A rare case of congenital aniridia with an unusual run-on mutation in PAX6 gene.

Congenital aniridia is characterized by partial or complete absence of the iris.[1] The disease manifestations extend to almost every part of the eye, including the cornea, anterior chamber angle, lens, fovea, and the optic nerve.[2] It has a global prevalence of approximately one in 40,000–100,000 live births.[2] Some of the clinical features like congenital nystagmus and foveal hypoplasia are present since birth, while others like cataract, aniridia-associated keratopathy (AAK), and glaucoma may develop later in life.[123] Two-third of the aniridia cases are familial and one-third are a result of de novo (sporadic) germline mutations in the parent of the affected individual.[1] The most common inheritance pattern is autosomal dominant (AD) with high penetrance and variable expression.[123] However, rare autosomal recessive forms have been reported.[4] Aniridia might present as a part of the WAGR syndrome (OMIM 194072; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) due to a contiguous gene deletion encompassing the PAX6 and WT1 genes.[1] Around 30% of individuals detected with sporadic aniridia are likely to develop symptoms of WAGR syndrome, making abdominal ultrasound screening among children mandatory up to 7 years of age.[56] Another phenotypically distinct disease entity called the Gillespie syndrome presents itself as a triad of partial aniridia, non-progressive cerebellar ataxia and intellectual disability.[7] However, a causative gene has recently been identified as ITPR1, an inosine triphosphate receptor with calcium channel activity which is genotypically distinct from PAX6-associated variations.[7]

Aniridia can broadly be divided into two subgroups: classic aniridia, encompassing mutations in the PAX6; and aniridia-like, which includes mutations in other genes like FOXC1, PITX2, CYP1B1, FOXD3 and TRIM44.[3] Although, non-PAX6 genes are increasingly being identified to be linked with aniridia, PAX6 defects remain the leading cause of congenital aniridia. Majority of aniridia cases (90%) result from haploinsufficiency in the PAX6, either due to mutations or chromosomal rearrangements and are inherited in AD manner.[26]

Genotype–phenotype correlations have been described based on the type of intragenic mutations in the PAX6 gene, wherein the premature termination codon and C-terminal extensions (CTE) lead to a more severe phenotype as compared to the missense mutations.[89] The present case report describes a rare run-on mutation in the PAX6 gene and the specific clinical phenotype of congenital aniridia linked with it. Ethical approval was obtained from the institutional review board of Dr Shroff’s Charity Eye Hospital and was consistent with the provisions of the Declaration of Helsinki.

A 21-year-old female presented at Dr Shroff’s Charity Eye Hospital in October 2018 with complaints of gradually increasing whitish lesion over the black part of both eyes (B/E) associated with photophobia since past few years. The proband (III: 5), her mother (II: 6), younger sister (III: 6), and younger half-brother (III: 3) [Fig. 1] had shaky eyeballs and poor vision in both the eyes since birth. The best corrected visual acuity (BCVA) of the proband was finger counting at 20 cm in right eye (R/E) and finger counting one meter in the left eye (L/E). Media opacity precluded refraction in the R/E. The refractive error was −18.0 D/−8.0 D at 180° in the L/E. Anterior segment examination revealed complete aniridia with only a thin stump of iris tissue in both eyes (B/E). There was centripetal growth of superficial vascularization from the limbus over the cornea, and adjacent stromal opacification. The severity of this AAK was more in the R/E, consequently involving the visual axis [Fig. 2a]. In the L/E, a central island of relatively clear cornea was present [Fig. 2b]. There was superior subluxation of the lens with zonular weakness in meridians 4–8 o’clock in the R/E and 4–7 o’ clock in the L/E [Fig. 2c and 2d]. There was diffuse lenticular haze and dot-like lenticular opacities in B/E. Gonioscopy (Ocular Sussman four mirror handheld gonioscope, Ocular Instruments, USA) showed open angles with increased pigmentation, and multiple thin strands of iris tissue bridging the angle [Fig. 2e and 2f]. The intraocular pressure (IOP) measured with rebound tonometry (IC 200, iCare, Finland) was 10 mmHg and 9 mmHg in the R/E and L/E, respectively. The details of the fundus were hazy in the R/E and only mild pallor could be appreciated. The retina findings of the L/E revealed disc pallor, normal cup/disc ratio and absent foveal reflex [Fig. 3]. The corneal thickness was 784 μm and 720μm in R/E and L/E, respectively. The younger sister had similar phenotype in B/E except that the AAK was less severe than the proband. The mother was not available for evaluation.

