Case series: Pyramidal cataracts, intact irides and nystagmus from three novel PAX6 mutations.

PURPOSE: To investigate the association between novel PAX6 mutations to bilateral anterior pyramidal congenital cataracts (APyC), complete and intact irides, and nystagmus. OBSERVATIONS: This is a retrospective observational case series in a multi-center setting with genetic testing. Three female patients were diagnosed with bilateral APyC, intact irides and nystagmus. Genetic testing identified the three patients had novel missense mutations in PAX6 - c.128C > T; p.Ser43Phe (S43F), c. 197T > A; p.Ile66Asn (I66N) and c.781C > G; p.Arg261Gly (R261G). CONCLUSIONS AND IMPORTANCE: This study demonstrates a novel phenotype of bilateral APyC, intact irides, and nystagmus in whom genetic testing for PAX6 identified novel missense mutations (S43F, I66N, R261G) in highly conserved DNA-binding domains. Implications of understanding why the iris is present in these cases is discussed.

Introduction

Pax6, a 422-amino-acid highly-conserved transcription factor, plays critical roles in the development of the eye, brain, olfactory system, pancreas, pituitary gland and spinal cord. Human PAX6 is located on chromosome 11p13, spanning 14 exons over 22 kb. PAX6 heterozygous mutations are commonly associated with an ocular phenotype involving complete or partial absence of iris, anterior polar cataract (APoC), aniridia-associated keratopathy, foveal and/or optic nerve hypoplasia, nystagmus, and glaucoma.2, 3, 4 Other ocular phenotypes reported with PAX6 heterozygous mutations include isolated foveal hypoplasia, Peters' anomaly,, autosomal dominant keratitis, microphthalmia,9, 10, 11, 12 and certain optic nerve defects. PAX6 homozygous mutations are extremely rare, as they generate debilitating central nervous system defects that lead to post-natal loss of life. Thus, PAX6 mutations producing primarily ocular phenotypes are likely inherited in an autosomal dominant pattern.

We herein describe three cases of bilateral anterior pyramidal cataracts (APyC), intact irides, and nystagmus due to novel missense mutations in PAX6. A thorough review of the literature suggests, to the best of our knowledge, that we are describing a novel phenotype. We speculate as to why this phenotype occurs with these mutations using molecular modeling and analysis of the PAX6 mutation database.

Findings

A total of three female patients with a mean age of 96.5 months (M) (Median = 88.5M; range = 5 months-17 years) with bilateral APyC, intact iris, and nystagmus were included.

Case 1

A 9-month old girl presented with fine vertical nystagmus. Her BCVA was 20/136 in the right eye and 20/200 in the left with a refraction of +2.5DS-3.0DC × 10° and +3.5DS-3.5DC × 180° D in the right and left eye, respectively (Table 1). On anterior segment evaluation she was noted to have bilateral APyC (Fig. 1A, B, C). The iris was intact and IOP was within normal limits. While posterior segment examination showed no evidence of foveal hypoplasia, given the nystagmus it was difficult to be sure. OCT was not successfully performed in this child. At 11 years, she underwent cataract extraction and posterior chamber intraocular lens (IOL) implantation in the left eye. At the last follow-up visit (12 months after cataract extraction), her BCVA was 20/125 in the right eye and 20/200 in the left.

Table 1

Clinical characteristics and genetic testing results.

