|Molecular Vision 2007;
Received 21 February 2007 | Accepted 26 July 2007 | Published 27 July 2007
Study of p.N247S KERA mutation in a British family with cornea plana
Pirro G. Hysi,4,5
John R. Ainsworth,7
Albert de la Chapelle,9
Stephen J. Tuft,2
Shomi S. Bhattacharya 1
1Division of Molecular Genetics, Institute of Ophthalmology, UCL, London, UK; 2Moorfields Eye Hospital NHS Foundation Trust, London, UK; 3Laboratory of the Biology and Pathology of the Eye and Ocular Tissue Bank, Institute of Inherited Metabolic Diseases, General Teaching Hospital and 1st Medical Faculty of Charles University, Prague, Czech Republic; 4Department of Epidemiology and Biostatistics, Institute of Child Health, UCL, London, UK; 5Department of Anesthesia, University of California at San Francisco, CA; 6Birmingham Women's Hospital, NHS Foundation Trust, Birmingham, UK; 7Eye Department, Birmingham Children's Hospital, NHS Foundation Trust, Birmingham, UK; 8Birmingham and Midland Eye Centre, City Hospital, NHS Foundation Trust, Birmingham, UK; 9Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, OH
Correspondence to: Petra Liskova,Division of Molecular Genetics,Institute of Ophthalmology,University College London,11-43 Bath Street,London, EC1V 9EL, UK; Phone: +44 2076086800; FAX: +44 2076086863; email: email@example.com
Purpose: To report clinical and genetic findings in a white British family with autosomal recessive cornea plana (CNA2) with a negative history for consanguinity. To look for evidence of a common ancestry with previously reported Finnish CNA2 patients by studying haplotypes.
Methods: Clinical examination and direct sequencing of the keratocan (KERA) gene was performed in two siblings affected with CNA2 and one unaffected parent. We also studied 22 single nucleotide polymorphisms distributed in the KERA genomic region by direct sequencing in this family as well as in one additional Finnish patient with CNA2 and 24 white British control subjects.
Results: Both siblings had the homozygous c.740A>G mutation leading to a p.N247S amino acid change originally reported as the founder mutation in 35 Finnish families. Genetic characterization of genomic regions surrounding the gene revealed large linkage disequilibrium, but the presence of shared extended haplotypes between affected individuals from Finland and the United Kingdom is consistent with a recent common ancestor.
Conclusions: This is the first description of recessive cornea plana in a white British family and it is the second report on the p.N247S change in the KERA gene. Extended haplotype analysis suggests that the two geographically remote occurrences of the c.740A>G mutation may have a common origin.
Cornea plana is a rare disorder in which the cornea is flattened with a low refractive power. Other features include microcornea, central corneal opacity, a widened corneal limbus, early arcus senilis, shallow anterior chamber, iris hypoplasia, corectopia, and peripheral anterior synechiae. Closed-angle glaucoma may also be present [1-3]. It can be inherited as an autosomal dominant (CNA1, OMIM 121400) or a clinically more severe autosomal recessive trait (CNA2, OMIM 217300) . CNA2 is found worldwide with a high prevalence among the Finnish population . Both CNA1 and CNA2 have been mapped to the long arm of chromosome 12 (12q21). Nonsynonymous or protein-truncating mutations of the keratocan (KERA) gene (OMIM 603288) have been identified as the cause of CNA2 but not CNA1 [4,5]. In some CNA1 families, linkage to the 12q21 locus was excluded .
KERA codes for keratocan, a small-sized highly conserved leucine-rich protein that is expressed in cornea as well as in other tissues [4,7]. In KERA knockout mice there is a thinner corneal stroma and a narrower cornea-iris angle than in the wild type with less organized packing and larger diameters of stromal collagen fibrils on transmission electron microscopy .
Overall, seven different mutations in the coding sequence of the KERA gene have been described in CNA2 families; 46 Finnish patients from 35 unrelated pedigrees were shown to have the c.740A>G (p.N247S) change inherited from a common founding ancestor . In the same report one Chinese-American patient, born to consanguineous parents, was homozygous for p.Q174X . Other sequence variants have also been reported, such as a consanguineous family originating from Bangladesh presenting with a combined phenotype of CNA2 and microphthalmia had a p.T215K change , one Hispanic consanguineous pedigree was shown to have p.N131D , while p.R313X, p.R279X, and p.C343fs were described in 14 consanguineous Arab families [11-13]. An affected member of one of the Arab families reportedly exhibited the cornea plana phenotype as well as superior pellucid marginal degeneration . Bilateral progressive corneal ectasia leading to the development of a presumed unilateral hydrops and bilateral high astigmatism has also been described in recessive cornea plana .
