Genotype/Phenotype Association in Indian Congenital Aniridia

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1 Special Article Genotype/Phenotype Association in Indian Congenital Aniridia Guruswamy Neethirajan 1,4,5, Abraham Solomon 1, Subbaiah Ramasamy Krishnadas 2, Perumalsamy Vijayalakshmi 3 and Periasamy Sundaresan 4 1 Department of Ophthalmology, Hebrew University Medical Center Jerusalem, Israel, 2 Department of Glaucoma, 3 Department of Pediatric Ophthalmology and Strabismus, Aravind Eye Hospital, Madurai, India, Department of Ophthalmology, Hebrew University Medical Center, 4 Department of Genetics, Aravind Medical Research Foundation, Madurai, 5 Department of Biotechnology, Alagappa, University, Karaikudi, Tamilnadu, India ABSTRACT The developmental birth eye disorder of iris is known as aniridia. Heterozygous PAX6 gene, which causes human aniridia and small eye in mice, is located on chromosome 11p13. The variability had been documented between the affected individuals within the families, is due to genotypic variation. Haploinsufficiency renders PAX6 allele non-functional or amorphic, however it presents hypomorphic or neomorphic alleles. India is not a well-studied ethnic group, hence the focus on congenital aniridia gene analysis supports the literature and the phenotypic association were analysed both in sporadic as well as familial. The consistent association of truncating PAX6 mutations with the phenotype is owing to non-sensemediated decay (NMD). It is presumed that the genetic impact of increased homozygosity and heterozygocity in Indian counter part arises as the consequence of consanguineous marriages. The real fact involved in congenital aniridia with other related phenotypes with PAX6 mutations are still controversial. [Indian J Pediatr 2009; 76 (5) : ] sundar@aravind.org Key Words: Aniridia; PAX6; Amorphic; Hypomorphic; Marfan's syndrome; Ectopia lentis; Sclerocornea The developmental eye disorders of aniridic patients usually have poor visions that are associated with other systematic disorders like congenital cataract, glaucoma, nystagmus etc. The prevalence of aniridia is 1:40,000 to 1:100,000 1 with no racial or sexual differences are recognized however the association of WAGR is unknown. Aniridia is caused by loss of function of one copy of human PAX6 located on chromosome band 11p13, 2,3 where as the homozygous mutations lead to lethal phenotypes. 4 Intragenic PAX6 mutations were identified in numerous non-syndromic aniridia patients, confirming the PAX6 as the candidate gene for congenital aniridia (MIM ). 2, 5, 6 The open reading frame (ORF) of PAX6 encodes two DNA binding domains, a paired domain, and a homeodomain. 7, 8 that are separated by a 79-amino acid linker peptide (LNK) and 3' end of the ORF encodes a proline, serine, threonine-rich (PST) domain that have transcriptional trans-activation function. Correspondence and Reprint requests : Dr. P.Sundaresan, Senior Scientist, Department of Genetics, Aravind Medical Research Foundation, Madurai , India, Phone: (ext.423) [DOI /s ] [Received July 26, 2007; Accepted August 20, 2008] The clinical descriptions that are associated with aniridia cases, express variable phenotypes. The variability had been documented between the affected individuals within the families, however the genotype/ phenotype correlations are not consistent among aniridic individuals. The PAX6 is expressed in the developing eye, brain, spinal cord, and pancreas. 9 PAX6 mutations are well documented in the human PAX6 allelic variant database (as on July 2007) contains 366 variations in PAX6 gene. Two hundred and ninety are associated with pathological mutations that cause congenital eye malformations, which includes 5 unique polymorphisms, and 12 compound mutations. 10 In most cases apart from iris abnormalities the nonaniridia phenotypes such as optic nerve defects, keratitis, microphthalmia, foveal hypoplasia and brain 11, 12 abnormalities were also well documented. In this short review, it has been shown that over 3 quarters of Indian aniridia cases were caused by mutations that introduced a premature termination codon on the ORF of PAX6. The distributions of mutations in PAX6 gene with other phenotypes (clinical/molecular pathological) were documented in familial as well as sporadic cases in India. Though, the PAX6 gene is given priority in western population, but Indian Journal of Pediatrics, Volume 76 May,

