are associated with extracellular matrix (ECM) turnover. TGFβ regulates collagen synthesis and deposition, and is activated by thrombospondin-1 [36,37]. Thrombospondins 1-3 were detected in the microarray analysis. Bone morphogenic protein (BMP-7) has been shown to regulate specific collagen deposition. Table 3 shows a list of transcription factors expressed in human sclera.
Collagen matrix organization is regulated by accessory proteins. Proteoglycans regulate collagen fibril spacing. Chondroitin 6-sulfotransferase-like protein was detected. Expression of this enzyme may be critical for maturation of the keratan sulfate proteoglycans, which are the major proteoglycans of the cornea and sclera. Several proteoglycans were identified, including glypicans 1 and 3-6, versican, dystroglycan 1, microfibril-associated glycoprotein-2, proline arginine-rich end leucine-rich repeat protein, lumican, aggrecan, and decorin. Another accessory protein identified was matrilin-3, which forms collagen dependent and independent fibrils, fibrillin, fibulin, various laminin subunits, fibronectin 1, fibromodulin, elastin, various cartilage matrix proteins, tenascin, and elastin microfibril interface located protein.
RT-PCR results confirmed expression in 7 of 7 genes examined (Figure 1). Genes tested were collagens 6A3 and 10A1, thrombospondins 2 and 4, and the proteoglycans dystroglycan, biglycan, and decorin. Biglycan and decorin are proteoglycans previously shown to be structural components of sclera .
These results have led to the identification of new scleral proteins that may be important in the maintenance of biochemical and biomechanical properties of the sclera. The list of genes reported here should not be considered comprehensive of all genes expressed in sclera, however. Limitations include those of the finite number of representative gene probe sets on the commercial chip itself, incomplete representation of all expressed genes in the mRNA, and differences in the developmental expression of various transcripts. Ideally, every gene that appears to be expressed should be confirmed with RT-PCR, immunostaining, and in situ hybridization. The present study chiefly serves to identify novel targets for future investigations. This is especially relevant for the extracellular matrix gene expression profile identified herein, which corresponds to other gene families under investigation that are known to be of major mechanistic interest.
Pathologic myopia can severely affect visual function and, at present, no effective pharmacologic agents are available to limit the development of this condition. It is clear, however, that in animal models of myopia the processes of biomechanical changes to produce axial elongation of the eye are regulated by the coordinated expression of genes encoding growth factors, cytokines, extracellular matrix molecules, metalloproteinases, specific glycoproteins, and glycosyltransferases. Investigators in past studies on gene expression during experimentally induced myopia have used traditional approaches, including quantitative reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, RNase protection assays, and western blot and immunohistochemistry analyses. The disadvantages of these approaches have been that only a few genes are detected per assay. A more global approach is needed to elucidate the many factors that may play a role in scleral physiology. In the present study, gene array technology was used to examine gene expression in human scleral donor tissue. We identified many genes for the first time to be expressed in human sclera. These genes are novel factors for further study.
Progress is underway to identify the hereditary basis of high myopia at the molecular genetic level. Mutations in extracellular matrix genes have been associated with several syndromic genetic disorders that include myopia as a consistent clinical feature. Collagen 2A1 and 11A1 mutations have been identified for Stickler syndromes type 1 and 2, respectively. Mutations in lysyl-protocollagen hydroxylase have been shown to be responsible for type VI Ehlers-Danlos Syndrome. Collagen 18A1 mutations have been identified in Knobloch syndrome, and fibrillin defects have been shown to be responsible for Marfan syndrome [39-43]. Each of these genes was detected in this microarray study of human sclera and supports the notion of increased scleral elasticity and ocular axial elongation due to faulty structural protein function when its gene is mutated, leading to the high myopia observed in these syndromes. Therefore, knowledge of gene expression of the membranous scleral wall is critical to our understanding of the mechanisms that regulate ocular size and shape, as well as to our understanding of the etiology of abnormal eye expansion and myopia.
Five chromosomal loci have been identified for non-syndromic high-grade myopia (MYP1 at Xq28, MYP2 at 18p11.31, MYP3 at 12q23-24, and loci at 17q21-22 and 7q36) suggesting significant genetic heterogeneity [44-49]. Despite these recent successes in mapping myopia loci and implicating specific extracellular matrix proteins with myopia development, no gene mutations for any loci have been identified to date, although transforming growth factor beta-induced has been implicated . The search for these disease-causing genes could be facilitated by knowledge of which expressed scleral genes reside at specific chromosomal loci. Table 4 displays a list of genes expressed in human donor sclera that map to chromosome loci with defined intervals (chromosomes 7q36, 18p11.31, 12q23-24, and 17q21-22) associated with non-syndromic high-grade myopia.
This study describes a reverse molecular genetic approach to identify genes involved in scleral composition. Microarray analyses of mRNA from 6 human donor scleral samples have identified several known genes, as well as previously uncharacterized novel genes expressed in this specialized connective tissue. Any of the genes identified in this cDNA library may serve as candidates for high myopia or other disorders of scleral growth and development, and may help to explain scleral involvement in a variety of heritable disorders.
These efforts are to our knowledge a first attempt using microarray analysis to obtain a broad picture of the composition of human scleral tissue. The results were used to create a preliminary, online database of genes expressed in normal human donor sclera.
This research was supported in part by the Macula Vision Research Foundation, Bala Cynwyd, PA (TLY and JAR), Mabel E. Leslie Research Funds, Children's Hospital of Philadelphia (TLY), Research To Prevent Blindness Career Development Award (TLY), National Center for Research Resources RR017703 (JAR), National Eye Institute CORE grant EY121291 (JAR), and the National Institutes of Health NEI-EY00376 (TLY), *and NEI-EY09391 (JAR).
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