Am. J. Hum. Genet. 57:539-548, 1995
Mutation of the PAX6 Gene in Patients with Autosomal Dominant Keratitis F. Mirzayans, W. G. Pearce, 1. M. MacDonald, and M. A. Walter Department of Ophthalmology, University of Alberta, Edmonton
Summary
Autosomal dominant keratitis (ADK) is an eye disorder chiefly characterized by corneal opacification and vascularization and by foveal hypoplasia. Aniridia (shown recently to result from mutations in the PAX6 gene) has overlapping clinical findings and a similar pattern of inheritance with ADK. On the basis of these similarities, we used a candidate-gene approach to investigate whether mutations in the PAX6 gene also result in ADK. Significant linkage was found between two polymorphic loci in the PAX6 region and ADK in a family with 15 affected members in four generations (peak LOD score = 4.45; 0 = .00 with D11S914), consistent with PAX6 mutations being responsible for ADK. SSCP analysis and direct sequencing revealed a mutation in the PAX6 exon 11 splice-acceptor site. The predicted consequent incorrect splicing results in truncation of the PAX6 prolineserine-threonine activation domain. The SeyI mouse results from a mutation in the Pax-6 exon 10 splicedonor site that produces a PAX6 protein truncated from the same point as occurs in our family with ADK. Therefore, the SeyN" mouse is an excellent animal model of ADK. The finding that mutations in PAX6 underlie ADK, along with a recent report that mutations in PAX6 also underlie Peters anomaly, implicates PAX6 broadly in human anterior segment malformations. Introduction
Autosomal dominant keratitis (ADK; MIM 148190) is a very rare ocular disorder presenting with congenital and progressive features predominantly involving the anterior segment of the eye (Kivlin et al. 1986; Pearce et al. 1995). The major clinical symptoms are anterior stromal corneal opacification and vascularization of the Received March 30, 1995; accepted for publication June 5, 1995. Address for correspondence and reprints: Michael A. Walter, Ocular Genetics Laboratory, 671 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. E-mail:
[email protected] X 1995 by The American Society of Human Genetics. All rights reserved. 0002-9297/95/5703-0002$02.00
peripheral cornea (fig. 1). Progression of the opacification and vascularization into the central cornea may occur with a corresponding reduction in visual acuity. In some eyes treated by penetrating keratoplasty, opacification and vascularization of the donor cornea occur rapidly. Other anterior segment features include variable radial defects of the iris stroma. In the family reported here, posterior segment involvement is characterized by foveal hypoplasia with minimal effect on visual acuity (Pearce et al. 1995). In all reported families, ADK is inherited as a fully penetrant autosomal dominant disease with variable clinical presentation (Kivlin et al. 1986; Pearce et al. 1995). Aniridia (MIM 106200) is a bilateral congenital anomaly that is defined by structural defects of the iris, frequently severe enough to cause an almost complete absence of the iris. This defect may be accompanied by other anterior segment manifestations, including cataract and keratitis. Posterior segment involvement in aniridia is characterized by foveal hypoplasia resulting in a highly variable impairment of visual acuity, and nystagmus. Aniridia is usually inherited as an autosomal dominant disease, although a rare autosomal recessive variant (Gillespie syndrome) is also known. Aniridia occurs in 1 in 64,000-96,000 live births (Shaw et al. 1960). The identification of the molecular basis of aniridia came from positional cloning experiments. Sporadic cases of aniridia with Wilms tumor, genitourinary anomalies, and mental retardation (acronym WAGR) were found to be associated with deletions at chromosomal band 11pl3 (Riccardi et al. 1978). Classical linkage studies also mapped aniridia to 11pl3 (Mannens et al. 1989). Candidate genes from this region were isolated, and one, PAX6, was found to be expressed in the fetal eye, cerebellum, and olfactory bulb (Ton et al. 1991). The murine homologue, Pax-6, was subsequently demonstrated to be mutated in mice with the semidominant small eye (Sey) phenotype (Hill et al. 1991). Homozygous Sey/Sey mice do not develop eyes and lack nasal structures and die soon after birth (Hill et al. 1991). Heterozygous Seyl+ mice have smaller-than-normal eyes and may present with a wide range of additional ocular defects, including iris hypoplasia, adhesions be539
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Am. J. Hum. Genet.