2 Guruswamy Neethiranjan et al India is not a well studied ethnic group, hence the focus on aniridia gene analysis pays more attention. BASIC OF THE ANALYSIS The inherited as well as congenital eye disorder of aniridia is infrequently reported in India, where the increased prevalence of the disease is high. According to the available literature, the causes of childhood blindness supports the etiology. Many children with congenital ocular anomalies, are also associated with systemic disorders which increases the morbidity and mortality ratio. Genetic studies that have identified specific mutations responsible for several ocular disorders, have been done extensively in the western population. Owing to the prevalent practices of consanguineous marriages in the Indian community might be one of the genetic causes for the mutation frequency. There is no statistical data available for the prevalence of sporadic as well as inherited eye diseases in India. Therefore, screening the families affected with aniridia for the allele variation would help for the early detection and genetic counseling to make an attempt to limit visual impairment. Genotype/Phenotype correlations in sporadic cases In sporadic aniridia, cases 10 out of 40 probands were having PAX6 mutations therefore only 25% mutations were identified where as in familial cases 5 out of 9 pedigrees (50.5%) were detected which includes frame shift, non-sense, and splice site. however few novel PAX6 gene mutations were detected in India. The overall mutation frequency statistical data reveals that 26.6% non-sense mutations, 6.6% splice mutation, 66.6% frame-shifting insertions or deletions were analysed. Unfortunately the mis-sense and run-on mutations were not detected in aniridia patients however the mis-sense mutations were detected in patients with ocular anomalies. 13 Clinical informations gathered for all sporadic probands are given in table 1. Aniridic patients had nystagmus 100%, kerotopathy 60% (9/15), cataract 80% (12/15), glaucoma 60% (9/15) and macular hypoplasia 26% (4/15). The detailed clinical descriptions with familial aniridia patients were sorted out in table 2. The proband with non-sense mutation c.1080c>t had shown bilateral aniridia, nystagmus and cataract where as the another case with novel mutation c.1180insa showed ectopia lentis and cataract besides bilateral aniridia. 14 The proband 28-1 with c.715ins5 mutation showed the presence of sclerocornea with nystagmus in both eyes. A similar phenotype (foveal hypoplasia) was observed in probands (27-1 and 21-1) with c.1201dela and c.482c>a. Proband 10-1 with c.901dela revealed ptosis, microcornea with dislocated cataractous lens. Marfan's syndrome and ectopia lentis was observed in both eyes of the proband 16-1 with c.830g>a. 15 The peripheral corneal pannus with bullous keratopathy and corneal ectasia was observed in IVS9-12C>T. The sporadic proband MAH with unusual 14bp deletion (c.728del14) in PAX6 gene was a 38yrs old male presented with a history of defective vision. Anterior segment examination revealed both eyes had corneal edema with total aniridia besides the upward subluxation of cataractous lens was also present. Intraocular pressure was high during prognosis and the fundus examination not possible since the patient had hazy media. Patient was started an anti-glaucoma medications. AJA proband with a single base deletion (c.537dela) in PAX6 was 10 yrs old male of sporadic total aniridia with history of defective vision since birth. Anterior segment examination revealed both eyes corneal pannus with corneal haze and upward subluxation of clear lens. Intraocular pressure was within normal limits. Fundus examination was not possible due to hazy media. 