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Year : 2018  |  Volume : 66  |  Issue : 2  |  Page : 229-232

Identification of a novel frameshift mutation in PAX6 gene and the clinical management in an Asian Indian aniridia family

1 Arasan Eye Hospital, Erode, India
2 Dualhelix Genetic Diagnostics, Chennai, Tamil Nadu, India

Date of Submission25-Apr-2017
Date of Acceptance07-Nov-2017
Date of Web Publication30-Jan-2018

Correspondence Address:
Dr. Madhavan Jagadeesan
Dualhelix Genetic Diagnostics, New No: 57, Panchali Amman Koil Street, Arumbakkam, Chennai - 600 106, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijo.IJO_311_17

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Purpose: This study aimed to characterize an Asian Indian aniridia family for both the phenotype and genotype of the disease for a better clinical management. Methods: The phenotype and genotype of the affected and unaffected individuals in the aniridia family were evaluated. The subjects underwent a standard ophthalmic evaluation followed by molecular screening of PAX6 gene in the peripheral blood for mutation detection. Results: The three affected individuals had aniridia with several common features and an uncommon presentation of bilateral congenital ptosis. Two affected siblings, a brother and a sister, had aniridia, nystagmus, ptosis, increase in central corneal thickness, cataract, and foveal hypoplasia. The sister had features of glaucoma. The offspring of the sister had all the features except cataract and rise in intraocular pressure. Mutation screening of PAX6 gene helped in identifying a novel heterozygous pathogenic variation g. 31801757dupG (c. 216-19dupG) that resulted in a frameshift mutation that extended into exon 7. Based on the evaluation and diagnostic testing, the family was clinically managed along with genetic counselling. Conclusion: Molecular diagnostic testing helps in genetic counseling of the family with aniridia to understand the nature of the disease and detection of complications early for better management.

Keywords: Aniridia, mutation screening, PAX6

How to cite this article:
Palayil I, Priya S G, Sivan N V, Madhivanan N, Venkatachalam PS, Jagadeesan M. Identification of a novel frameshift mutation in PAX6 gene and the clinical management in an Asian Indian aniridia family. Indian J Ophthalmol 2018;66:229-32

How to cite this URL:
Palayil I, Priya S G, Sivan N V, Madhivanan N, Venkatachalam PS, Jagadeesan M. Identification of a novel frameshift mutation in PAX6 gene and the clinical management in an Asian Indian aniridia family. Indian J Ophthalmol [serial online] 2018 [cited 2020 Aug 12];66:229-32. Available from: http://www.ijo.in/text.asp?2018/66/2/229/224078

Aniridia (MIM 106210) occurs at a frequency of around 1 in 60,000–100,000 in general population.[1] The eye and visual defects associated with aniridia include some or all of the following: corneal opacification, cataract, glaucoma, lens dislocation, ciliary body hypoplasia, foveal hypoplasia, strabismus, and nystagmus.[2],[3],[4] The disease occurs as an autosomal dominant trait with high degree of penetrance but with variable expressivity. Two-thirds of aniridia cases are familial, while the remaining third is sporadic. A small number of patients have Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome.

The aniridia gene, a paired box transcriptional factor localized on chromosome 11p13 PAX6, which is considered the master control gene for morphogenesis of the eye, was found responsible for 95% of aniridia cases.[5],[6],[7],[8],[9] PAX6 gene spans 22 kb, which is divided into 14 exons and encodes by alternatively splicing two proteins of 422 and 436 amino acids.[10] WAGR syndrome occurs due to deletion of 11p13 region, which encompasses both the PAX6 and WT1 genes.[11] Aniridia results due to defect in one copy (heterozygous) of the PAX6 gene. Most of the PAX6 mutations identified so far have resulted in premature protein termination. Two-thirds of PAX6 mutations were identified in familial aniridia cases. Here, we report mutational analysis of PAX6 gene and the management strategy in a family with three affected individuals.

  Methods Top

This study was approved by Institutional Ethics Committee and the guidelines of Declaration of Helsinki were followed. Patients were recruited for the study after obtaining a written informed consent.

