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Correspondence and reprintrequests to:Dr. Subhabrata ChakrabartiBrien Holden Eye Research CentreHyderabad Eye ResearchFoundationL.V. Prasad Eye InstituteL.V. Prasad Marg, Banjara HillsHyderabad – 500034Andhra PradeshIndiaTel: 91-40-23608262, 23548267Fax: 91-40-23548271E-mail: [email protected]
Acknowledgements:The authors wish to thank all thepatients and their families forparticipating in this study, Prof. D.Balasubramanian for his valuablecomments on the manuscript, Dr.G.N. Rao for encouragement andsupport, Dr. Seyed E. Hasnain ofCDFD for his kind help in gettingthe primers, the ClinicalBiochemistry Services and J.V.Ramanamma Children’s Eye Care
Abstract In order to understand the underlying molecular geneticdefect causing aniridia in India, eight probands from sporadic caseswere screened for all 14 exons of the PAX6 gene using polymerasechain reaction-single strand conformation polymorphism (PCR-SSCP). Direct sequencing of the SSCP variants revealed a nonsensemutation (R317X) in the eleventh exon leading to a premature termi-nation of the PAX6 protein in the proline-serine-threonine (PST)-richdomain in two probands. Another proband exhibited an intronic poly-morphism (IVS 9–12 C-T). The mutation resulted in loss of function ofthe PAX6 protein along with variable phenotypic manifestations in theprobands. This is the first report describing a PAX6 gene mutation inaniridia cases from India and highlights the variable expressivity inphenotypes due to haploinsufficiency.
Key words PAX6 gene; aniridia; mutation; haploinsufficiency;phenotype
Introduction Aniridia is a bilateral, panocular, congenital disordercharacterized by iris hypoplasia, often associated with cataract, glau-coma, nystagmus or optic nerve hypoplasia.1 With an incidence of onein 50,000–100,000 individuals, about two-thirds of the aniridia casesexhibit an autosomal dominant inheritance with high penetrance. Therest are sporadic.2 Mutations in the transcription factor gene PAX6 onchromosome 11p13 have been identified to be primarily responsible foraniridia and also for Peter’s anomaly, autosomal dominant keratitis,
PAX6 gene mutations in Aniridia 161
Ophthalmic Genetics 1381-6810/03/US$ 16.00
Ophthalmic Genetics – 2003, Vol. 24No. 3, pp. 161–165© Swets & Zeitlinger 2003
Accepted 14 November 2002
PAX6 gene mutations and genotype-phenotype correlations in sporadic cases of
aniridia from India
Neeraja Dharmaraj1
Aramati B.M. Reddy1
Velamakanni S. Kiran1
Anil K. Mandal2
Shirly G. Panicker1
Subhabrata Chakrabarti1
1Brien Holden Eye Research Centre, Hyderabad Eye ResearchFoundation, and 2Jasti V. Ramanamma Children’s Eye Care
Centre, L.V. Prasad Eye Institute, Hyderabad, India
Research report
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Centre staff at LVPEI for theirassistance in sample collection, andDr. K.R. Rajyashree of AvesthaGengraine for her help insequencing the DNA samples. Thestudy was supported in part bygrants from the Department ofBiotechnology, Government of India(# BT/PR 1357/MED/13/046/98).
and foveal dysplasia.1,2 The human PAX6 gene spans 22 kilobases andconsists of a paired and a homeobox DNA-binding domain and aproline-serine-threonine (PST)-rich transactivation domain that hasbeen well conserved through evolution.2
Over 200 mutations in the PAX6 gene have been reported foraniridia,3 80% of which are nonsense mutations, splicing errors, orinsertion-deletions leading to premature truncation of the protein.1,4,5
Haploinsufficiency of the gene product is thought to result in theaniridia phenotype due to reduction in the levels of functional PAX6protein needed for iris development.1,4 Considerable phenotypic vari-ability is seen due to different mutations and genetic modifiers in thePAX6 gene in different patients.1 Classical aniridia occurs mainly dueto deletions or premature truncations of the PAX6 protein.1,3 While theclinical etiology of aniridia is well characterized, the genetic causeshave yet to be identified in the Indian populations. This study, there-fore, is aimed at screening mutations in the PAX6 gene in sporadiccases of aniridia to understand the underlying genetic mechanism andits role in the manifestation of the phenotype.