Cases		1		2		3
Age at presentation		9 months		5 months		17 years
		OD	OS	OD	OS	OD	OS
Vision (BCVA)		20/136	20/200	20/1200	20/1200	20/80	20/200
Refraction		+2.50−3.00×10°	+3.50−3.50×180°	+7.50−2.00×45°	+7.00−1.00×170°	−12.25−4.75×87°	−12.25−4.50×105°
IOP		normal		normal		18 mmHg	19 mmHg
Foveal hypoplasia		no		yes(PVEP abnormal)		yes
Nystagmus		fine vertical		yes		high frequency, low amplitude pendular
Iris present		yes	yes	yes	yes	yes	yes
Pyramidal cataract		yes	yes	yes	yes	yes	yes
Other observations and clinical notes		Performed cataract extraction and posterior chamber IOL. Vision after surgery was 20/125 OD and 20/200 OS		persistent pupillary membranes		inferior corneal pannus, persistent pupillary membranes, small optic nerves
PAX6 mutation	NucleotideTranscript variant 1, mRNA NCBI Reference Sequence: NM_000280.4	c.128C > Texon 5		c. 197T > Aexon 6		c. 781C > Gexon 10
	Protein	p.Ser43Phe (S43F)		p.Ile66Asn (I66N)		p.Arg261Gly (R261G)
	Structural location (refer to Fig. 4)	PAI - α2		linker 1 between PAI and RED sub-domains		HD - α9
	Impact on helical structure and DNA-binding stability	undisrupted		undisrupted		undisrupted

Abbreviations: BCVA, best-corrected visual acuity; HD, homeodomain DNA-binding domain; IOL, intraocular lens; IOP, intraocular pressure; NCBI, National Center for Biotechnology Information; PAI, PAI DNA-binding subdomain; PVEP, pattern visual evoked potential; RED, RED DNA-binding subdomain.

Fig. 1

Clinical examination and genetic testing of case 1. A: APyC through the undilated pupil with intact iris. B: APyC through the dilated pupil. C: Slit-lamp biomicroscopy clearly demonstrating the morphology of APyC with conical protrusion into the anterior chamber and some amount of posterior cortical opacification. No evidence of corneal opacity. D: Missense mutation, p.Ser43Phe (S43F; denoted by ˆ), is in the α2 helix of the DNA-binding PAI domain. The protein structure of human PAX6 is shown at the top. Amino acid sequence alignments from several vertebrate species in the region of the missense mutation is at the bottom. The numbers refer to amino acid positions for human PAX6. This shows that the serine which in this mutation has been replaced by a phenylalanine amino acid, is highly conserved across six species, including human. Abbreviations: APyC, Anterior Pyramidal Cataracts; PAI, first subunit of the PAIRED DNA-binding domain; RED, second subunit of the PAIRED DNA-binding subunit; HD; homeodomain DNA-binding domain; TAD, transactivation domain; Chk, chicken; Gor, gorilla; Hu, human; M, mouse; XL, Xenopus leavis (frog); Zf, zebrafish.

Genetic testing revealed a novel PAX6 missense mutation for this young patient. The proband was heterozygous for a c.128C > T mutation in exon 5 that changes the amino acid at codon 43 from serine to phenylalanine (p.Ser43Phe). (Fig. 1D). No other family members were affected and, therefore, genetic testing was not carried out on the parents.

Case 2

A 5 months old girl was presented with vision of 20/1200 both eyes open, with no fixation preference, and nystagmus. Her cycloplegic refraction was +7.50DS-2.00DC × 45° in the right and +7.00DS −1.00DC × 170° in the left eye (Table 1). Prior to dilation, the child had mid-dilated pupils revealing APyC in both eyes (Fig. 2A). On EUA, upon pupil dilation, the APyC became more apparent and she was noted to have persistent pupillary membrane (Fig. 2B). The iris was slightly hypoplastic. IOP was 13 mmHg in both eyes with applanation tonometry. Ultrasound biomicroscopy (UBM) of the right eye revealed a more accurate morphology of the cataracts, where a conical shape was observed arising from a break in the lens capsule (Fig. 2C). Fundoscopy revealed good optic nerve size in both eyes and the possibility of foveal hypoplasia. The electrophysiological evaluation using flash electroretinogram (ERG) showed normal retinal responses, but the pattern evoked pattern visual evoked potentials (PVEP) indicated macular pathway dysfunction confirming the foveal hypoplasia suspected on fundus examination.