Clinical evaluation

The affected and unaffected individuals in the family were evaluated. All individuals underwent a standard ophthalmic evaluation that included testing of best-corrected visual acuity for near and distance visual acuity (Snellen's visual acuity chart), color vision (Ishihara pseudo isochromatic color vision plates), intraocular pressure (IOP) (Applanation Tonometer, Topcon Medical Systems, NJ, USA), clinical biomicroscopy, and a dilated fundus examination using slit lamp/90D and indirect ophthalmoscopic examination.

All the affected individuals underwent molecular characterization. Two milliliters of peripheral venous blood was collected and DNA extraction was done using a Qiagen mini kit (Qiagen India, Chennai, India). Polymerase chain reaction was performed using primers designed in house. The primers and the annealing temperatures were provided in [Table 1]. The coding exons and flanking intronic regions of PAX6 gene were amplified via polymerase chain reaction and sequenced by the Sanger sequencing method (ABI 3500 genetic analyzer, Applied Biosystems, India). Any changes observed were confirmed bidirectional. The pathogenicity of novel variation was confirmed through family segregation analysis and by screening 100 normal control chromosomes.
Table 1: Heading: Primer sequences for polymerase chain reaction amplification of PAX6 gene

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  Results Top

In total, three affected and two unaffected individuals were evaluated and the pedigree of the family is provided in [Figure 1]. Clinical summary of the affected individuals is given below:
Figure 1: Pedigree of the aniridia family. All the affected individuals in the family carried a heterozygous g.31801757dupG (c.216-19dupG), while the unaffected individuals did not carry the variation. *Evaluated and genotyped patients

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Case 1: The proband

A 29-year-old male presented with the complaints of drooping of eyelids since birth and defective vision in both eyes with rowing eye movements since childhood. The best-corrected vision in both eyes was 6/24. On examination, he had bilateral ptosis, peripheral corneal vascularization, aniridia, opacification of the lens, and nystagmus in both the eyes [Figure 2]. IOP by Goldmann applanation tonometry was 18 mmHg in the right eye and 26 mmHg in the left eye. Gonioscopy showed rudimentary iris stump with angle dysgenesis and pigment deposition. The fundus examination of both the eyes revealed normal disc with absent foveal reflex [Figure 3]. Central corneal thickness was 0.617 mm in the right eye and 0.654 mm in the left eye. Humphrey visual field testing was within the normal limits. Complete systemic evaluation and investigations to rule out any association, especially renal anomalies, were done. He was given best-corrected visual acuity with photochromic lenses and preservative-free lubricants. He was started on topical beta-blocker (timolol) in the left eye for ocular hypertension. At present, his IOP is under control and has been advised periodic follow-up.
Figure 2: Anterior segment examination. (a) (II-2), (b) (II-3), and (c) (III-1) showing the presence of bilateral ptosis in all the affected individuals. (d) (II-2), (e) (II-3), (f) (III-1) depicting the aniridia and cataract status in lens: (d) Both eyes: Early nuclear changes; (e) right eye: posterior chamber intraocular lens implantation, left eye: dense nuclear cataract Grade IV; (f) No lens changes in both eyes. The numbers in bracket represent pedigree numbers of individuals

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Figure 3: Posterior segment examination. (a) (II-2), (b) (II-3), and (c) (III-1) showing the presence of foveal hypoplasia with the absence of foveal reflex and foveal dip. Figure 3b shows a cup:disc ratio of 0.8:1 with retinal nerve fiber layer defect in superotemporal and inferotemporal quadrants in the right eye. Fundus view is hazy in the left eye due to dense nuclear cataract

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Case 2: Sister of the proband

The 33-year-old affected sister had dense nuclear cataract in both the eyes along with bilateral ptosis, aniridia, and peripheral corneal vascularization. Her best-corrected vision in the right eye was 6/60 and 6/36 in the left eye. IOP was 38 and 28 mmHg and central corneal thickness was 0.600 mm in both the eyes. Fundus examination in the right eye showed a cup disc ratio of 0.8:1 with inferior neural retinal rim thinning, superotemporal and inferotemporal wedge-shaped retinal nerve fiber layer defect, and absence of foveal reflex. The left eye had a cup disc ratio of 0.65:1. Cataract surgery with posterior chamber intraocular lens (IOL) implantation was done in her right eye and IOP was kept under control in both the eyes with a combination of brimonidine (0.2%) and timolol (0.5%) eye drops.

