Molecular Vision 2006; 12:499-505 <http://www.molvis.org/molvis/v12/a58/> ©2006 Molecular Vision 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 Giuliana Silvestri,2 Anne E. Hughes1 Departments of 1Medical Genetics and 2Ophthalmology, Queen’s University of Belfast, Belfast, United Kingdom 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, FA H , 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 candi- date 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, al- though a pathogenic genomic rearrangement or exonic deletion would not have been detected. Keratoconus (KC; OMIM 148300), the most common Mutations in the VSX-1 transcription factor (OMIM corneal dystrophy, is a bilateral, noninflammatory progressive 605020) were identified in 4.7% of patients with keratoconus corneal ectasia. Clinically, the cornea becomes progressively (KTCN1; OMIM 148300) and also in posterior polymorphous thin and conical which leads to myopia, irregular astigma- corneal dystrophy (PPCD1; OMIM 122000) [11]. However, tism, and corneal scarring. The transparency and refractive traditional mapping methods to identify the genetic basis of state of the cornea is a prerequisite for normal vision. The KC have been limited by the lack of large multigeneration disease usually arises in the teenage years, eventually stabi- families for study. Genome-wide scans to localize the KC gene lizing in the third and fourth decades [1]. It occurs with no or genes had not been reported until our group identified a ethnic or gender preponderance and causes significant visual large three-generation family with KC and anterior polar cata- impairment in young adults. No specific treatment exists ex- ract and reported linkage to chromosome 15q22.32-24.2 [12]. cept to replace the corneal tissue by surgery (corneal trans- Recently loci for autosomal dominantly inherited keratoco- plantation) when the visual acuity can no longer be corrected nus have been mapped to chromosomes 21 [13], 16q22.3-q23.1 by contact lenses. In the Western world, KC is the most com- (KTCN2; OMIM 608932) [10], 3p14-q13 (KTCN3; OMIM mon indication for corneal transplantation [2]. The underly- 608586) [14], 2p24 (KTCN4; OMIM 609271) [15] and 5q14.1- ing biochemical processes and pathobiology of keratoconus q21.3 (NCBI). remain poorly understood. The keratoconus with cataract locus on chromosome 15q The incidence of KC is 1 in 2,000 in the general popula- was identified in a large Northern Irish family of three gen- tion [1,3]. A family history is present only in a minority of erations affected by combined autosomal dominant early on- cases, however, one of the major etiological factors is genetic set anterior polar cataract and clinically severe keratoconus [1,4-6]. KC is believed to be inherited autosomally, because [12]. The disease gene in this family was successfully mapped of familial occurrence [7], a higher concordance rate of the to chromosome 15q22.32-24.2 within a 6.5 Mb region flanked trait in monozygotic twins then dizygotic twins [8] and its by markers CYP11A and D15S211 [12]. CTSH, CRABP1, prevalence in first degree relatives is 15-67 times higher than IREB2, and RASGRF1 were excluded previously as the caus- the general population [6]. Most studies describe autosomal ative gene for keratoconus with cataract [12]. The purpose of dominant inheritance, with incomplete penetrance or variable the present study was to narrow the critical region of linkage expressivity [1,9,10]. of keratoconus with cataract by genotyping novel microsatellite markers and single nucleotide polymorphisms identified dur- Correspondence to: Durga Prasad Dash, Department of Ophthalmol- ing sequencing of candidate genes. Fine mapping of a kerato- ogy, Institute of Clinical Science, Block A, Queen’s University of conus with cataract locus on 15q has reduced the linked re- Belfast, Royal Victoria Hospital, Grosvenor Road, Belfast, BT12 gion to 5.5 Mb, thereby excluding 28 potential candidate genes. 