Fig. 2

Clinical examination and genetic testing of case 2. A: APyC through the undilated pupil with intact iris. B: Dilated eye exam showing a persistent pupillary membrane. C: Ultrasound biomicroscopy demonstrating the morphology of the APyC. D. Missense mutation, p.Ile66Asn (I66N; denoted by ˆ), in the region after the α3 helix of the PAI domain. The isoleucine at this position, which in this mutation is replaced by asparagine, is highly conserved across 6 species, including human. Amino acid sequence alignments from several vertebrate species in the region of the missense mutation is at the bottom. The numbers refer to amino acid positions for human PAX6. Abbreviations: APyC, Anterior Pyramidal Cataracts; PAI, first subunit of the PAIRED DNA-binding domain; Chk, chicken; Gor, gorilla; Hu, human; M, mouse; XL, Xenopus leavis (frog); Zf, zebrafish.

Genetic testing revealed a novel missense PAX6 mutation. She was heterozygous for a c.197T > A mutation in exon 6 that changes the amino acid at codon 80 from isoleucine to asparagine (p.Ile66Asn) (Fig. 2D). In order to determine the pathogenicity of this novel amino acid substitution, 100 controls were tested for this sequence variation and shown to be negative suggesting that this could be the disease-causing mutation. The proband's parents and maternal uncle, who were all clinically unaffected, were negative for the mutation in blood-derived genomic DNA.

Case 3

A 17 year-old girl presented with bilateral APyC and nystagmus. Her BCVA was 20/80 with −12.25 DS/+4.75 DC in the right eye and 20/200 with −12.25 DS/+4.50 DC in the left eye. She exhibited high frequency, low amplitude pendular nystagmus (Table 1). On examination of the anterior segment, APyC, inferior corneal pannus and persistent pupillary membrane was noted in both eyes. The iris pattern was intact. Pneumotonometry was 18  mmHg in the right eye and 19 mmHg in the left eye. On fundus examination, she had small optic discs in both eyes suggestive of optic nerve hypoplasia. There was no history of consanguinity, but the family history was significant for cataract, nystagmus and iris coloboma in the father and paternal aunt. There was a history of nystagmus and coloboma in the paternal uncle. The maternal family history was negative for significant ocular disease. The girl's clinical features and family history led us to suspect a PAX6 mutation.

On genetic testing, she was found to be heterozygous for a novel variation in PAX6, namely c.781C > G (Fig. 3A) resulting in the amino acid substitution at position 261 of arginine for glycine, p.Arg261Gly (Fig. 3B). No other family members were affected and, therefore, genetic testing was not carried out on the parents.

Fig. 3

Genetic testing of case 3 A: Chromatogram of the patient showing the c.781C > G missense mutation in PAX6. B: Missense mutation, p.Arg261Gly (R261G; denoted by ˆ), in the α9 helix of the human PAX6 DNA-binding homeodomain (HD). Amino acid sequence alignments from several vertebrate species in the region of the missense mutation is at the bottom. The numbers refer to amino acid positions for human PAX6. Abbreviations: Chk, chicken; Gor, gorilla; Hu, human; M, mouse; XL, Xenopus leavis (frog); Zf, zebrafish.

Discussion

We report three unrelated patients who shared a common phenotype consisting of APyC, intact irides and nystagmus from novel heterozygous missense mutations in PAX6, specifically at S43F, I66N and R261G.

To understand why the three novel but different mutations caused the iris to remain intact, we must briefly understand: (1) the structure of the PAX6 protein, (2) type of mutations in this protein, (3) the location of previous missense mutations and their effect on iris formation, and (4) protein stability as a result of the mutation. Structurally, the PAX6 protein is composed of three domains - two DNA-binding domains and an activation domain. The two DNA-binding domains are the PAIRED domain and the homeodomain, where the former is split into 2 subunits, the PAI and RED subdomains. The most common types of mutations seen in PAX6 are deletions, frameshifts, splicing errors, and formation of nonsense codons, all of which result in premature translational termination (PTT) with loss of normal protein function and, in the vast majority, total or near total absence of development of the iris. The three novel mutations we describe do not result in PTT and, instead, are all missense mutations resulting in protein structure changes.

