|Molecular Vision 2006;
Received 30 September 2005 | Accepted 26 April 2006 | Published 12 May 2006
Fine mapping of the keratoconus with cataract locus on chromosome 15q and candidate gene analysis
Durga Prasad Dash,1
Anne E. Hughes1
Departments of 1Medical Genetics and 2Ophthalmology, Queen's University of Belfast, Belfast, United Kingdom
Correspondence to: Durga Prasad Dash, Department of Ophthalmology, Institute of Clinical Science, Block A, Queen's University of Belfast, Royal Victoria Hospital, Grosvenor Road, Belfast, BT12 6BA, United Kingdom; Phone: 0044-7754404969; FAX: 0044-2890632699; email: firstname.lastname@example.org
Purpose: To report the fine mapping of the keratoconus with cataract locus on chromosome 15q and the mutational analysis of positional candidate genes.
Methods: Genotyping of two novel microsatellite markers and a single nucleotide polymorphism (SNP) in the critical region of linkage for keratoconus with cataract on 15q was performed. Positional candidate genes (MORF4L1, KIAA1055, ETFA, AWP1, REC14, KIAA1199, RCN2, FAH, IDH3A, MTHFS, ADAMTS7, MAN2C1, PTPN9, KIAA1024, ARNT2, BCL2A1, ISL2, C15ORF22 (P24B), DNAJA4, FLJ14594, CIB2 (KIP2), C15ORF5, and PSMA4) prioritized on the basis of ocular expression and probable function were screened by PCR-based DNA sequencing methods.
Results: We report the refinement of the linkage region for keratoconus with cataract to an interval of approximately 5.5 Mb flanked by the MAN2C1 gene and the D15S211 marker on chromosome 15q. Mutational analysis of positional candidate genes detected many sequence variations and single nucleotide polymorphisms. None of the sequence variants were considered pathogenic as they were also found in unaffected family members and normal control DNA samples.
Conclusions: Fine mapping of the keratoconus with cataract locus on 15q has reduced the linked region to 5.5 Mb, thereby excluding 28 candidate genes. A further 23 candidate genes were excluded by direct sequencing methods, although a pathogenic genomic rearrangement or exonic deletion would not have been detected.
Keratoconus (KC; OMIM 148300), the most common corneal dystrophy, is a bilateral, noninflammatory progressive corneal ectasia. Clinically, the cornea becomes progressively thin and conical which leads to myopia, irregular astigmatism, and corneal scarring. The transparency and refractive state of the cornea is a prerequisite for normal vision. The disease usually arises in the teenage years, eventually stabilizing in the third and fourth decades . It occurs with no ethnic or gender preponderance and causes significant visual impairment in young adults. No specific treatment exists except to replace the corneal tissue by surgery (corneal transplantation) when the visual acuity can no longer be corrected by contact lenses. In the Western world, KC is the most common indication for corneal transplantation . The underlying biochemical processes and pathobiology of keratoconus remain poorly understood.
The incidence of KC is 1 in 2,000 in the general population [1,3]. A family history is present only in a minority of cases, however, one of the major etiological factors is genetic [1,4-6]. KC is believed to be inherited autosomally, because of familial occurrence , a higher concordance rate of the trait in monozygotic twins then dizygotic twins  and its prevalence in first degree relatives is 15-67 times higher than the general population . Most studies describe autosomal dominant inheritance, with incomplete penetrance or variable expressivity [1,9,10].
Mutations in the VSX-1 transcription factor (OMIM 605020) were identified in 4.7% of patients with keratoconus (KTCN1; OMIM 148300) and also in posterior polymorphous corneal dystrophy (PPCD1; OMIM 122000) . However, traditional mapping methods to identify the genetic basis of KC have been limited by the lack of large multigeneration families for study. Genome-wide scans to localize the KC gene or genes had not been reported until our group identified a large three-generation family with KC and anterior polar cataract and reported linkage to chromosome 15q22.32-24.2 . Recently loci for autosomal dominantly inherited keratoconus have been mapped to chromosomes 21 , 16q22.3-q23.1 (KTCN2; OMIM 608932) , 3p14-q13 (KTCN3; OMIM 608586) , 2p24 (KTCN4; OMIM 609271)  and 5q14.1-q21.3 (NCBI).
The keratoconus with cataract locus on chromosome 15q was identified in a large Northern Irish family of three generations affected by combined autosomal dominant early onset anterior polar cataract and clinically severe keratoconus . The disease gene in this family was successfully mapped to chromosome 15q22.32-24.2 within a 6.5 Mb region flanked by markers CYP11A and D15S211 . CTSH, CRABP1, IREB2, and RASGRF1 were excluded previously as the causative gene for keratoconus with cataract . The purpose of the present study was to narrow the critical region of linkage of keratoconus with cataract by genotyping novel microsatellite markers and single nucleotide polymorphisms identified during sequencing of candidate genes. Fine mapping of a keratoconus with cataract locus on 15q has reduced the linked region to 5.5 Mb, thereby excluding 28 potential candidate genes. A further 23 positional candidate genes were prioritized for analysis based on expression studies, molecular and biochemical evidence (Table 1) and excluded by PCR-based DNA sequencing.