57:539-548, 1995
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Figure I Photograph of the left eye of a patient (C7) with ADK, showing superior corneal vascularization and opacification. Pedigree numbering is as delineated in fig. 2.
tween the lens and/or iris and the cornea, polar opacities, and corneal vascularization (Favor et al. 1988; Hanson et al. 1994; Hill et al. 1991). The similarities of the Sey/+ and aniridia/+ phenotypes, and the additional evidence that the regions containing the aniridia and Sey genes are syntenic (Glaser et al. 1990; van der Meer-de Jong et al. 1990), suggested that the gene involved in the Sey mutation is the mouse homologue of the human aniridia gene. PAX (paired box) genes are expressed in many different tissues undergoing differentiation and morphogenesis (for reviews see Gruss and Walther 1992; Hill and Hanson 1992; and Strachan and Read 1994). The paired-box region that defines this gene family (in addition to the homeobox domain that many PAX genes also contain) has been shown to bind DNA, consistent with the suggestion that PAX genes are regulators of early mammalian embryogenesis (Gruss and Walther 1992). Molecular genetic studies have revealed that autosomal dominant aniridia results from mutations in the PAX6 gene (Glaser et al. 1992; Jordan et al. 1992; Davis and Cowell 1993; Hanson et al. 1993; Martha et al. 1994). Recent reports indicate that while PAX6 mutations do not underlie the autosomal recessive Gillespie aniridia variant (partial aniridia, cerebellar ataxia, and
mental retardation) (Glaser et al. 1994), disturbances in PAX6 do cause some forms of Peters anomaly (corneal opacity, corneal defects, and adhesions between the iris and cornea) (Hanson et al. 1994). The finding that PAX6 mutations result in some forms of Peters anomaly besides aniridia, together with the overlapping clinical findings and similar patterns of inheritance in aniridia and ADK, led us to investigate whether ADK is a variant of the same genetic disorder. The linkage and sequence analyses results of this candidate-gene approach indicate that the ADK in one family results from a mutation in the PAX6 gene. Subjects, Material, and Methods Subjects The family with ADK studied in this report has 15 affected individuals in four generations (fig. 2). The clinical findings in this family have been described in detail by Pearce et al. (1995). All affected individuals presented with, at a minimum, a circumferential 1-2-mm-wide band of corneal opacification and vascularization at the level of Bowman's membrane and in continuity with the limbus. There was considerable variation in the severity of the corneal changes, with generally more progressive
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Figure 2 Investigation and haplotype analysis of two polymorphic loci flanking the PAX6 gene in the ADK family. a, Analysis of the CA-repeat polymorphisms D11S914 and DllS907 within the ADK family. Square brackets indicate deceased individual Al, on whose DNA analyses were not conducted. Blackened circles and squares indicate affected female and males, respectively. Asterisks (*) indicate two individuals with recombinations between the ADK phenotype and the DllS907 locus. b, Schematic diagram of the DllS914-ADK-DlS907 haplotypes present in the ADK family. Square brackets indicate the inferred DllS914-ADK-DllS907 haplotypes of the deceased individual Al. Asterisks (*) indicate two individuals displaying recombination between the ADK phenotype and the DllS907 locus.
