Molecular Vision 2003; 9:508-514 <>
Received 4 November 2002 | Accepted 30 September 2003 | Published 7 October 2003

Identification of genes expressed in a human scleral cDNA library

Terri L. Young,1,2 Xiaoshan D. Guo,1,2 Richard A. King,3 Janell M. Johnson,4 Jody A. Rada4,5

Divisions of 1Ophthalmology and 2Pediatrics, Children's Hospital of Philadelphia and the University of Pennsylvania, Philadelphia, PA; 3Department of Medicine, University of Minnesota, Minneapolis, MN; 4Department of Anatomy and Cell Biology, University of North Dakota, Grand Forks, ND; 5Department of Cell Biology, University of Oklahoma Health Science Center, Oklahoma City, OK

Correspondence to: Terri L. Young, MD, 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:


Purpose: Clones established from a human scleral cDNA library were systematically sequenced. Public database sequence comparisons were performed to generate a profile of genes expressed in the human sclera and identify candidate genes for inherited diseases with scleral involvement.

Methods: A directionally cloned pCMV-PCR cDNA library was constructed from RNA isolated from scleras of human donor eyes with known plano refractive history. Plasmid DNA was extracted from randomly selected cDNA clones, and the insert sequences were determined by 5' end single-pass sequencing. Expressed sequence tags (ESTs) were generated and analyzed with the GenBank BLASTN program to identify sequence homologies to known genes.

Results: A total of 609 ESTs underwent BLAST analysis. Of these, 341 (56%) matched 228 known human genes and 4 non-human genes, 252 matched uncharacterized ESTs, and 16 showed no significant homology to human or non-human known sequences. The most redundant connective tissue-related genes were αA-crystalline, Xα-1 collagen, and β-5 integrin. Other extracellular matrix gene matches were biglycan, syndecan, decorin, fibromodulin, proline arginine-rich end leucine-rich repeat protein, transgelin, TIMP-1, and fibulin 1. Human scleral expression of all but decorin and biglycan has not previously been reported.

Conclusions: This effort provides the first partial list of genes expressed in human sclera. Identification of genes expressed in the 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 coat of the eye, is a typical connective tissue that provides the structural framework that defines the shape and therefore the axial length of the eye. The sclera consists largely of collagenous lamellae in close association with proteoglycans and glycoproteins [1,2]. Located between the lamellae are scleral fibroblasts that synthesize and remodel the scleral extracellular matrix. Alterations in the extracellular matrix components of the sclera, or in any molecules required for the synthesis and degradation of the scleral matrix, are likely to lead to significant changes in the biomechanical properties of the sclera and are likely to result in changes in scleral shape, ocular size, and the refractive state of the eye [3-6].

Myopia is the most common of all ocular problems, affecting 25% of the US adult population [7]. The most common structural abnormality associated with myopia is excessive lengthening of the posterior segment of the ocular globe (axial myopia) [8]. Many of the pathological changes seen in highly myopic human eyes are a consequence of extracellular matrix reorganization and gross scleral thinning, particularly at the posterior pole of the eye [8,9].

Several gene products have been shown to change their levels of expression in the sclera during the development of experimentally induced myopia in animal studies. The synthesis and accumulation of total protein [10], the scleral proteoglycans decorin, biglycan, and aggrecan [11-14], the matrix metalloproteinase, gelatinase A [15-17], inhibitors of metalloproteinases TIMP-1 and TIMP-2 [16,18], and collagen [18] have all been shown to be altered in the sclera during form deprivation myopia development in chicks, tree shrews, and primates. Moreover, the expression of proteoglycans, gelatinase A and TIMP-2, have been shown to normalize following restoration of normal (unrestricted) vision [19,20], indicating a direct relationship between the expression of these extracellular matrix constituents and the rate of ocular elongation.

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 Ehler Danlos Syndrome; collagen 18A1 mutations have been identified in Knobloch syndrome; and fibrillin defects have been shown to be responsible for Marfan syndrome [21-25]. Since the sclera is a connective tissue consisting largely of extracellular matrix, it is likely that each of these genes is expressed in the sclera, and may partially explain the scleral involvement and high myopia observed with 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) [26-31], suggesting significant genetic heterogeneity. 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 implicated to date. This is partially due to the fact that many families in which myopia segregates are small, with an insufficient number of informative recombination events to allow narrowing of the genetic interval to determine where individual myopia candidate genes map. Thus, in many cases, several megabases of genomic DNA must be analyzed to identify candidate genes for each of the loci.