Correlation of a database of all reported PAX6 missense mutations to date and location of our mutations in the PAX6 DNA-binding sites with the phenotype seen (Fig. 4 and Table 2) reveals that some missense mutations in the DNA-binding site lead to drastic protein structure changes. Such structural events prevent correct binding to DNA and, therefore, no activation of the protein causing absence of iris. However, some missense mutations lead to subtle protein changes that do not affect binding to DNA and enable intact iris formation. Two of the three missense mutations in our patients (I66N in case 2 and R261G in case 3) are indeed in those sites where intact irides have been reported (Fig. 4), in addition to symptoms of typical aniridia (e.g. autosomal dominant keratitis and foveal hypoplasia only). However, one mutation (S43F) in case 1 was in a site where you would expect total aniridia. To explain why the iris was intact in case 1, we looked at protein stability through three independent molecular modeling simulations (Fig. 5). The results from these simulations demonstrate that iris formation is dependent not only on the region of mutation with respect to more/less sensitivity for iris formation (Fig. 4), but also to disruption of the DNA-binding specificity because of reduced protein stability that leads to significant reduction in protein function. While the S43F mutation (S1) is in a highly sensitive region for iris formation that would be expected to result in a no iris phenotype, its effect on secondary and tertiary protein structure is minimal resulting in a stable protein with decreased DNA-binding but no loss of DNA-binding specificity and, therefore, protein function (Fig. 5). We speculate this result explains the intact irides phenotype. Therefore, to have an intact iris certain regions of DNA-binding domain within the PAX6 protein must be intact and the DNA-binding protein domains must be stable.

Fig. 4

Schematic of PAX6 DNA-binding domains with all reported human missense mutations to date resulting in varying degrees of iris hypoplasia shown at their positions and the resulting iris phenotypes. The β-turn (purple designation comprising of 20 amino acids; ) consists of two β-turn motifs (β1, β2) at the N-terminus of the protein. The PAI subdomain secondary structure consists of three α-helices (α1-, α2-and α3-; blue rectangles, 40 amino acids). The RED subdomain secondary structure also consists of three α-helices (α4-, α5-and α6-; pink rectangles, 52 amino acids). Linker 1 connects the PAI subdomain to the RED subdomain () and linker 2 connects the RED-subdomain to the HD (). The HD is shown following the linker region after the RED subdomain and consists of 3 α-helices (α7-, α8-and α9; yellow rectangles, 57 amino acids). Amino acid residues are denoted by their standard letter abbreviations (i.e. L = leucine; see Key). Secondary structure of the α-helices is represented by residue positioning in the α-helix boxes (blue, pink, yellow rectangles; see Key). The complete amino acid residue chain for linker 2 () are not shown due to space limitations, but the iris hypoplasia phenotypes for the only missense mutations in this region are shown as residues Gln178 (Q178) and Arg208 (R208).

Data from Fig. 4 and Table 2 shows that two of our novel missense mutations (I66N (S2) and R261G (S3)) are in DNA-binding regions that are less critical to iris formation. This is because missense mutations in these regions lead more often to intact irides/intact irides with iris hypoplasia (brown circles/broken brown circles) than total aniridia (red circles). For example, linker 1 (where the S2 mutant lies) has 5 mutations resulting in intact irides, 3 to intact irides with iris hypoplasia and only 1 with total aniridia (see Table 2), therefore the intact irides phenotype seen for S2 would be expected in linker 1. However, one of our missense mutations (S43F (S1)) is in a critical region to iris formation (PAI:α2-helix), where a mutation would be expected to give a total aniridia phenotype. This is clearly observed in Table 2 where we observe six missense mutations in PAI:α2-helix that lead to total aniridia compared to 3 missense mutations leading to intact irides. An explanation for this unexpected phenotype is given in Fig. 5. Abbreviations: PAI, first subunit of the PAIRED DNA-binding domain; RED, second subunit of the PAIRED DNA-binding subunit; HD; homeodomain DNA-binding domain. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Table 2

Compendium of Mutations, expressed as amino acid substitutions, and associated phenotypes extracted from the PAX6 mutation database (http://lsdb.hgu.mrc.ac.uk/home.php? select_db = PAX6).