We describe a white British CNA2 family with two affected members. One sibling showed typical cornea plana phenotype and the other, in addition, had high simple astigmatism. Marker haplotypes in the KERA genomic region were studied to determine if the observed p.N247S mutation was identical by descent to those in the previously described Finnish families .
Patients and clinical examination
The research complied with the tenets of the Declaration of Helsinki. Appropriate consent was obtained from participating subjects. The family was white British with no known foreign ancestry. Family members reported a negative family history for consanguinity in at least two preceding generations. The proband was a 26-year-old female (individual II:3) with poor vision since birth. Her 30-year-old sister (individual II:1) had similar phenotype, but the proband's brother, nephews, and parents were all normal (Figure 1A). Standard ophthalmic examination, which included best corrected visual acuity, intraocular pressure measurement, and slit lamp biomicroscopy, was performed in the two affected siblings and their mother. Anterior segment photographs were taken of the affected siblings. Corneal horizontal diameters (white-to-white), corneal topography, pachymetry, and anterior chamber depth (ACD) measurements were performed using an Orbscan II operating under software version 3.12 (Bausch & Lomb, Rochester, NY).
DNA preparation and keratocan mutation screening
Genomic DNA was extracted from venous blood samples using NucleonTM BACC3 genomic DNA extraction kit (GE Healthcare, Bucks, UK). The coding regions and exon-intron boundaries of the KERA gene were amplified by polymerase chain reaction (PCR) using sets of four primers (Table 1). Primers were designed with Primer 3 program. Each PCR reaction consisted of approximately 50 ng of genomic DNA, 50 pmol of each primer and 12.5 μl of ReddyMixTM PCR master mix (1.5 mM MgCl2; ABgene, Epsom, UK) and water added up to a volume of 25 μl. After purification with Montage PCR96 Cleanup Kit (Millipore, Billerica, MA), samples were sequenced on an automated sequencer using dye-terminator chemistry under standard conditions with primers identical to those for genomic amplification (Applied Biosystems, Foster City, CA). Montage SEQ96 Sequencing Reaction Cleanup Kit (Millipore) was used for purification according to the manufacturer's instructions. Sequence data were aligned and analyzed by DNASTAR Lasergene sequence analysis software (DNAStar, Inc., Madison, WI) followed by manual inspection for base changes and comparison with database reference sequence NCBI accession NM_007035. Ninety-four unrelated white British control individuals were screened for the identified disease-causing mutation by direct sequencing. The DNA of these subjects was purchased from the European Collection of Cell Cultures (ECCAC, Porton Down, UK).
Selection of polymorphisms and genotyping
Thirty-three variations found on the Single Nucleotide Polymorphism dbSNP database across 7.64 kb of the KERA genomic sequence were genotyped in the proband, an unaffected parent, and one Finnish subject with CNA2 previously described by Pellegata et al. . All alleles were determined by direct sequencing of 290-492 bp long PCR fragments as described above. A list of the polymorphisms and appropriate primer sequences are shown in Table 2. NCBI accession NT_019546 was used as the reference sequence.
To characterize linkage disequilibrium (LD) blocks in the KERA genomic region, we performed extended genotyping of 22 polymorphisms (one insertion/deletion polymorphism and 21 single nucleotide polymorphisms) by direct sequencing, not only in the British CNA2 family and Finnish CNA2 patient, but also in a subset of 24 white British control individuals (the same subjects were used for mutation screening). This was followed by haplotype comparison of a region spanning both sides of the KERA genomic sequence for a total of almost 280 kb. The genotyped polymorphic variants, their position, and the primers used for amplification of PCR fragments are shown in Table 3. Selection of these polymorphisms was made to provide haplotypic information for wider regions of the chromosome and was made in ever increasing distances from KERA using the information from the HapMap publicly accessible databases.
Based on information about individual genotypes, haplotypes for each subject were reconstructed using PHASE , whereas the phylogenic tree was obtained using PHYLIP (version 3.2)  software and the upgraded package (version 3.6) as distributed by the author.