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keratitis amongst older affected individuals. Foveal hypoplasia was found in all affected individuals. There was no evidence of skin or mucous membrane involvement in patients with ADK, as has been shown to occur with keratitis in patients suffering from epidermolysis bullosa (Granek and Baden 1980), ectrodactyly-ectodermal dysplasia-clefting syndrome (Mawhorter et al. 1985), or keratitis-ichthyosis-deafness syndrome (Wilson et al. 1991). Keratoendotheliitis fugax hereditaria is distinct from ADK, as it characterized by self-limiting intermittent attacks of keratoendotheliitis affecting one or other eye (Ruusuvaara and Setala 1987). The study and collection of blood samples from ADK patients were approved by the research ethics board of the Faculty of Medicine of the University of Alberta. Genotypic and Molecular Studies
Blood samples were collected in EDTA tubes, and DNA was prepared from isolated white blood cells by standard organic solvent extraction procedures. Oligonucleotide primers for D 1S907 and D 1S914 were obtained from Research Genetics. PCR primers for the amplification of all 14 exons of the PAX6 gene were as described elsewhere (Glaser et al. 1992). PAX6 oligonucleotides were synthesized by Genosys Biotech. PCR amplification for linkage and SSCP analyses was done with 35S-dATP incorporated directly into the PCR product. PCR reactions were carried out in thin-walled microtiter plates (Fisher Scientific) in an MJ Research thermocycler. PCR reactions were performed with 20 ng DNA containing 20 mM Tris-HCI (pH 8.4 at 230C); 50 mM KCI; 1.5 mM MgCl2; 200 FM each dCTP, dTTP, and dGTP; 25 gM dATP; 5 jiCi S35-dATP; and 60 ng each primer. Each reaction was overlaid with 40 g1 of light mineral oil to prevent evaporation. PCR specificity was increased through a "hot-start" step in which samples were subjected to a denaturing step of 5 min at 95°C, during which time 1 U of Thermus aquaticus DNA polymerase and sufficient H20 were added for a final PCR reaction volume of 25 p1. This was followed by 35 cycles of denaturing at 94°C for 30 s, annealing for 30 s, and extending at 72°C for 30 s (2 min for SSCP), with a final elongation step of 72°C for 7 min. PCR primers for D11S907 and D11S914 were annealed at 55°C. Annealing temperatures for each PAX6 exon were as described elsewhere (Davis and Cowell 1993). PCR products for linkage analysis were separated on 6% denaturing polyacrylamide gels. SSCP analysis was conducted using 6% nondenaturing polyacylamide gels containing glycerol with specific conditions for each PAX6 exon as described (Davis and Cowell 1993). After electrophoresis, gels were dried and used for autoradiography. PCR products were directly sequenced on an ABI automated sequencer.
Am. J. Hum. Genet. 57:539-548, 1995
Linkage Analysis
Linkage analysis was conducted with a DOS-compatible Power Macintosh 6100/66 computer. The Hypercard-based program Linkage Interface (Nichols et al. 1993) was used to store and export information for linkage analysis with the DOS-computer program Linkage (Ott 1991). LOD score values were calculated using the MLINK option of the Linkage program. The allele frequencies were assumed to be equal for the two markers used in this study. The phenotype in this family was analyzed as an autosomal dominant trait with complete penetrance and the gene frequency of .0001. Significance of linkage was evaluated using standard criteria (Zmax > 3). Results Linkage Studies with ADK
Linkage studies were conducted on the ADK family to test the hypothesis that ADK results from mutations in the aniridia/PAX6 gene. Two microsatellite markers, D11S907 and D11S914, were selected, which are close to and flank the PAX6 gene (James et al. 1994). D15907 and Dl1S914 have been estimated to be separated by 5 cM (Gyapay et al. 1994). The segregation patterns of these two markers in the ADK family are shown in figure 2a. Linkage results are summarized in table 1. D11S914 (located 16.4 cRays [cR] distal of PAX6) (James et al. 1994) does not recombine with ADK (peak LOD score 4.45; 0 = .00). Two recombinants between the ADK phenotype and D11S907 (located 71.8 cR proximal of PAX6) were observed, resulting in a peak LOD score of 3.41 at 9% recombination. These results are consistent with mutations of a gene in the PAX6 region being responsible for ADK. Mutations in the PAX6 Gene Associated with ADK
To identify mutations in the PAX6 coding region, all 14 exons of the PAX6 gene were PCR-amplified from affected and nonaffected individuals in the ADK family and were analyzed by the SSCP technique (Orita et al. 1989). An altered migration pattern in exon 11 was detected by SSCP analysis (fig. 3a). No other alterations involving PAX6 exons in the ADK family were detected. Direct sequencing of the PCR product revealed no alterations in the exon 11 coding sequence; however, an Ato-T transversion in the exon 11 splice-acceptor site was discovered (fig. 3b). This mutation results in the abolition of a normally present DdeI restriction site that segregates with the ADK phenotype in four generations (data not shown). The A-to-T mutation in the PAX6 exon 11 splice-acceptor site is predicted to result in aberrant splicing and in the skipping of exon 11. In addition, the direct joining of exons 10 and 12 would result in
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Mirzayans et al.: ADK Caused by a PAX6 Mutation
Table I ADK Versus PAC Region Polymorphic Markers
LOD SCOREb
COMPARISON (Recombinantsa) ADK versus D11S914 (0/16) ADK versus D11S907 (2/22)
.........