Mutation	Iris phenotype	Mutation	Iris phenotype	Mutation	Iris phenotype
β-turn		Gly51Arg	II	linker 2
Gly7Arg	TAN	Cys52Arg	TAN	Gln178His	TAN
Asn9Ile	IH	Val53Leu	II	Ala208Gln	TAN
Gly12Arg	TAN	Lys55Thr	TAN	Ala208Trp	PAN
Asn17Ser	TAN	Lys55Arg	IH	HD:α7-helix
Gly18Trp	PAN	Ile56Thr	IH	Arg214Gly	TAN
Arg19Pro	II	Thr63Pro	IH	Arg214Ser	TAN
PAI:α1-helix		linker 1		Ser216Pro	PAN
Arg26Pro	IH	Gly64Val	IH	HD:α8-helix
Ile29Ser	TAN	Ile66Asn	II	Arg242Thr	PAN
Ile29Val	TAN	Pro68Ser	II	HD:α9-helix
Leu32Val	TAN	Gly72Ser	IH	Val256Ala	IH
Ala33Pro	PAN	Gly73Asp	TAN	Phe258Ser	IH
Gly36Trp	TAN	Ser74Gly	II	Arg261Gly	II
Gly36Arg	IH	Pro76Arg	II
Gly36Ala	II	Val78Ala	II
PAI:α2-helix		Ala79Glu	IH
Arg38Trp	TAN	RED:α4-helix
Asp41Gly	PAN	Ile87Arg	TAN
Ile42Ser	PAN	Ile87Lys	TAN
Ser43Pro	TAN	Glu93Lys	TAN
Ser43Phe	II	RED:α5-helix
Arg44Gln	TAN	Ala99Pro	II
Leu46Arg	IH	Arg105Ser	IH
Leu46Pro	II	Pro118Arg	IH
Leu46Val	II	Pro118Gln	IH
Gln47Arg	TAN	Ser119Arg	TAN
Gln47Glu	TAN	RED:α6-helix
Val48Gln	TAN	Ser121Pro	PAN
Ser49Phe	IH	Ser121Leu	TAN
PAI:α3-helix		Val126Asp	PAN
Asn50Lys	II	Arg128Cys	II
Gly51Ala	II	Arg128Pro	IH

Abbreviations: TAN, total aniridia; PAN, partial aniridia; IH, intact irides with some iris hypoplasia; II, intact irides; PAI, PAI DNA-binding subdomain; RED, RED DNA-binding subdomain; HD, homeodomain DNA-binding domain.

Terms in bold refer to the structural regions of the PAX6 protein (see Fig. 4).

Fig. 5

Molecular model simulations to understand differences in phenotype between the intact irides-S43F mutant (S1) and the total aniridia-S43P mutant. Three results of molecular stability studies from three independent simulations are shown in this figure. All show that the mutant S43F (our mutation) is more stable than the aniridic S43P mutant, thereby generating complete intact irides in the former mutant and total aniridia in the latter mutant. A: Heat map of the percentage helicity (stability of structure) of each amino acid residue within the PAI domain in the wild-type (Wt, normal), S43F mutation (S1, intact irides) and S43P mutation (classic aniridia with no iris). Darker shading indicates higher percentage helicity for the amino acid residue. Therefore, by looking at this simulation, particularly the α2-helix chain, helicity is highest for the wild-type allele, followed by the S1 mutant, and then the S43P total aniridia mutant. B: Average and standard deviation of the Root Mean-Square Deviations (RMSD) for the alpha-carbon (in the carboxyl chain of an amino acid residue). This is a measure of each alpha-carbon distance for an amino acid residue between alignments of the PAI domain helical motif (from the crystal structure of PAX6) for PAI domain wild-type (blue), S43F mutation (S1, green) and S43P mutation (red) simulations. Cartoon of the secondary structure of the PAI domain of PAX6 is shown below the RMSD values. This demonstrates that S1 (green; intact irides) and Wt (blue) are aligned tightly, but the S43P (total aniridia) is not (red). C: Backbone RMSD of the helical motifs of the PAI domain (tertiary structure) in simulations of the Wt (blue), S43F mutation (S1, green) and S43P mutation (red). Backbone RMSD considers all the atoms over the protein chain backbone, not particular atoms in an amino acid (like alpha-carbon atom above in B). This shows disruption of the protein backbone in the S43P mutant resulting in total aniridia, but not in S1 (irides intact) or in the Wt (normal). Abbreviations: PAI, first subunit of the PAIRED DNA-binding domain. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