Genomic DNA was extracted from the peripheral blood leukocytes of all available family members using Puregene DNA purification kit (Gentra Systems, Inc., Minneapolis, MN).
Single nucleotide polymorphism typing
The single nucleotide polymorphism (SNP) rs1128933 in exon 20 of MAN2C1 (GenBank NM_006715) was typed in key recombinants using the forward primer 5'-CTG GAG ACA CGG TAT AGG CTG G-3' and the reverse primer 5'-AGT GTA CCT GGG AGT GGG AAG G-3'. Cycle sequencing was performed with the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) on PCR products using the forward PCR primer and analyzed on the ABI Prism 3100 DNA Sequencing System (Applied Biosystems).
Identification and typing of microsatellite markers
Two novel microsatellite markers on chromosome 15 located at 73.8 Mb between MAN2C1 and ETFA and at 78.4 Mb (within ARNT2) were identified from the human genome database (HGD). PCR was performed on genomic DNA from family members using the following primers: 5'-Fam-CTG TAT GCA GCG ATC AGT GAG C-3' and 5'-GTA GGA GAC AGA GAC ACT CAG TCC-3' for the marker at 73.8 Mb and 5'-Fam-TTA CAT CAC TTG CAT TGC TTC C-3' and 5'-AAT CAC AGC CAA ACA TAG ATG C-3' for the ARNT2 intragenic marker. Diluted PCR products were electrophoresed on an ABI 3100 genetic analyzer. Alleles were sized by Genotyper software (ABI) and scored using in-house software, C-allele (C-ALLELE program, Dr. Shane Mckee, Department of Medical Genetics, Queen's University of Belfast, UK). Linkage analysis was performed using the Fastlink linkage program version 5.1.
PCR-based DNA sequencing
Analysis of positional candidate genes was carried out by PCR-based DNA sequencing using dye terminator chemistry (ABI). Primers were designed using Primer Detective, version 1.01 (Clontech Labs Inc., CA) for all individual exons plus at least 50 bp of flanking intronic sequence, and 5' and 3' untranslated regions. For ADAMTS7, long-range PCR using Elongase (Invitrogen, Paisley, UK) was carried out as a preliminary step, using gene-specific primers that did not amplify related pseudogenes. After dilution of product (1 in 1000) nested PCR was performed before sequencing as already described. All primer pairs and PCR conditions are available on request.
One SNP and two novel microsatellite markers were identified toward the extremities of the previously linked interval on chromosome 15q  in a family with autosomal dominant keratoconus and cataract. Utilizing these markers, family members were genotyped to clarify the position of the recombination in key individuals. Typing of rs1128933 (Figure 1) within exon 20 of MAN2C1 showed III23 is recombinant at the MAN2C1 locus and more proximal markers, whereas III21, previously shown to be recombinant at CYP11A, is not recombinant at MAN2C1. The novel microsatellite markers located at 73.8 and 78.4 Mb (ARNT2) were fully informative for tracking haplotypes inherited by the crucial individuals in this family; however, none showed recombination at these loci. The further haplotype analysis described here has reduced the candidate interval of the keratoconus with cataract locus by 1.0 Mb, to the region flanked by rs1128933 (within MAN2C1) and D15S211.
Candidate genes positioned within this 5.5 Mb region (Figure 2) were identified and prioritized for analysis based on expression studies, molecular, and biochemical evidence (Table 1). Numerous sequence variations were found (Table 2) in positional candidate genes (MORF4L1, KIAA1055, ETFA, AWP1, REC14, KIAA1199, RCN2, FAH, IDH3A, MTHFS, ADAMTS7, MAN2C1, PTPN9, KIAA1024, ARNT2, BCL2A1, ISL2, C15ORF22 (P24B), DNAJA4, FLJ14594, CIB2 (KIP2), C15ORF5, and PSMA4). None of the sequence variants were considered pathogenic as they were also found in unaffected family members and unrelated, ethnically matched normal control DNA samples.