Analysis of expressed sequence tags (ESTs) has proven useful in identifying positional candidate genes for human disease [32,33]. ESTs provide short nucleotide sequences that act as unique identifiers of both novel and known genes, and can be used as probes to clone genes from appropriate cDNA libraries. The profiling of gene expression in specific tissues provides useful information to characterize gene function and tissue physiology [34-36]. Partial/single-pass sequencing of clones from cDNA libraries to generate ESTs is a convenient and efficient means of identifying genes expressed in certain tissues. On the basis of sequence similarity, information from single-pass sequencing of cDNA clones is sufficient to allow the description of the profile of expressed genes in cell types of particular tissues. Greater than 90% of positionally cloned genes associated with mutations in human diseases are represented by exact sequence matches to ESTs in the EST division of GenBank. This indicates that the vast majority of disease genes are already present within the GenBank EST collection. Greater than 50,000 human sequence-tagged sites (STSs), many of them derived from ESTs, have been assigned a chromosomal locus. Map positions of ESTs from dozens of cDNA libraries are a crucial resource for determining positional candidates for disease genes.

To potentially identify positional candidate genes for human scleral disorders, and to gain a more fundamental understanding of human sceral biology at the molecular level, we constructed a human scleral cDNA library. We partially sequenced and characterized randomly isolated clones from this library of human sclera. The generation of ESTs from this human sclera cDNA library is, to our knowledge, the first effort to establish an extended list of expressed genes involved in scleral development and composition.


Construction of cDNA library

A cDNA library was commercially constructed from RNA isolated from sclera of 9 pairs (18 scleras) of human donor eyes, with known non-myopic refractive history. 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, and treated by submersion in RNAlater Solution (Ambion, Austin, TX) within 12 h post mortem. The entire sclera except the lamina cribosa was used for extraction of total RNA. Frozen scleral tissue was cryogenically ground into a powder form using a freezer/mill (6750 SPEX CertiPrep, Metuchen, NJ). Total RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA). A high salt (0.8 M sodium citrate and 1.2 M NaCl) precipitation step was added to increase yield and improve quality of the total RNA. The quantity and qualilty of RNA was assessed by obtaining the ratio of absorbance values 260 at 280 nm using a Beckman spectrophotometer, and by visualization of the 28 and 18S rRNAs on denaturing formaldehyde agarose gels, respectively. The yield of total RNA varied from donor to donor, ranging from 4.2 μg/sclera to 50.4 μg/sclera. The RNA was then submitted to Stratagene (La Jolla, CA) for commercial preparation of a cDNA library. The library was constructed from 584 μg of total RNA.

Following cDNA synthesis, cDNA inserts were annealed with the pCMV-PCR vector (Stratagene, La Jolla, CA) using the Ligation Independent Cloning method to generate a plasmid library. The resulting library had an estimated unamplified titer of 1.8x104 cfu/ml, an estimated background of <17% nonrecombinants, and an approximate average insert size of 0.9 kb (range from 0.25 to 3.5 kb). The directionally cloned library was not subtracted or normalized for this analysis due to the titer amount and the relative mono-cellular nature of sclera.

Plasmid preparation and DNA sequencing

Colonies from the sclera cDNA library were plated at low density on LB plates containing 50 μg/ml kanamycin and grown overnight at 37 °C. Well-spaced colonies were selected randomly and cultured overnight in 2 ml of LB broth containing 50 μg/ml of kanamycin. After growth, 2 ml of each culture were used to isolate plasmid DNAs using the Wizard SV 96 Plasmid DNA Purification System (Promega, Madison, WI).