While APyC have been considered morphologically a subgroup of anterior polar cataracts (APoC), certain characteristics make considering APyC a separate entity a reasonable proposal. These characteristics include: asymmetry, association with surrounding cortical opacification necessitating removal, and extension anteriorly into the anterior chamber with corneal fusion and rarely detachment into the anterior chamber. In the largest series of 15 patients with APyC by Wheeler and colleagues, 9 were found to have bilateral APyC with only 2 patients having nystagmus with poor vision. Surprisingly they did not find irregular astigmatism in any of the patients. The authors also did not describe the iris details nor have they reported any genetic analysis related to these patients. While we are describing a specific genotype-phenotype correlation we feel that the phenotype may have been incompletely described in the previous two reports, but we cannot be sure.

A review of the literature suggests that previous causes of APyC have been considered to be acquired or congenital with acquired causes being varied, including corneal ulcer.16, 17, 18 Congenitally, the morphology of the APyC suggests that they could arise from remnants of prior attachment to the presumptive cornea during lens vesicle separation from the surface ectoderm in the fifth week of embryonic development. This could possibly explain the irregular astigmatism seen in our patients. Furthermore, even though refractive errors in aniridia do vary, anterior congenital cataracts are usually associated with variable astigmatism.

While the association of congenital cataracts with nystagmus has been previously reported, they were not noted to be APyC and no genetic analysis was done. In a study by Hingorani et al., in a cohort of 43 patients with identified PAX6 mutations, only 3 patients with identified PAX6 mutations were found to have intact iris, cataract, irregular astigmatism and nystagmus (2 with foveal hypoplasia). It is possible that these three cases represent a similar phenotype seen in our study, though no lenticular phenotype was mentioned.

If pyramidal cataracts arise from abnormal detachment of the lens vesicle to the overlying presumptive cornea, then our results suggest that PAX6 may have a role in lens vesicle detachment. This has been documented before in cases of Peters' anomaly. In mouse models, genes downstream from Pax6 have been identified that have a specific role in lens vesicle separation defect when mutated.23, 24, 25, 26, 27, 28, 29 We speculate that Pax6 has a role in the expression of the above genes and the missense mutations reported in this study mildly affect their expression leading to APyC.

Conclusions

We define a novel phenotype with bilateral anterior pyramidal cataract, intact irides, and nystagmus due to missense mutations in PAX6. To the best of our knowledge this precise genotype-phenotype has not been recognized as one seen in PAX6 mutations previously, and we show for the first time a molecular explanation at the protein level as to why iris may remain intact despite mutations or deletions in PAX6.

Patient consent

The Institutional Review Board approval was waived as this study was considered a retrospective case series.

Genetic testing was performed after the informed consent from the parents. The patients provided oral consent for publication of this case series.

Funding

This work is supported by the National Institutes of Health CORE Grant P30 EY008098, Eye and Ear Foundation of Pittsburgh, Pennsylvania, USA and an unrestricted grant from Research to Prevent Blindness, New York, New York, USA.

Authorship

All authors attest that they meet the current ICMJE criteria for authorship.

Conflicts of interest

No financial disclosures.