The refinement of the linkage region for the keratoconus with cataract locus on chromosome 15q  to 5.5 Mb between MAN2C1 and D15S211 has excluded 28 positional candidate genes. CTSH, CRABP1, IREB2, and RASGRF1 which remained within the refined disease interval were excluded previously by direct sequencing . The reduced disease gene interval is exceedingly gene rich containing a total of 75 positional candidate genes, of which 27 have now been screened without identifying a causative mutation. There is limited scope for further refinement of the linkage region, which cannot be reduced below a core interval of 4.6 Mb with the present family members. One family has been reported in the literature with corneal pathology and congenital cataract which maps to chr15q . The authors of this study have proposed the term EDICT syndrome for the clinical findings of endothelial dystrophy, iris hypoplasia, congenital cataract, and stromal thinning (EDICT) which were inherited as an autosomal dominant trait in their family. The locus for EDICT syndrome maps to a 26 Mb region of chromosome 15q which overlaps our region, and may therefore be allelic, however the small size of this family restricts its potential for improved mapping. Although the clinical phenotype in this family shows considerable differences from the EDICT syndrome , it is possible that both families share a common genetic basis. The EDICT syndrome includes features of significant anterior dysgenesis such as microcornea, iris hypoplasia, endothelial abnormalities and importantly the snydrome been reported to have congenital anterior polar cataract. Our family differs in that those affected have been documented as having no abnormalities or signs of anterior segment dysgenesis at birth, with the corneal and lens changes developing after the age of 5 years. Sequential examination of our family has indicated that the anterior polar cataract and keratoconus are developmental and not congenital. The ocular phenotype in our family is also unusual as the occurrence of keratoconus and developmental anterior polar cataract has not to our knowledge been previously described. Isolated congenital cataracts are usually inherited in an autosomal dominant fashion with mutations predominantly reported in the lens crystallins and connexins . Anterior polar cataracts are developmental lens opacities and have been mapped to 14q24-qter (CTAA1; OMIM 115650)  and 17p13 (CTAA2; OMIM 601202) . Corneal guttata (focal thickenings of the Descemet's membrane of the corneal endothelium) have been reported in association with anterior polar cataract (OMIM 121390) [20,21]. Developmentally the human lens is derived from surface ectoderm and the cornea from surface ectoderm and neuroectoderm. The ocular phenotype in this family may reflect a single developmental anomaly. Pathogenic mutations have been reported in a number of transcription factors which result in ocular phenotypes combining cataract, anterior segment dysgenesis and specific iris defects (aniridia, coloboma) [22-24].
Human VSX-1 (OMIM 605020) is a member of the Vsx1 group of vertebrate paired-like homeodomain transcription factors [25,26]. Mutations in the VSX-1 transcription factor were identified in 7%  of keratoconus patients . To date, five pathogenic heterozygous missense mutations have been reported in VSX-1 associated with keratoconus: L17P, R166W, L159M, D144E, and P247R [11,27]. The aryl hydrocarbon receptor nuclear translocator (ARNT2) in our candidate interval is also member of a novel transcription factor family consisting of a conserved basic helix-loop-helix (bHLH) structural motif contiguous with a PAS domain. A W278X mutation in the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) gene on 17p was found in a Pakistani family with Leber's congenital amaurosis and anterior keratoconus . We have not found any pathogenic mutation in ARNT2 or in ISL2 or MORF4L1 (also called MRG15) which also code for transcription factors.
ADAMTS7 was a strong candidate gene as the ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type 1 motif) families are secreted enzymes metalloproteases with a prominent role in extracellular matrix proteolysis  The proteolytic activity of this gene may be related to development of keratoconus in which there is evidence implicating matrix metalloproteinases in the pathogenesis . Other ADAMTS7 family members such as ADAMTS18 (16q22.3-q23.1) and ADAMTS9 (3p14-q13) have been mapped within other reported keratoconus loci [10,13]. ADAMTS7 presented sequencing difficulties because of its extremely close homology to several genes located on chromosome 15q and elsewhere, necessitating the use of long-range PCR to ensure specificity. Although no causative mutation was found in ADAMTS7, several novel SNPs were identified.
The most interesting of the remaining positional candidate genes are RNUT1, IMP3 (C15orf12), CSPG4, FBXO22, PSTPIP1, HMG20A, LRRN6A, ACSBG1 (BG1), CHRNA5, CHRNA3, CHRNB4, transcription factor genes ZNF291 and SIN3A1 and will be studied in the future.
The pathogenic mutation for keratoconus with cataract in this family may be in one of the lower priority genes that have not yet been screened, however, the possibility of a mutation in a high priority gene that is not amenable to detection by PCR-based sequencing must also be considered. Exonic deletions, duplications and other rearrangements would fall into this category. We have applied multiplex ligation-dependent probe amplification (MLPA) [31-34] to three genes (data not shown) and have found no evidence of copy number change. The mutation may also be located in a conserved intergenic region of regulatory importance. Chromosome 15q shows a high frequency of neocentromeres, which harbour a high density of clinically important duplicons, indicating a significant evolutionary history of this region . Rearrangements in this region have previously been associated with diseases [36-41].
Quantitative analysis of SNP genotypes at numerous loci spanning the critical region of the keratoconus with cataract locus is included in future research plans. This will enable more precise mapping of the linkage interval and also provide evidence of any anomalous dosage effects that may be of relevance to the disease process. Identification of genes implicated in the pathogenesis of keratoconus is vital for the development of novel treatments and early preventative strategies for this common corneal disorder.
This work was supported by grants from The Guide Dogs for the Blind Association, The Northern Ireland HPSS Research and Development Office, and The Royal College of Surgeons of Edinburgh. We are most grateful to the family who participated in this study. We are thankful to Mr. C. E. Willoughby, Department of Ophthalmology and Vision Sciences, Queen's University of Belfast, UK for discussions and suggestions regarding the project.
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