.........
AT 0 =
.00
.01
.02
.03
.04
.05
.075
.10
.20
.30
.40
4.816
4.747 2.535
4.677 3.051
4.605 3.313
4.533 3.473
4.464 3.580
4.275 3.696
4.026 3.702
3.264 3.284
2.336 2.476
1.264 1.386
-00
a No. of recombinants/total no. of informative meioses. b Zmax values are underlined.
exon 12 being read out of frame, producing a short nonsense peptide and premature stop. Cryptic splicing to potential intronic splice-acceptor sites 5' of exon 11 might be considered as a means of recovering functional portions of exons 11-13. This can be ruled out, as an in-frame stop codon (with respect to exon 11) occurs at position -28 from the start of exon 11 and no potential splice-site sequences occur between this stop codon and the normal exon 11 splice-acceptor site (fig. 4). Therefore, if such cryptic splicing did occur, the PAX6 protein would be prematurely truncated before reaching exon 11, producing a PAX6 protein that is normal until the end of exon 10 but that is followed by a short nonsense protein and is prematurely truncated. No suitable cryptic splice sites appear within exon 11. A mutant PAX6 protein truncated for 117 amino acids from the C terminus PAX6 proline-serine-threonine (PST) domain is therefore expected to be produced in individuals with ADK in this family (fig. 5). Genotype/Phenotype Correlations ADK, like aniridia and the Sey mouse, displays considerable phenotypic variation between individuals with the same mutant allele. The multigeneration ADK family allowed us to investigate whether different normal PAX6 alleles contributed to the varied expression of the ADK phenotype. Three pairs of affected siblings (B2 and B4, B6 and B10, and C7 and C8) inherited the same
normal PAX6 allele (different normal alleles for each pair of individuals). Investigation of the ocular history of each individual, including age at onset and symptoms present, indicated that the normal allele does not account for the variation in the expression of ADK within members of the same family (table 2). For example, while siblings C7 and C8 share the same normal PAX6 allele (and mutant PAX6 allele), C7 presented with photophobia since childhood and vascular progression centrally into the cornea in both eyes and had a right penetrating keratoplasty at age 17 years. In contrast, C8 had no history of photophobia and only minimal corneal changes with little effect upon visual acuity. Therefore,
other factors in the genetic background of these individuals, distinct from the PAX6 gene, must modify the presentation of the ADK phenotype. Discussion
Linkage and direct sequencing have demonstrated that patients in a family with ADK have a mutation in the exon 11 splice-acceptor site of the PAX6 gene. The mutated base is invariably an A in all normal genes hereto examined, as shown in a sequence survey of >3,700 5' splice sites (Senapathy et al. 1990). Several analogous A-to-T mutations involving this normally invariant A in splice-acceptor sites at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary cells have been shown to abolish correct splicing and to result in