Restriction enzyme reactions using EcoR I and Xho I were performed to cut the plasmid DNA, and products were examined by agarose gel to exclude clones containing short (less than 300 bp) or no inserts (17%), and those which gave multiple bands. The vector size is 4.3 kb, and samples showing only this band size and one other band were sequenced after gel electrophoresis. Since sequencing of 5' ends is more likely to yield DNA sequences that encode amino acids, 5' end single pass sequencing using T3 primer was performed with the Thermo Sequenase Dye terminator Cycle Sequencing Pre-Mix kit (Amersham Pharmacia Biotech, Piscataway, NJ) and analyzed on an automated sequencer (Model 373A, Applied Biosystems, Inc., Foster City, CA).

Homology to known sequences in databases

The insert sequences were examined for similarities to human genes and ESTs in the GenBank database of expressed genes. This was conducted using the "basic local alignment search tool" program (BLASTN), available through the National Center for Biotechnology Information (NCBI, Bethesda, MD).

Homology comparisons of the cDNA sequences with known genes in the Genbank database were searched by the BLAST program. All searches were performed in the period May 2001 to March 2002. Poly (A), vector sequences, and sequences with many ambiguities were manually removed from the sequence data, and only sequences longer than 300 bp were analyzed further. Sequence identity was considered significant to a database entry with a match of p <1 x 10-35 (smallest sum probability) and aligned region >85% over the entire length of the EST. The majority of scleral sequences were >95% identical to the query sequence, disregarding 1 bp frame shifts and ambiguous (N) nucleotides. When a BLAST search revealed a significant match to more than one known gene, only the highest scoring hit was included in the data set.

Scleral ESTs that did not match a known gene subsequently were BLASTed against the Genbank EST, non-human (i.e. rodent, insect, microbial, etc.) and mitochondrial databases. A scleral EST nucleotide sequence was considered to be a match to an EST, non-human or mitochondrial sequence when p <1 x 10-30, the aligned region was >50 nucleotides and >85% over the entire EST, or p<1 x 10-20, the aligned region was >50 nucleotides and >90% over the entire EST.


We have developed a cDNA library that represents a significant resource for the expression profile in human sclera. We examined DNA sequences obtained from 640 clones selected randomly from the human sclera cDNA library for sequence homology or identity to genes whose DNA sequences had been deposited in the database. Of the 640 total scleral ESTs generated, nucleotide sequences from 31 were either too short or of insufficient quality to yield useful data for this study. Thus, 609 sclera ESTs (609 clones) were of adequate length and quality to continue with further analysis.

Each of the 609 scleral ESTs was compared by BLAST search. With noted redundancy, 337 scleral EST sequences matched 228 known human genes. Four scleral ESTs showed sequence homology to 4 non-human genes. Of the remaining 268 scleral ESTs, 252 showed significant homology to ESTs from other cDNA libraries in GenBank. Sixteen transcripts did not match any sequences in GenBank (non-redundant database, human and mouse EST databases, mitochondrial database), and are possibly novel genes. (Table 1). A summary of the known human genes that are identical to at least one scleral EST are listed in Table 2. Matches to 54 mitochondrial genes were seen (data not shown). Among the 252 sequences that matched to human ESTs from other cDNA libraries, 176 ESTs matched to 166 different BAC/PAC/cosmid clones.

A list of redundant genes and their chromosome location, if identified, and the number of clones are shown in Table 3. Genes of interest with the greatest redundancy among the scleral clones were humanin (HN1; 8 clones), light polypeptide ferritin (FTL; 5 clones), apolipoprotein D gene exon 4 (APOD; 5 clones), cytochrome c oxidase II (MTCO2; 5 clones), crystallin, alpha A (CRYAA; 3 clones), collagen, type X alpha 1 (COL10A1; 2 clones), and integrin beta 5 (ITGB5; 2 clones).

A list of extracellular matrix/connective-tissue genes with their chromosomal loci and GenBank accession numbers are shown in Table 4. Seventeen extracellular matrix/connective tissue genes are represented among the scleral ESTs including laminin beta 1(LAMB1), collagen type X alpha 1(COL10A1), crystallin alpha A (CRYAA), integrin beta 5 (ITB5), Vimentin (VIM), human biglycan BGN, syndecan-1 gene (exon 2-5; SDC1), transgelin (TAGLN), fibulin 1 (FBLN1), actinin alpha 1(ACTN1), fibulin-1 isoform D precursor, decorin (DCN), actin beta (ACTB), fibromodulin (FMOD), tissue inhibitor of metalloproteinase 1 (TIMP1), proline arginine-rich end leucine-rich protein (PRELP), and matrix Gla protein (MGLAP). As part of a separate study [37] the expression of fibromodulin and PRELP in the human sclera has been verified by qualitative and quantitataive RT-PCR.