Case 3: Niece of the proband

The 7-year-old girl child had defective vision, photophobia, and nystagmus, ptosis and absent foveal reflex. Her best-corrected vision in both eyes was 6/36. Her IOP and lens were normal.

In common, all the three affected individuals had defective vision, nystagmus, ptosis, aniridia, and foveal hypoplasia.

Mutation screening

Screening for PAX6 in the affected individuals revealed a novel mutation and a single base-pair duplication ENST00000419022.6 – PAX-209 “g. 31801757dupG (c. 216-19dupG) [Table 1] and [Figure 1],” resulting in a frameshift mutation [Figure 4]. Family segregation analysis showed that all the affected individuals carried the pathogenic variation. None of the 100 chromosomal controls screened showed this variation.
Figure 4: Genetic defect in exon 7 of PAX6 gene. (a, c, and e) Sequence intron 6 and exon 7 of PAX6 gene in unaffected individual (III-2). (b) A heterozygous g.31801757dupG (c.216-19dupG) nucleotide variation (arrow) at intron 6 of PAX6 gene resulting in a frameshift mutation in the affected individual (III-1). (d) Frameshift mutation along exon 7 of the affected individual. (f) End of frameshift mutation of exon 7 (arrow) in the affected individual

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  Discussion Top

PAX6 is expressed in many parts of the developing eye. The eye tissues that develop from neuroectoderm, surface ectoderm, and mesenchyme (mesoderm invaded by cranial neural crest cells) have specific dosage requirements of the PAX6 gene. It has been demonstrated previously that there has been PAX6 dosage-sensitive maturation of the iris and ciliary body.[12]PAX6 activity is essential in the ectoderm for lens placode formation as well. Eye lids develop from mesenchyme and surface ectoderm. It should be noted that the mesenchyme gives rise to levator and tarsal muscles, while the levator aponeurosis is developed from neural crest.[13],[14],[15] Finally, PAX6 expression continues in the adult retina, lens, and cornea. The developmental role of PAX6 demonstrates the occurrence of aniridia phenotype and ptosis, with acquired corneal, lens, and glaucoma changes following its absence.[16] The insertion of a nucleotide in exon 7 noted in the present study caused a frameshift mutation that resulted in haploinsufficiency and the developmental anomaly.

Management of a patient with aniridia is mainly aimed at symptomatic relief, identifying and preventing the complications, and genetic counseling to prevent transmission of the disease to the next generation. Clinical management includes refractive error correction and low vision aid for poor vision. Tinted or photochromic lens should be prescribed to reduce glare. Periodic screening for glaucoma should be performed throughout the lifetime that should include measurement of IOP, gonioscopy, and optic disc evaluation. Medical management is the initial choice of treatment, but usually it is not sufficient and may require trabaculectomy or aqueous drainage devices or cyclophotocoagulation. Cataract surgery is indicated in dense cataracts and the improvement in vision is subject to various factors including foveal hypoplasia. Black diaphragm aniridic IOLs are preferred as they reduce glare postoperatively. Aniridia-associated keratopathy can be managed by preservative-free lubricants and amniotic membrane graft in initial stages and by limbal stem cell transplantation and penetrating keratoplasty in advanced cases. Periodic follow-up and complete systemic evaluation should be done to rule out the associated systemic abnormalities.