16,17 It is worthwhile to note that all the mutations observed in this study are associated with other phenotypes table 3. Genotype/Phenotype in hereditary congenital cases In ANF 6-1 family the proband was a 10 years old female during prognosis presented rolling movements of eyeball since birth, cataractous lens with macular hypoplasia with c.1080c>t genotye. The threegeneration family (ANF 2-1) who were heterozygous for PAX6 mutant genotype (c.1174del TG), passed on to the 2 children from the affected mother, which manifested autosomal dominant aniridia. In a 13-year-old female eye, appeared in the opacity in both eyes. Anterior segment analysis showed ectopic coloboma. Fundus examination revealed glaucomatous cupping and underwent medical surgery for trabeculectomy with lensectomy. The other affected member of this family was a 11 year old male who had defective vision of aniridia since birth with cataractous lens. In twogeneration family ANF 9-1 the proband with c.406deltt was a 5-year-old female with defective vision since birth, aniridia was observed in both eyes with nystagmus. The proband had cataract with foveal hypoplasia. 18 The 12-year male had sudden loss of vision since 8 months and the anterior segment examination revealed both eyes had microcornea with aniridic keratopathy in ANF8-1 family. Another affected 8-years female also had microcornea with post polar cataract in the right eye. In 3 generation family pedigree ANF8-1 with frameshift mutation (c.393instcagc) identified, was a de novo mutation of PAX6. The proband ANF 7-1 was a 6months old male child during diagnosis, hence no other examinations were done however the both eyes had nystagmus with genotype c.710delc. The mother 514 Indian Journal of Pediatrics, Volume 76 May, 2009

3 Genotype/Phenotype Association in Indian Congenital Aniridia TABLE 1. Clinical and Common Phenotypes Associated with the Sporadic Aniridic Probands Probands Age Best Refractive / Sex Vision Error Nystagmus Keratopathy Iris Cataract Glaucoma Optic nerve Macular Treatment Hypoplasia Hypoplasia RE LE RE LE RE LE RE LE RE LE RE LE RE LE Medical treatment Surgical treatment RE LE RE LE yrs/m 6/24 6/36-4.0, , R x 180 x yrs/m 6/24 6/ A yrs/f 6/24 6/ NA + + A NA NA + + 4,1 1, yrs/m 6/60 6/60-1.0, A x yrs/m PL HM NI NI A + + NA NA NA mon/m R yrs/m 6/60 H M NI NI R yrs/m PL PL NI NI R NA NA MHA 38 yrs/m 6/60 1/ A NA NA NA NA AJK 10 yrs/m PL NOPL A NA NA NA NA HM - Hand Movement; PL - Perception of Light, A - Absent; R - Remnant; NA - Not Available, NI - No Improvement 1 - Trabeculectomy, 2 - Cyclocryocoagulation, 3 - Awaiting cataract surgery, 4 - Cataract surgery, 5 - Surgery for Retinal Detachment. TABLE 2. Clinical Information of Familial Aniridia Patients Patient Patient Best Refractive Nystagmus Keratopathy Iris Cataract Glaucoma Foveal Macular Treatment Vision Error Hypoplasia Hypoplasia study Sex Age RE LE RE LE RE LE RE LE RE LE RE LE RE LE Medical treatment Surgical treatment No RE LE RE LE ANF2-1 F 5 CF 6/ , R , ANF2-3 M 11 PL 5/ R ,2 ANF2-2 F 28 PL 3/ A ANF6-1 F 10 4/60 6/ A ANF6-2 F 24 3/60 3/ A ANF7-1 M 6months NA NA R ANF7-2 F 22 6/18 PL A ANF9-1 F 5 6/24 6/ A ANF9-2 M 39 3/60 3/ R ANF8-1 M 12 PL PL A ANF8-2 M 45 6/36 6/ A ANF8-3 F 8 PL PL PL - Perception of Light, CF - Counting fingers, A - Absent; R - Remnant; NA - Not applicable, 1 - Trabeculectomy, 2 - Cataract surgery Indian Journal of Pediatrics, Volume 76 May,

4 Guruswamy Neethiranjan et al TABLE 3. Review of PAX6 Mutation with their Variable Phenotypes in Indian Congenital Aniridic Patients S.No. Patient Status Mutation Phenotype Associated Phenotypes Sporadic c.1080c>t Aniridia Nystagmus, Cataract Sporadic c.1180insa Aniridia Ectopia lentis, Cataract Sporadic c.715ins5 Aniridia Sclerocornea with Nystagmus Sporadic c.1201dela Aniridia Foveal hypoplasia Sporadic c.901dela Aniridia Ptosis, Microcornea, Cataractous lens Sporadic c.482c>a Aniridia Foveal hypoplasia Sporadic c.830g>a Aniridia Marfan syndrome, Ectopia lentis Sporadic IVS9-12C>T Aniridia Corneal Pannus, Bullous keratopathy, Corneal ectasia 9 MAH Sporadic c.728del114 Aniridia Corneal edema, Cataract lens 10. AJA Sporadic c.537dela Aniridia Corneal pannus with Corneal haze 11. AHF6-1 Familial c.1080c>t Aniridia Nystagmus, Cataractous lens with macular hypoplasia. 