Figure 1

Pedigree of an aniridia family of Indian origin harboring a heterozygous c.1268A>T; p.*423L variant in PAX6. Proband (III: 5) is highlighted with an arrow. Star is used to denote members who gave their blood samples and participated in genetic testing

Figure 2

Clinical photographs of anterior segment of proband. (a and b) show the aniridia-associated keratopathy, (c and d) show superior subluxation of the lens (arrow), and (e and f) show the gonioscopic view of the inferior angle

Figure 3

Fundus photograph of the left eye of proband (III: 5) showing average-sized disc with pallor, a normal cup/disc ratio and the absence of foveal reflex

The proband was visually rehabilitated with photochromatic glasses. Preservative-free artificial tears eye drops (carboxymethylcellulose, Allergan) were prescribed for the AAK. She was advised for simple limbal stem cell transplantation followed by penetrating keratoplasty for the right eye. However, she denied any surgery considering the high rates of recurrences. She was referred for low vision and assistive devices. Given the unique aniridia phenotype and positive family history, members of the family (III: 5, III: 6, II: 10) were counselled about importance of molecular diagnosis and assessing recurrence risk to offer prenatal diagnostic options. The proband was a young, married individual planning to start a family and her younger sister was unmarried. Pre-conceptional counselling for the proband, and pre-marital counselling for her sister was carried out as they were keen to have unaffected children. Post counselling, they consented for the genetic investigation. Subsequently, molecular genetic investigations were carried out for individuals III: 5, III: 6 and II: 10.

Blood samples were collected in EDTA solution. Clinical exome analysis was carried out for the proband (III: 5) by next generation sequencing.[10] Genomic DNA was isolated and in-solution hybridization of the coding exons and flanking intronic regions within the genes tested was performed on the DNA. Illumina NextSeq system was used for paired read sequencing of the amplified captured regions. Sequence obtained was aligned to the human reference genome (GRCh37/hg19) and variant calling was performed using BWAGATK. The analyzed region included all the coding exons and ±10 base pairs (bp) of flanking intronic region on both sides. Only variants in the coding region and the flanking intronic regions with a minor allele frequency of <5% were evaluated. The mutation annotation was carried out using published variants in literature and set of disease databases such as ClinVar, OMIM, GWAS, HGMD and SwissVar. Based on results of the above-mentioned bioinformatics analysis, the mutation identified in the proband was a heterozygous variant (c.1268A>T; p.*423L) causing a stop loss change in PAX6. Sanger sequencing was performed for individuals II: 10 and III: 6. High-fidelity polymerase chain reaction (PCR) was performed on their genomic DNA for amplification of PAX6 gene covering the variant identified in individual III: 5 using separate primer sets. The amplified product was sequenced bi-directionally as described elsewhere by Vanita et al. in 2006.[11] The nucleotide sequence was extracted in FASTA format and aligned using CodonCode Aligner to identify variants and was verified for quality using ChromasLite software. The final result confirmed c.1268A>T; p.*423L in the affected sister (III: 6) too and the unaffected maternal uncle (II: 10) showed wild-type (AA) genotype.

