Broad Phenotypic variability in a Single Pedigree With a Novel 1410delC Mutation in the PST Domain of the PAX6 Gene

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1 HUMAN MUTATION Mutation in Brief #537 (2002) Online MUTATION IN BRIEF Broad Phenotypic variability in a Single Pedigree With a Novel 1410delC Mutation in the PST Domain of the PAX6 Gene Michèle M. Sale 1,2, Jamie E. Craig 3,4, Jacinta C. Charlesworth 2, Liesel M. FitzGerald 2, Isabel M. Hanson 5, Joanne L. Dickinson 2, Sarah J. Matthews 4, Veronica van Heyningen 6, John H. Fingert 7, and David A. Mackey 4 1 Center for Human Genomics, Wake Forest University School of Medicine, Winston-Salem NC, USA; 2 Menzies Centre for Population Health Research, University of Tasmania, Hobart, Australia; 3 Department of Ophthalmology, Flinders Medical Centre, Adelaide, Australia; 4 Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Australia; 5 Department of Medical Sciences, University of Edinburgh, Western General Hospital, Edinburgh, UK; 6 MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK; 7 Department of Ophthalmology, The University of Iowa College of Medicine, Iowa City, Iowa *Correspondence to: Dr Michèle M. Sale, Center for Human Genomics, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem NC 27157, USA; Tel.: ; Fax: ; msale@wfubmc.edu Grant sponsors: Glaucoma Research Foundation, NIH; Grant number: EY ; Grant sponsors: Clifford Craig Medical Research Trust, Royal Hobart Hospital Research Foundation, UK Medical Research Council. Communicated by Henrik Dahl The PAX6 mutation present in an individual with aniridia was determined and phenotypic features of immediate relatives carrying the same mutation investigated. Mutation analysis revealed a novel single base deletion 1410delC in the PAX6 gene in ten affected individuals. Clinical features ranged from total aniridia to very mild anterior segment findings. Other findings included partial aniridia, iris stromal hypoplasia, keratitis, cataract, glaucoma, optic disc anomalies and foveal hypoplasia. It appears that independent modifying factors may underlie the variability of the different phenotypic features of the PAX6 mutation Wiley-Liss, Inc. KEY WORDS: development; ocular; gene expression; genetic diseases; iris; glaucoma; PAX6; aniridia INTRODUCTION Aniridia (MIM# ) is a rare panocular developmental disorder typically diagnosed postnatally by bilateral absence of the iris, although iris remnants may be observed. About two thirds of cases are familial, with autosomal dominant inheritance, high penetrance and variable expressivity [Hanson and van Heyningen, 1995]. Foveal hypoplasia associated with nystagmus results in reduced visual acuity. Congenital cataracts, typically anterior polar, may be present, and vision may progressively deteriorate due to corneal opacification, cataracts and glaucoma. Aniridia is caused by mutations of the PAX6 gene (MIM# ; GenBank: M77844) [Ton et al., 1991; Jordan et al., 1992] located on chromosome 11p13. The PAX6 protein contains paired box and homeobox DNA-binding domains and a transcriptional activating proline-serine-threonine-rich (PST) domain. The PAX6 gene is expressed in the developing eye, and is mutated in the mouse small eye phenotype [Hill et al., 1991]. Received 21 June 2002; accepted 18 July WILEY-LISS, INC. DOI: /humu.9066

2 2 Sale et al. Homozygosity for mutant alleles is lethal [Glaser et al. 1994]. Considerable phenotypic variability has been associated with PAX6 mutations. The over-representation of mutations resulting in nonsense codons suggests that there has been an ascertainment bias in screening for PAX6 mutations primarily in aniridia cases, and that there may be additional phenotypes associated with missense mutations [Prosser and van Heyningen, 1998]. Phenotypes reported in the absence of classical aniridia include Peters anomaly [Hanson et al., 1994; Azuma et al. 1996], corneal opacification with minimal iris hypoplasia [Epstein et al., 1994], autosomal dominant keratitis [Mirzayans et al., 1995], congenital cataracts [Glaser et al., 1994; Gupta et al., 1998], foveal hypoplasia [Azuma et al., 1996], ectopia pupillae [Hanson et al., 1999] and congenital nystagmus [Hanson et al., 1999; Sonoda et al. 2000]. We have detected a novel PAX6 mutation in a large pedigree from the Australian state of Tasmania. This mutation is associated with considerable variability in phenotype and severity, even among family members possessing an identical mutation. METHODS Ascertainment and clinical examination This study was approved by the Royal Hobart Hospital and University of Tasmania Human Research Ethics Committees. Three generations of a Tasmanian pedigree, shown in Figure 1, were ascertained through proband III:3 who possessed iris abnormalities. Examination of the proband s daughter IV:3 and nephew IV:1 revealed aniridia. Eleven relatives (II:2, III:2, III:3, III:6, IV:1, IV:3, IV:4, IV:5, IV:6, IV:7, IV:8) and 4 marriage partners (II:3, III:4, III:5, III:7) were examined in detail. Anterior segment and fundus photographs were taken. Humphrey 24-2 visual field testing (Humphrey, San Leadro CA) and intra-ocular pressure (IOP) measurement were performed. I:1 I:2 I:3 II:1 II:2 II:3 III:1 III:2 III:3 III:4 III:5 III:6 III:7 III:8 III:9 III:10? IV:1 IV:2 IV:3 IV:4 IV:5 IV:6 IV:7 IV:8 IV:9 IV:10 Mutation screening Figure 1. The Tasmanian pedigree indicating family members with aniridia or other iris anomalies (black shading).? : Mutation carrier unavailable for examination. Buccal mucosa swabs were collected and genomic DNA isolated using Puregene DNA isolation kits (Gentra Systems, Inc.). All coding exons of PAX6 (exons 4-13) were screened in the proband III:3 using single-stranded conformational polymorphism (SSCP) [Love et al., 1998]. A novel mutation of the PAX6 gene was identified and confirmed by sequencing. Segregation of the mutation was investigated by direct automated sequencing using ABI PRISM BigDye Terminator Cycle Sequencing Kits (Applied Biosystems) and the exon 12 primers of Love et al. [1998].