Materials and methods A total of eight unrelated sporadic casesof aniridia were recruited. These individuals had different ethnic back-grounds and no previous family history. The guidelines of the Decla-ration of Helsinki were strictly followed and clearance was obtainedfrom the institutional review board prior to study commencement.The cases were selected after a thorough clinical evaluation (by AKM) including slit-lamp biomicroscopy, gonioscopy, measurement ofintraocular pressure (IOP), fundus examination, and visual acuitytesting. The mean age of these patients was 10.2 years and all had acongenital onset of the disease. Around 5–10ml of venous blood wasdrawn from the probands and their parents after prior informedconsent.
mutation screening by pcr-sscp and direct sequencingMutation screening was carried out in the entire coding region of thePAX6 gene, which consists of 14 exons.3 Genomic DNA was extractedand amplified with primers for all of the exons using polymerase chainreaction (PCR). The primer sequences and PCR-cycling conditionshave been reported earlier.2 Amplified products were screened for vari-ations using single-strand conformation polymorphism (SSCP). Elec-trophoresis was carried out on an 8% nondenaturing polyacrylamidegel with and without glycerol, under constant current (10mA) at 4°Cand room temperatures, and visualized by silver staining. On detectionof an SSCP variant due to mobility shift, the amplicon was character-ized for mutation/polymorphism by direct sequencing on an automatedDNA sequencer (ABI 3700, PE-Applied Biosystems, USA).
Results
identification of mutation and polymorphism Twoprobands (AG6 and AG8) exhibited SSCP variants in the eleventh
162 N. Dharmaraj et al.
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exon of PAX6 (Figure 1a). Direct sequencing of this exon revealed aCGA Æ TGA substitution at codon 317, causing an amino acid change from arginine to stop codon (Figure 1b) and resulting in a non-sense mutation (R317X) in the PST-rich domain. Another proband(AG3) exhibited a C Æ T substitution in the ninth intronic region(IVS9-12C-T); this was previously reported as a polymorphism. Thesemutations were not observed in the normal parents. The otherprobands did not exhibit any SSCP variants. Haplotype analyses withintragenic markers of PAX6 exhibited different sets of haplotypes inAG6 and AG8 probands that were not observed in their parents (datanot shown).
genotype-phenotype correlation Probands AG6 and AG8exhibited variable expression of phenotype for the R317X mutation.Both of them had poor vision, persistent corneal haze, and high cup-to-disc (CD) ratios with marked iris and anterior segment abnormali-ties along with secondary glaucoma at presentation (Table 1).However, the prognosis in proband AG8 was relatively better after asingle medical treatment (IOP was controlled and visual acuityimproved to 20/80, left eye), while no improvement was seen inproband AG6 despite multiple treatments. Proband AG3 had a fairprognosis with controlled IOP, normal CD ratios, and a visual acuity of20/80 in the left eye. His poor vision in the right eye is surmised as beingdue to cataract.
Discussion To the best of our knowledge, this is the first reportdescribing a PAX6 gene mutation in aniridia cases from India. Wefound the R317X mutation in 25% of the sporadic cases (2/8 patients).Inactivation of one copy of the gene for phenotypic expression or hap-loinsufficiency has been reported in different studies on aniridia.1,2,4 Aprevious report described 28% of the PAX6 mutations to be C Æ Tchanges at hypermutable CpG dinucleotide repeats, due to the methy-lation of cytosine residues which are thought to be hot spots for muta-tions.2,5 Another study identified 11 mutations leading to prematuretruncation of the protein in 23 aniridia patients.3 Nonsense mutationsleading to loss of function have also been described in over 80% of the
PAX6 gene mutations in Aniridia 163
Fig. 1. (a) An SSCP gel on silverstaining showing variant bands dueto mobility shifts in lane 6 (AG6)and lane 9 (AG8) for the eleventhexon of the PAX6 gene. This was an8% nondenaturing polyacrylamidegel with glycerol, run at 4°C underconstant current (10mA). An arrowindicates the variant bands. Lane 1was the control sample and lanes2–5, 7, and 8 the other aniridiaprobands. (b) Electropherogram of asense strand of exon 11 of the PAX6gene, showing the heterozygouschange from C Æ T resulting innonsense mutation R317X in theAG6 proband. An arrow indicatesthe point of mutation at codon 317.
