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The proteoglycans lumican and fibromodulin regulate collagen fibril assembly and show expression in ocular tissues. A recent mouse knockout study implicates lumican and fibromodulin as functional candidate genes for high myopia. Lumican maps within the chromosome 12q21-q23 autosomal dominant high grade myopia-3 (MYP3) interval, and fibromodulin maps to chromosome 1q32. We screened individuals for lumican and fibromodulin sequence alterations from the original MYP3 family, and from a second high grade myopia pedigree that showed suggestive linkage to both the MYP3 interval and to chromosome 1q32.A total of 10 affected (average spherical refractive error was -16.13 D) and 5 unaffected individuals from the 2 families were screened by direct DNA sequencing. Six primer pairs spanning intron-exon boundaries and coding regions were designed for the 3-exon 1804 base pair (bp) lumican gene. Two primer pairs for the 2-exon 2863 bp fibromodulin gene were designed. Polymerase chain reaction products were sequenced and analyzed using standard fluorescent methods. Sequences were quality scored and aligned for polymorphic analysis.Direct DNA sequencing of lumican amplicons yielded the expected sequence with no evidence of polymorphism or pathologic mutation. Sequencing of fibromodulin amplicons revealed 6 polymorphisms, 1 of which was novel. One polymorphism was a silent mutation, and five were in the 3' untranslated region. No polymorphism segregated with high myopia.Although null and double null Lum and Fmod mouse models have been developed for high myopia, our human cohort did not show affected status association with these genes. Sequencing of the human lumican and fibromodulin genes has excluded them as candidate genes for MYP3 associated high grade myopia.

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.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.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.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.

Purpose To map the gene(s) associated with autosomal dominant (AD) high-grade myopia. Methods A multigeneration English/Canadian family with AD severe myopia was ascertained. Myopes were healthy, with no clinical evidence of syndromic disease, anterior segment abnormalities, or glaucoma. The family contained 22 participating members (12 affected). The average age of diagnosis of myopia was 8.9 years (range, birth to 11 years). The average refractive error for affected adults was -13.925 D (range, -5.50 to -50.00). Microsatellite markers for genotyping were used to assess linkage to several candidate loci, including three previously identified AD high-myopia loci on 18p11.31, 12q22-q23, and 7q36. Syndromic myopia linkage was excluded by using intragenic or flanking markers for Stickler syndrome types 1, 2, and 2B; Marfan syndrome; Ehlers-Danlos syndrome type 4; and juvenile glaucoma. A full genome screening was performed, with 327 microsatellite markers spaced by 5 to 10 cM. Two-point linkage was analyzed using the FASTLINK program run at 90% penetrance and a myopia gene frequency of 0.0133. Results Linkage to all candidate loci was excluded. The genome screening yielded a maximum two-point lod score of 3.17 at theta = 0 with microsatellite marker D17S1604. Fine mapping and haplotype analysis defined the critical interval of 7.71 cM at 17q21-22. Conclusions A novel putative disease locus for AD high-grade myopia has been identified and provides additional support for genetic heterogeneity for this disorder.

Objective Bornholm eye disease (BED) consists of X-linked high myopia, high cylinder,optic nerve hypoplasia, reduced electroretinographic flicker with abnormalphotopic responses, and deuteranopia. The disease maps to chromosome Xq28and is the first designated high-grade myopia locus (MYP1). We studied a second family from Minnesota with a similar X-linkedphenotype, also of Danish descent. All affected males had protanopia insteadof deuteranopia. Methods X chromosome genotyping, fine-point mapping, and haplotype analysisof the DNA from 22 Minnesota family individuals (8 affected males and 5 carrierfemales) and 6 members of the original family with BED were performed. Haplotypecomparisons and mutation screening of the red-green cone pigment gene arraywere performed on DNA from both kindreds. Results Significant maximum logarithm of odds scores of 3.38 and 3.11 at thetas;= 0.0 were obtained with polymorphic microsatellite markersDXS8106andDXYS154,respectively, in theMinnesota family. Haplotype analysis defined an interval of 34.4 cM at chromosomeXq27.3-Xq28. Affected males had a red-green pigment hybrid gene consistentwith protanopia. We genotyped Xq27-28 polymorphic markers of the family withBED, and narrowed the critical interval to 6.8 cM. The haplotypes of the affectedindividuals were different from those of the Minnesota pedigree. Bornholmeye disease–affected individuals showed the presence of a green-redhybrid gene consistent with deuteranopia. Conclusions Because of the close geographic origin of the 2 families, we expectedaffected individuals to have the same haplotype in the vicinity of the samemutation. Mapping studies, however, suggested independent mutations of thesame gene. The red-green and green-red hybrid genes are common X-linked colorvision defects, and thus are unrelated to the high myopia and other eye abnormalitiesin these 2 families. Clinical Relevance X-linked high myopia with possible cone dysfunction has been mappedto chromosome Xq28 with intervals of 34.4 and 6.8 centimorgan for 2 familiesof Danish origin.