3 Phenotypic Variability in a Pedigree With a PAX6 Mutation 3 RESULTS Mutation analysis revealed a novel single base deletion 1410del C in exon 12, codon 350, of the PAX6 gene. This mutation in the PST domain produces a STOP codon 15 codons later, resulting in premature truncation of the protein. No other PAX6 variants were found in the proband, III:3. The 1410delC PAX6 mutation was detected in nine family members with anterior segment anomalies: II:2, III:2, III:3, III:6, IV:1, IV:3, IV:4, IV:5, and IV:8. The mutation was also present in individual IV:2 who was unavailable for ophthalmic examination. All individuals with the mutation were heterozygous. The mutation was not present in individuals II:1, II:3, III:1, III:4, III:5, III:7, III:9, III:10, IV:6, IV:7, IV:9 and IV:10. II:2 IV:1 III:2 IV:5 III:6 IV:8 Figure 2. Iris photographs of right and left eyes of affected individuals, showing ocular abnormalities (see text). Numbering of individuals is as in the pedigree shown in Figure 1. Clinical features of affected individuals ranged from total aniridia through partial aniridia to minimal stromal hypoplasia. Other findings included keratitis, cataract, glaucoma, optic disc anomalies and foveal hypoplasia. One woman (II:1) was reported to have an abnormal iris due to injury, but was not able to be examined and did not possess the PAX6 1410delC mutation. One family member (IV:6) believed to be mildly affected by the family on the basis of mildly reduced visual acuity of right 6/9 and left 6/7.5, was found not to carry the mutation. Figure 2 shows iris photos from family members, illustrating some of the observed abnormalities. Bilateral classical aniridia with anterior polar cataracts is illustrated by individual IV:8. Partial aniridia with anterior polar cataracts is exemplified by individual IV:1; note the characteristic appearance of the sphincter pupillae muscle (see Discussion). Sectoral full thickness iris defects were observed infertemporally in IV:5. Individuals II:2 and III:6