In the present study, the duplication of a single nucleotide in intron 6 of PAX6 gene resulted in a frameshift mutation affecting the exon 7 of the gene. This highly pathogenetic mutation resulted in severe phenotype with high penetrance in the family. Molecular diagnostic testing helped in genetic counseling of the family to understand the nature of the disease and detection of complication early for better management. In addition, as the risk of transmission of the defective allele to the offspring is 50%, identification of the defective allele may be useful to prevent the disease transmission. The proband presented late for medical evaluation. Screening of the family identified the same defect in his niece. As the disease was picked up early, managing her appropriately may prevent complications due to increase in IOP and corneal decompensation. Further, as the proband is on his family way, identification of the mutation will help to prevent the risk of his offspring getting the disease.

  Conclusion Top

We demonstrated duplication of a single nucleotide in intron 6 of PAX6 gene resulting in a frameshift mutation affecting the exon 7 of the gene in our patient with aniridia. This highly pathogenetic mutation resulted in severe phenotype with high penetrance. Molecular diagnostic testing helps in genetic counseling and risk-stratified intensive monitoring of the patient.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Shaw MW, Falls HF, Neel JV. Congenital aniridia. Am J Hum Genet 1960;12:389-415.  Back to cited text no. 1
Nelson LB, Spaeth GL, Nowinski TS, Margo CE, Jackson L. Aniridia. A review. Surv Ophthalmol 1984;28:621-42.  Back to cited text no. 2
Hanson IM, Fletcher JM, Jordan T, Brown A, Taylor D, Adams RJ, et al. Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including peters' anomaly. Nat Genet 1994;6:168-73.  Back to cited text no. 3
Prosser J, van Heyningen V. PAX6 mutations reviewed. Hum Mutat 1998;11:93-108.  Back to cited text no. 4
Gessler M, Simola KO, Bruns GA. Cloning of breakpoints of a chromosome translocation identifies the AN2 locus. Science 1989;244:1575-8.  Back to cited text no. 5
Davis LM, Everest AM, Simola KO, Shows TB. Long-range restriction map around 11p13 aniridia locus. Somat Cell Mol Genet 1989;15:605-15.  Back to cited text no. 6
Rose EA, Glaser T, Jones C, Smith CL, Lewis WH, Call KM, et al. Complete physical map of the WAGR region of 11p13 localizes a candidate Wilms' tumor gene. Cell 1990;60:495-508.  Back to cited text no. 7
Lyons LA, Martha A, Mintz-Hittner HA, Saunders GF, Ferrell RE. Resolution of the two loci for autosomal dominant aniridia, AN1 and AN2, to a single locus on chromosome 11p13. Genomics 1992;13:925-30.  Back to cited text no. 8
Compton DA, Weil MM, Jones C, Riccardi VM, Strong LC, Saunders GF, et al. Long range physical map of the Wilms' tumor-aniridia region on human chromosome 11. Cell 1988;55:827-36.  Back to cited text no. 9
Glaser T, Walton DS, Maas RL. Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene. Nat Genet 1992;2:232-9.  Back to cited text no. 10
Chao LY, Huff V, Strong LC, Saunders GF. Mutation in the PAX6 gene in twenty patients with aniridia. Hum Mutat 2000;15:332-9.  Back to cited text no. 11
Davis N, Yoffe C, Raviv S, Antes R, Berger J, Holzmann S, et al. Pax6 dosage requirements in iris and ciliary body differentiation. Dev Biol 2009;333:132-42.  Back to cited text no. 12
Hayashi Y, Liu CY, Jester JJ, Hayashi M, Wang IJ, Funderburgh JL, et al. Excess biglycan causes eyelid malformation by perturbing muscle development and TGF-alpha signaling. Dev Biol 2005;277:222-34.  Back to cited text no. 13
Dutton JJ. The eyelids and anterior orbit. In: Dutton JJ, editor. Atlas of Clinical and Surgical Orbital Anatomy. Philadelphia, PA: W.B. Saunders Company; 1994. p. 113-38.  Back to cited text no. 14
Barishak YR. Embryology of the eye and its adnexae. Dev Ophthalmol 1992;24:1-42.  Back to cited text no. 15
van Heyningen V, Williamson KA. PAX6 in sensory development. Hum Mol Genet 2002;11:1161-7.  Back to cited text no. 16


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1]


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