12. ANF2-1 Familial c.1174deltg Aniridia Ectopic coloboma, Foveal hypoplasia. 13. ANF7-1 Familial c.710delc Aniridia Small cornea 14. ANF9-1 Familial c.406deltt Aniridia Nystagmus, cataract, Foveal hypoplasia 15 ANF8-1 Familial c.393ins5 Aniridia Microcornea with aniridic keratopathy of the proband had bilateral aniridia, nystagmus, kerotopathy on left eye and underwent cataract surgery in right eye with foveal hypoplasia, but no glaucoma was observed. 18 It is worthwhile to note that all the mutations observed in this study are associated with other phenotypes table 3. DISCUSSION New PAX6 mutations are described in patients with aniridia presumably related to the disease causing phenotypes. The variant forms of genes (alleles) initially defined by their phenotypic effects by their nucleotide sequences. The wild type allele at any locus is the predominant allele in the population and produces the functional gene products, where as the mutant allele product may differs in quality, quantity or distribution. It is convenient to classify the mutant allele by comparing their phenotypes. PAX6 haploinsufficiency through loss of function intragenic mutation, larger deletions, or occasional chromosomal rearrangements at nearby regulatory elements produces congenital aniridia Although the phenotype can be variable within a family, individuals usually show little difference between 2 eyes. The reason for this varying phenotype among individuals with the same mutation is unknown. However the down or up regulated genes of PAX6 are unknown hence PAX6 plays a pleiotropic role in variable phenotypes among individuals. 21,22 In general, the genotype/phenotype correlations such that exists in all PAX6 mutations in patients with congenital aniridia cannot be amorphic, few represents hypomorphic or neomorphic alleles. 23 PAX6 proteins with altered structure would have different biological effects. C-terminal truncation mutations are rarely associated congenital aniridia with less severe phenotypes. In contrast, the N-terminal mutations leads to severe phenotypes, which were observed in Indian congenital aniridia. The frequencies of consanguineous marriages being very high in India within various religious groups perhaps might be the one among the causes of more novel frame shift mutations. The real fact involved with other related phenotypes with PAX6 mutation is still controversial. The consistent association of truncating mutations with the congenital aniridia phenotype suggests that non-sense-mediated decay acts on PAX6 mutant alleles. 24 MANAGEMENT AND GENETIC COUNSELING One-third of sporadic aniridia cases have a deletion of the WT1 and PAX6 genes have chance to develop Wilms tumor. 25 This emphasizes the importance of performing chromosomal deletion analysis in a sporadic aniridic newborn baby. Children undergo eye examination for refractive errors spectacle usually recommended to evaluate correction of errors. Early diagnosis of any disease will help in treatment of severe complications. Whenever possible the measurement of intra-ocular pressure, optic disc examination, and visual field assessment should be done. Initial glaucoma was treated with typical anti-glaucoma medication. Surgery must be essential for eyes that do not respond to medical therapy. Children with aniridia and a WT1 deletion require regular renal ultrasound examinations. Genetic counseling provides sound knowledge to the affected individual as well as the family members on the nature and inheritance patterns of the disease. The information about the family history will help the genetic risk assessment and the gene test analysis will provide accurate genetic status of the disease. CONCLUSION In conclusion, the review suggested that the genotype/ 516 Indian Journal of Pediatrics, Volume 76 May, 2009