Discussion

PAX6 (OMIM: 607108) is a crucial transcription factor gene for oculogenesis that is expressed during developmental stages of eye.[12] Several types of mutations in the PAX6 gene such as frameshifts, nonsense, splice site, insertions, deletions, and CTE have been reported for aniridia with other co-occurring ocular anomalies (PAX6 webpage - MRC Human Genetics Unit LOVD at MRC IGMM - Leiden Open Variation Database, 2022)).[13] C-terminal extensions in PAX6 cause the reading frame to continue protein coding into the 3’ UTR and leads to gain-of-function and severe aniridic phenotype.[2] The c.1268A>T substitution observed in present study is a CTE mutation leading to loss of the stop codon at the 3’ end and leading to addition of 21 to 24 Poly-dAs, as also previously reported by Singh et al.[14] c.1268A>T has previously been reported in few individuals with aniridia in different studies across the globe [Table 1]. Compared to the earlier reports, the unique clinical features of the affected individuals with aniridia in the current study are superior subluxation of the lens, high myopia and optic atrophy. These features are similar to those reported by Souzeau et al. in a Cambodian family.[15]

Table 1

Aniridia cases reported to be linked with c.1268A>T; p.*423L in PAX6

Author/year	Origin of the family	Number of families	Familial/Sporadic case	Phenotype
Baum, 1999[16]	Chinese (Hong Kong)	1	NM	Bilateral affection, vision of 20/200, aniridia, nystagmus, keratopathy, cataract, macular hypoplasia
Singh, 2001[14]	Hispanic	1	Sporadic	Bilateral poor vision, nystagmus, moth-eaten appearance of pupil due to absence of inner part of iris, anterior capsular plaque type cataract
Chao, 2003[17]	NM	1	Sporadic	Aniridia, strabismus, ptosis, corneal pannus, glaucoma, preauricular ear pits
Bobilev, 2016[18]	NM	2	Sporadic	Aniridia†
Vasilyeva, 2017[19]	Russian	1	Sporadic	Aniridia†
Souzeau, 2018[15]	Cambodia	1	Familial, AD	Five individuals affected in the family. All had similar features of total aniridia, nystagmus, foveal hypoplasia, poor vision (ranging from 20/200 to HM), high myopia, high CCT (ranging from 584 to 779) and no glaucoma. Additionally, one family member had mild keratopathy, three had cataract, one had lens subluxation and two had chorio-retinal atrophy due to high myopia
Present study	Indian	1	Familial, AD	Four individuals (II: 6, III: 3, III: 5, III: 6) affected in the family. III: 5 had total aniridia, nystagmus, foveal hypoplasia, keratopathy, high CCT, superior lens subluxation, high myopia, and optic atrophy.

NM, Not mentioned; AD, Autosomal dominant; HM, Hand movements; CCT, Central corneal thickness. Only English language literature has been mentioned here. †Detailed description of phenotype not mentioned

The minor allele frequency for c.1268A>T substitution is not reported in the general population. In silico analysis of c.1268A>T supports its deleterious effect. Mutation Taster, CADD, DANN, FATHMM, Eigen and GenoCanyon predicted it to be disease causing/damaging. Validation by Sanger sequencing confirmed c.1268A>T substitution in PAX6 in the proband as well her sister in heterozygous form, while their unaffected maternal uncle had wild-type genotype. Affected mother of proband could not be tested for c.1268A>T in PAX6 due to geographical limitations. Based on the segregation of variant with the phenotype in the present study and previously published reports [Table 1], c. 1268A in PAX6 seems to be a mutation hot-spot for aniridia.

The treatment of aniridia entails treating its various clinical manifestations individually.[13] The foveal hypoplasia and the nystagmus do not have any definitive treatment. Cataract surgery may benefit those with significant lenticular opacities or lens subluxation.[20] Since the lenticular opacity and subluxation were mild in this patient, they did not warrant any treatment. Cataract extraction, when indicated, may be combined with iris reconstruction with various iris prosthetic devices.[21] AAK may benefit in early stages with artificial tears and autologous serum.[1] However, in advanced stages when the visual axis is affected, restoration of the limbal stem cell niche followed by keratoplasty has been shown to have limited success.[1]

To conclude, through this case report, we have described the detailed phenotype of the c.1268A>T; p.*423L run-on mutation in the PAX6 gene in an Indian family. We have demonstrated the likely pathogenic modality of this mutation by segregation analysis among affected and unaffected members of this family, as well as in silico analysis. To the best of our knowledge, this mutation has not been reported previously in the Indian population.

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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.