The clinical characteristics of the proband were consistent with cornea plana (Table 4). Both corneas appeared thin and flat, but simulated keratometry could only be obtained from the left eye (Figure 2C). There was axial full thickness scarring in the right eye and bilateral mild peripheral corneal vascularization around a widened corneal limbus. Both anterior chambers were shallow. There was no other evidence of anterior segment dysgenesis. Intraocular pressures and fundus examination were normal. Individual II:1 (Figure 1A) also had poor vision since birth. She had less severe hypermetropia than her sister but higher astigmatism (Table 4). Both of her eyes had an abnormally broad limbus zone and arcus with more pronounced corneal thinning than her sister and mild bilateral anterior stromal scarring (Figure 2D,E). The rest of the clinical examination was normal.
Sequencing of the KERA gene in the affected individuals revealed homozygous c.740A>G substitution in exon 2 leading to asparagine to serine amino acid change at codon 247 (Figure 1B). Consistent with autosomal recessive cornea plana, the parent was heterozygous for this change (Figure 1B). The mutated allele was absent from 94 control subjects (188 chromosomes). Genotyping of 33 known simple genetic polymorphisms across the KERA gene (Table 2) in both the British and Finnish patients, as well as the unaffected parent, revealed that these polymorphisms were identical except for the disease-causing mutation. Further characterization of alleles in the KERA genomic region in the patients, their available parent, and healthy controls is shown in Table 3 while the reconstructed haplotypes are shown in Table 5.
LD was strong over the extended genomic region. All three affected individuals shared exactly the same haplotype (Table 5) for a 279 kb long genomic interval between the rs6144808 and rs826778 markers. Unaffected controls typically had a lower degree of LD with haplotype continuity broken beyond rs2701166 and rs516115. Organization of haplotypes in phylogenic trees (Figure 3) shows the haplotype from the parent containing the mutation and the CNA2 patients forming a separate branch. The individual alleles in the diseased individuals remain in phase over the entire genomic interval studied. LD is not as strong and shows clear signs of decay at the extremities of the same interval in the controls' haplotypes (the pairwise LD between the two furthermost markers in our genotyped interval is only a modest D'=0.13; Figure 4). Although not fully conclusive proof, this contrast is suggestive of a relatively recent common ancestor of all CNA2 cases of Finnish and British origin.
Autosomal recessive cornea plana is a very rare disorder. It is most common in Finland where 78 cases have been identified. This represents the majority of cases reported worldwide . We describe a new CNA2 family of British descent exhibiting a nonsynonymous amino acid substitution at codon 247 in the KERA gene. Prior to this study, this mutation has only been described in a cohort of 35 Finnish pedigrees (Figure 1B) . To our understanding, this is the first case of a p.N247S substitution outside Finland. The clinical characteristics of the two affected patients were consistent with CNA2. High astigmatism associated with superior pellucid marginal degeneration has previously been observed in one patient out of six affected members from a Saudi Arabian family with CNA2 . Although we are unsure of the mechanism leading to the atypical topography pattern in patient II:1, there were no clinical features of pellucid marginal degeneration. Both patients had considerably less than average central corneal thickness measurements, but this did not increase toward the periphery. High astigmatism has also been reported in another Arab patient with progressive bilateral corneal ectasia . Since serial observations have not been performed, we cannot comment whether there has been a progression of the corneal changes to possible ectasia in patient II:1.
As patients with p.N247S changes have previously only been reported from a confined geographical area, we tried to determine whether there was a recent shared ancestry between the affected individuals from Finland and the United Kingdom. For this purpose, we attempted to collect haplotype information through the genotyping of single nucleotide polymorphisms. This class of molecular markers has the advantage of being more frequent than variable number tandem repeats and is transmitted from one generation to another with few, if any, modifications. The entire genomic region around KERA shows a generally high level of LD. By examining the single nucleotide polymorphisms and their combination in haplotypes, we found haplotypes containing the disease-causing change were perfectly conserved across the studied interval while the majority of haplotypes in healthy controls were not. A differential conservation of haplotypes adjacent to susceptibility loci and their susceptibility to natural selection has been observed in many occasions [17-21]. There may be two explanations for the observed conservation of both the structure and length of a specific haplotype: recent common ancestry or positive selection. Positive selection often results in extension of the size of the haplotype containing the beneficial allele. This possibility is unlikely since the extended haplotypes were observed in people affected with serious vision impairment.
Although no conclusive proof can be produced with regards to the identity of descent by the cases examined here, the presence of haplotype sharing between affected individuals of both British and Finnish origin is suggestive of a common ancestor.
This work was supported by the Special Trustees of Moorfields Eye Hospital and research project MSM 0021620806-VZ-206100-11 (Ministry of Education of the Czech Republic).
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