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exonic substitutions.5 The different affected haplotypes in probandsAG6 and AG8 indicate that there might be independent origins of this mutation in different ethnic groups. Moreover, as the parents ofAG6 and AG8 had a normal phenotype and genotype constitution,the R317X mutation represents a new dominant mutation in these families.
The intronic change (IVS 9–12 C-T) observed in proband AG3has been described as a polymorphism in earlier studies,2,4 and wasobserved in 10% of our normal population. However, the phenotypeof the affected individuals described in a previous study2 is somewhatsimilar to that in our AG3 proband except that he did not present withmild corneal dystrophy (Table 1).
The frequency of the PAX6 mutation in the present study was rela-tively low compared to the mutations observed in sporadic aniridia casesin some other studies.2,4 This could be due to unidentified mutations inthe promoter region or in intronic sequences away from the splice sites.2
Genotype-phenotype correlations in probands AG6 and AG8 indi-cate relatively severe and less severe phenotypes, respectively, withrespect to the glaucoma symptoms and visual outcomes, suggesting avariable expressivity of the R317X mutation and implying that thisvariation is due to haploinsufficiency.2 Note, the proband with a poly-morphism had a better prognosis.
Currently, an array of upstream and downstream enhancers, regula-tors, and promoters of PAX6 is being functionally assessed, along with other interacting genes, for the expression of the aniridia pheno-type.1 Mice models are also being devised to characterize the develop-mental pathways.1 Knowledge of these underlying factors may throwmore light on the role of the PAX6 gene in the manifestation ofaniridia.
Ethnic and geographical variants play a major role in the manifesta-tion of genetic determinants in different populations. This study pro-vides some insight into the nature of PAX6 gene mutations in apreviously uncharacterized patient cohort from India. Whether R317Xis an important type of mutation in aniridia patients from India remainsto be seen. Analysis of more aniridia families will be useful in identi-fying the full range of mutations in this gene along with their functionalcharacteristics.
164 N. Dharmaraj et al.
Case IOP on Last CD Last recorded Iris Anterior Corneal Associated Treatment Mutation/no. treatment ratios vision abnormality segment clarity anomaly polymorphism
OD; OS OD; OS OD; OS abnormality
AG6 25; 20 0.8; 0.9 20/200; HM A + Haze Nystagmus Medical OU R317XAG8 16; 18 0.8; 0.4 20/125; 20/80 A + Haze EL Medical OU R317XAG3 10; 08 0.3; 0.3 CF-1m; 20/80 A + Clear Cataract & Medical OU IVS 9-12-C-T
nystagmus
IOP, intraocular pressure; CD ratio, cup-to-disc ratio of the optic nerve; OD, right eye; OS, left eye; OU, both eyes; HM, hand movements closeto face; CF, counting finger; EL, ectopia lentis; A, total absence of iris clinically; +, present.
table 1. Clinical findings ofprobands exhibiting nucleotidechanges.
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PAX6 gene mutations in Aniridia 165
References1 van Heyningen V, Williamson KA.
PAX6 in sensory development. HumMol Genet. 2002;11:1161–1167.
2 Gronskov K, Rosenberg T, Sand A,Brondum-Nielsen K. Mutationalanalysis of PAX6: 16 novelmutations including 5 missensemutations with a mild aniridiaphenotype. Eur J Hum Genet.1999;7:274–286.
3 http://pax6.hgu.mrc.ac.uk/4 Axton R, Hanson I, Danes S, Sellar
G, van Heyningen V, Prosser J. Theincidence of PAX6 mutation inpatients with simple aniridia: anevaluation of mutation detection in12 cases. J Med Genet.1997;34:279–286.
5 Prosser J, van Heyningen V. PAX6mutations reviewed. Hum Mutat.1998;11:93–108.
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