4 4 Sale et al. demonstrate no regions of full thickness iris absence, but do demonstrate iris stromal hypoplasia with an intact posterior pigment epithelium. In addition, II:2 also demonstrates the typical vascularizing keratitis previously described in aniridia. Optic disc findings (Figure 3) ranged from within normal limits (individual IV:1 s left optic disc) or mildly hypoplastic (IV:1 s right optic disc) to moderate optic nerve hypoplasia with vertically oval appearing optic nerve heads with aberrant vascular branching (III:6 and IV:4). Individual III:2 demonstrated the most striking disc anomalies: a grossly tilted and hypoplastic left optic nerve head with peripapillary atrophy, with a similarly hypoplastic but less tilted right optic nerve head. Interestingly, the severity of optic nerve abnormalities seemed to be independent of the severity of iris abnormalities as the most abnormal disc appearances were not found in the individuals with total aniridia. The clinical findings of the eleven examined relatives in the aniridia branch of the pedigree are summarised in Table 1. The four marriage partners examined were normal. Patient Aniridia Other iris abnormality Table 1. Ocular Findings in Examined Individuals from the Tasmanian Pedigree Keratitis Cataract Glaucoma Anomalous discs Foveal hypoplasia Visual acuity II:2 X X X X X 6/12 6/60 III:2 X X X 6/60 6/18 III:3 X X X X X X HM HM III:6 X X X X 6/18 6/18 IV:1 X X X X X 6/12 6/24 IV:3 X X X X 6/36 6/36 IV:4 X X 6/12 6/12 IV:5 X X X 6/18 6/18 IV:6*?? 6/9 6/7.5 IV:7* 6/6 6/6 IV:8 X X X X X 3/60 3/60 Numbering of individuals as in Figure 1. HM: hand movements visual acuity;? : equivocal. *IV:6 and IV:7 did not carry the PAX6 1410delC mutation. DISCUSSION The majority of aniridia mutations lead to premature truncation [Hanson and van Heyningen, 1995] and are predicted to be associated with complete loss of function of the mutant allele. However in vitro expression studies of nonsense mutation S353X close to the C-terminus of the PAX6 protein have shown about 5-10% of the wild type activity may be retained [Glaser et al. 1994]. Mutations in this region may therefore result in reduced transactivation activity [Hanson et al., 1993; Glaser et al. 1994; Mirzyans et al., 1995] rather than total loss of function. Although certain individuals and families with classical aniridia possess mutations in the PST domain [Davis and Cowell, 1993; Hanson et al., 1995], other families with variable and atypical ocular findings and mutations in this region have been described [Mirzayans et al., 1995; Gupta et al., 1998; Azuma and Yamada, 1998; Syagailo et al., 1998; Negishi et al. 1999]. From the clinical observations of these families, it has been suggested that small C-terminal truncations may result in less severe and more varied phenotypes compared with more proximal mutations of the PAX6 PST domain [Negishi et al., 1999]. All four exons of the PST domain act synergistically in activating transcription and none can function independently [Tang et al., 1998]. Certain mutant PAX6 proteins lacking transactivating activity may act as dominant-negative repressors [Singh et al., 1998], outcompeting wild-type protein. This may at least partly explain why mutations in different regions of the PST domain result in diverse phenotypes. As all individuals from the single pedigree described in this report possess an identical PAX6 mutation, the broad spectrum of intra-pedigree phenotypic variability gives a clear indication that other as yet unidentified factors must also contribute to disease expressivity. While conceivable that unscreened PAX6 variations in the exon sequence, promoter or introns may be responsible since the coding exons were screened in the proband alone, only two human compound heterozygotes have been reported to date [Glaser et al., 1994; Gronskov et al., 1999]. Downstream targets of PAX6 transcriptional activation, genes that interact with PAX6 positive and negative cis-

5 Phenotypic Variability in a Pedigree With a PAX6 Mutation 5 regulatory promoter elements, other genes involved in eye development, and/or environmental exposures may contribute to phenotypic expression. It is interesting to note that the presence of and severity of abnormalities in one ocular tissue (e.g. iris or lens) did not necessarily correlate well with abnormalities in another ocular tissue. Thus, mild iris abnormalities could be found with severe disc anomalies (III:2). This raises the possibility that putative modifying genes could be multiple and segregate independently. III:2 IV:1 III:6 IV:4 Figure 3. Optic disc photographs of right and left eyes of affected individuals, illustrating the range of disc anomalies (see text). Numbering of individuals is as in Figure 1. As well as optic nerve anomalies, some patients in this pedigree have iris changes superficially resembling those in the Axenfeld-Rieger spectrum of anomalies [Alward, 2000]. These findings re-emphasize the importance of considering investigating possible mutations in the PAX6 gene in phenotypes other than aniridia. Many clinicians are unaware of the association of partial aniridia to PAX6 mutations. In this pedigree and others with similar findings of individuals with partial aniridia or even just iris stromal hypoplasia we have noted a characteristic appearance of a hypoplastic sphincter pupillae muscle, which is absent in the areas of partial aniridia (E. Traboulsi, personal communication). This may be a useful discriminating feature from cases of Axenfeld- Rieger anomaly in which the sphincter pupillae muscle often appears prominent and unusually well demarcated due to the stromal hypoplasia. None of the anterior segment anomalies in this pedigree exhibited posterior embryotoxon. Ultimately, large pedigrees such as this with wide variation of phenotypic severity are likely to be most informative in identifying other anterior segment and optic nerve developmental genes that interact with PAX6 to fully define the ocular phenotype. ACKNOWLEDGEMENTS We wish to thank family members for their participation. Thanks to Dr RJ Mac Gardner, the Tasmanian ophthalmologists, Susan Stanwix and Danielle Healey for assistance in ophthalmic examination and DNA collection, and Andrew Bell, Michele Brown and James Love for technical assistance. Grant support for this project was received from the Glaucoma Research Foundation, NIH Grant EY , Clifford Craig Medical Research Trust, Royal Hobart Hospital Research Foundation, and UK Medical Research Council. REFERENCES Alward WL Axenfeld-Rieger syndrome in the age of molecular genetics. Am J Ophthalmol 130:

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