6BA, United Kingdom; Phone: 0044-7754404969; FAX: 0044- A further 23 positional candidate genes were prioritized for 2890632699; email: [email protected] 499 Molecular Vision 2006; 12:499-505 <http://www.molvis.org/molvis/v12/a58/> ©2006 Molecular Vision analysis based on expression studies, molecular and biochemi- purification kit (Gentra Systems, Inc., Minneapolis, MN). cal evidence (Table 1) and excluded by PCR-based DNA se- Single nucleotide polymorphism typing: The single nucle- quencing. otide polymorphism (SNP) rs1128933 in exon 20 of MAN2C1 (GenBank NM_006715) was typed in key recombinants us- METHODS ing the forward primer 5'-CTG GAG ACA CGG TAT AGG Genomic DNA was extracted from the peripheral blood leu- CTG G-3' and the reverse primer 5'-AGT GTA CCT GGG kocytes of all available family members using Puregene DNA AGT GGG AAG G-3'. Cycle sequencing was performed with TABLE 1. LIST OF POSITIONAL CANDIDATE GENES IN KERATOCONUS WITH CATARACT LOCUS SCREENED FOR MUTATION Gene Full name of the gene OMIM -------------- ----------------------------------------------------------- ------ MORF4L1(MRG15) Mortality factor 4 like 1 607303 KIAA1055 Transmembrane protein 2-like gene ETFA Electron-transfer-flavoprotein, alpha polypeptide (glutaric 608053 aciduria II) AWP1 Protein associated with PRK1 REC14 Recombination protein REC14 KIAA1199 KIAA1199 608366 RCN2 Reticulocalbin 2, EF-hand calcium binding domain 602584 FAH Fumarylacetoacetate hydrolase (fumarylacetoacetase) 276700 IDH3A Isocitrate dehydrogenase 3 (NAD+) alpha 601149 MTHFS 5,10-methenyltetrahydrofolate synthetase 604197 (5-formyltetrahydrofolate cycloligase) ADAMTS7 A disintegrin-like and metalloprotease (reprolysin type) 605009 with thrombospondin type 1 motif, 7 MAN2C1 Mannosidase, alpha, class 2C, member 1 154580 PTPN9 Protein tyrosine phosphatase, nonreceptor type 9 600768 KIAA1024 KIAA1024 protein ARNT2 Aryl-hydrocarbon receptor nuclear translocator 2 606036 BCL2A1 BCL2-related protein A1 601056 ISL2 ISL2 transcription factor, LIM/homeodomain, (islet-2) C15ORF22(P24B) Chromosome 15 open reading frame 22 DNAJA4 DnaJ (Hsp40) homolog, subfamily A, member 4 FLJ14594 Leucine rich repeat neuronal 6A CIB2 (KIP2) Calcium and integrin binding family member 2 605564 C15ORF5 Chromosome 15 open reading frame 5 PSMA4 proteasome (prosome, macropain) subunit, alpha type, 4 Gene name and corresponding OMIM (wherever possible) is given in the disease linked region of keratoconus with cataract on chromosome 15q. 500 Molecular Vision 2006; 12:499-505 <http://www.molvis.org/molvis/v12/a58/> ©2006 Molecular Vision the BigDye Terminator Cycle Sequencing kit (Applied PCR-based DNA sequencing: Analysis of positional can- Biosystems, Foster City, CA) on PCR products using the for- didate genes was carried out by PCR-based DNA sequencing ward PCR primer and analyzed on the ABI Prism 3100 DNA using dye terminator chemistry (ABI). Primers were designed Sequencing System (Applied Biosystems). using Primer Detective, version 1.01 (Clontech Labs Inc., CA) Identification and typing of microsatellite markers: Two for all individual exons plus at least 50 bp of flanking intronic novel microsatellite markers on chromosome 15 located at 73.8 sequence, and 5' and 3' untranslated regions. For ADAMTS7, Mb between MAN2C1 and ETFA and at 78.4 Mb (within long-range PCR using Elongase (Invitrogen, Paisley, UK) was ARNT2) were identified from the human genome database carried out as a preliminary step, using gene-specific primers (HGD). PCR was performed on genomic DNA from family that did not amplify related pseudogenes. After dilution of members using the following primers: 5'-Fam-CTG TAT GCA product (1 in 1000) nested PCR was performed before sequenc- GCG ATC AGT GAG C-3' and 5'-GTA GGA GAC AGA GAC ing as already described. All primer pairs and PCR conditions ACT CAG TCC-3' for the marker at 73.8 Mb and 5'-Fam- are available on request. TTA CAT CAC TTG CAT TGC TTC C-3' and 5'-AAT CAC AGC CAA ACA TAG ATG C-3' for the ARNT2 intragenic RESULTS marker. Diluted PCR products were electrophoresed on an ABI One SNP and two novel microsatellite markers were identi- 3100 genetic analyzer. Alleles were sized by Genotyper soft- fied toward the extremities of the previously linked interval