Four genes were found that corresponded to mapped myopia loci. These included biglycan (BGN), which maps to MYP1 locus at Xq28, myosin regulatory light chain 2 (MYL2), which maps to chromosome 12q23-24, and keratin 13 (KRT 13) and transducer 1 of avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2; TOB 1), both of which map to the chromosome 17q21-22 locus. No gene matches were found for the MYP2 locus at chromosome 18p11.31 or the chromosome 7q36 locus. Eleven EST-BAC matches mapped specifically to the MYP3 locus at chromosome 12q23-24.


This study describes a reverse molecular genetic approach to identify genes involved in controlling scleral growth and development. Sequencing 609 human scleral cDNAs to create ESTs has resulted in the identification of 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 the scleral involvement in a variety of heritable disorders.

Obtaining high quality RNA from eye bank donor eye tissue can be difficult. There is a requisite delay time for serological testing (e.g. HIV and hepatitis) before the tissue can be handled safely. A prolonged interval between patient death and the collection of dissected ocular tissues may take several hours to days. RNA degradation occurs within this time period, compromising quality and yield. Many ocular tissues contain melanin, a substance known to co-purify with RNA and inhibit reverse transcription, as well as polymerase chain reaction methods [38].

To our knowledge, there are no known cDNA libraries specifically of sclera, nor are there cDNA libraries for cartilage, joint, or cardiac valvular tissue. This, we suspect, may in part be due to the difficulty in homogenization of the tissue. Conventional methods of tissue disruption have proven to be ineffective for cell membrane lysis with this tough, fibrous tissue. We found that freezing the sclera to make the tissue brittle, and then pulverizing to a powder form allowed for optimal intracellular RNA release. Nonetheless, RNA yields were still small, requiring combining multiple scleral samples. Consequently, the cDNA library titer was low, and for this reason we elected not to perform subtraction or normalizing techniques for this directionally cloned library.

In general, the number of clones derived from an individual gene from a non-subtracted or non-normalized cDNA library approximates the expression level of that gene in that certain tissue. Since the extracellular matrix is largely responsible for the biochemical and biomechanical properties of the sclera, we have focused our attention on connective tissue elements in the sclera. The most redundant connective tissue-related genes were αA-crystalline, Xα-1 collagen, and β-5 integrin. Other extracellular matrix gene matches were biglycan, syndecan, decorin, fibromodulin, proline arginine-rich end leucine-rich repeat protein, transgelin, TIMP-1, and fibulin 1. Human scleral expression of all but decorin and biglycan has not previously been reported.

This study has identified message in human scleral mRNA for a number of proteins, many of which have not been previously reported in sclera, and some of which are not currently catalogued in GenBank. However, the list of gene reported here should not be considered comprehensive of all genes expressed in the sclera. Indeed, genes such as collagen type I and elastin, known constituents of the sclera, were not identified in our library screening procedure. Indeed, estimates of the level of gene expression indicate that at any one time, a human cell expresses approximately 10-20,000 genes [39]. Therefore the genes that were identified in the present study represent only 1-2% of the total number of mRNA species potentially expressed by scleral fibroblasts. This most likely reflects the incomplete representation of all expressed genes in our cDNA library and/or differences in the developmental expression of various transcripts. These results are a first attempt to obtain a broad picture of genes expressed in scleral tissue, the results of which could potentially be integrated into more comprehensive analyses using microarray technologies.


We thank Dr. Beverly Emmanuel for helpful discussions and comments on the manuscript. We also thank the Minnesota Lions Eye Bank (Minneapolis, MN) for assistance in obtaining eye tissues.

Grant/Financial Support: 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), and the National Institutes of Health-NEI-EY00376 (TLY), and NIH-NEI-EY09391 (JAR).


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