5 Genotype/Phenotype Association in Indian Congenital Aniridia phenotype association in Indian congenital aniridia is predominant disorder among pediatric ophthalmology. Congenital aniridia and the related phenotypes were documented for the first time in Indian counter part. However, it s worthwhile to explore the properties of PAX6 in relation to the expression of phenotypes. Acknowledgements The authors are very thankful to the participants in the present study. Thanks to Indian Council of Medical Research (ICMR, New Delhi, India) for the financial support for the project and the authors are also thankful to all the reviewers. Contributions: GN, Worked and detected the findings; AS, corrections done on the manuscript; KSR and PV identified the clinical diagnosis for the works; PS, drafted the final version of the manuscript. Conflict of Interest: None. Role of Funding Source: ICMR, New Delhi. REFERNCES 1. Shaw MW, Falls HF, Neel JV. Congenital aniridia. Am J Hum Genet 1960; 12: Glaser T, Walton DS, Maas RL. Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene. Nat Genet 1992; 2: Churchill A, Booth A. Genetics of aniridia and anterior segment dysgenesis. Br J Ophthalmol 1996; 80: Glaser T, Jepeal L, Edwards JG et al. PAX6 gene dosage effect in a family with congenital cataracts, aniridia, anophthalmia and central nervous system defects. Nat Genet 1994; 7: Ton CC, Hirvonen H, Miwa H et al. Positional cloning and characterization of a paired-box and homeobox-containing gene from the aniridia region. Cell 1991; 67: Hanson IM, Seawright A, Hardman K et al. PAX6 mutations in aniridia. Hum Mol Genet 1993; 2: Wilson DS, Guenther B, Desplan C et al. High resolution crystal structure of a paired (PAX) class cooperative homeodomain dimer on DNA. Cell 1995; 82: Xu HE, Rould MA, Xu W et al. Crystal structure of the human PAX6 paired domain-dna complex reveals specific roles for the linker region and carboxy-terminal subdomain in DNA-binding. Genes Dev 1999; 13: Simpson TI, Price DJ. PAX6, a pleiotropic player in development. Bioassays 2002; 24: Tzoulaki I, White IMS, Hanson IM. PAX6 mutations: genotype-phenotype correlations. BMC Genetics 2005; 6: Azuma N, Yamaguchi Y, Handa H et al. Mutations of the PAX6 gene detected in patients with a variety of optic-nerve malformations. Am J Hum Genet 2003; 72: Song SJ, Liu YZ, Cong RC et al. PAX6 mutation caused brain abnormalities in humans. Beijing Da Xue Xue Bao 2005; 37: Nallathambi J, Neethirajan G, Shashikant S et al. PAX6 missense mutations associated in patients with optic nerve malformation. Mol Vis 2006; 12: Neethirajan G, Hanson IM, Krishnadas SR et al. A novel PAX6 gene mutation in an Indian aniridia patient. Mol Vis 2003; 9: Nelson LB, Spaeth GL, Nowinski TS et al. Aniridia: a review. Surv Ophthalmol 1984; 26: Neethirajan G, Krishnadas SR, Vijayalakshmi P et al. PAX6 gene variations associated with aniridia in south India. BMC Med Genet 2004; 5: Neethirajan G, Collinson JM, Krishnadas SR et al. De novo deletions in the paired domain of PAX6 in south Indian aniridic patients. J Hum Genet 2004; 49: Neethirajan G, Nallathambi J, Krishnadas SR et al. Identification of novel mutant PAX6 alleles in Indian cases of familial aniridia. BMC Ophthalmol 2006; 6: van Heyningen V, Williamson KA. PAX6 in sensory development. Hum Mol Genet 2002; 11: Chao LY, Mishra R, Strong LC et al. Missense mutations in the DNA-binding region and termination codon in PAX6. Hum Mutat 2003; 21: Negishi K, Azuma N, Yamada M. Various phenotypic expressions of familial aniridia with a PAX6 mutation. Br J Ophthalmol 1999; 83: Chao LY, Mishra R, Strong LC et al. Missense mutations in the DNA-binding region and termination codon in PAX6. Hum Mutat 2003; 21: Gupta SK, De Becker I, Tremblay F et al. Genotype/ phenotype correlations in aniridia. Am J Ophthalmol 1998; 126: Byers PH. Killing the messenger: new insights into nonsensemediated mrna decay. J Clin Invest 2002; 109: Gronskov K, Olsen JH, Sand A et al. Population-based risk estimates of Wilms tumor in sporadic aniridia: a comprehensive mutation screening procedure of PAX6 identifies 80% of mutations in aniridia. Hum Genet 2001; 109: Indian Journal of Pediatrics, Volume 76 May,