Abstract
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.
Keywords: Pyramidal cataracts, Intact irides, Nystagmus, Novel PAX6 missense mutations
1. 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.1 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,5 Peters' anomaly,6,7 autosomal dominant keratitis,8 microphthalmia,9, 10, 11, 12 and certain optic nerve defects.13 PAX6 homozygous mutations are extremely rare, as they generate debilitating central nervous system defects that lead to post-natal loss of life.14 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.
2. 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.
2.1. 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.
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 | Nucleotide Transcript variant 1, mRNA NCBI Reference Sequence: NM_000280.4 |
c.128C > T exon 5 |
c. 197T > A exon 6 |
c. 781C > G exon 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.
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.
2.2. 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.
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.
2.3. 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.
3. 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.15 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.
Table 2.
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).
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.16 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.19 Furthermore, even though refractive errors in aniridia do vary, anterior congenital cataracts are usually associated with variable astigmatism.20
While the association of congenital cataracts with nystagmus has been previously reported,21 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).22 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.6 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.
4. 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.
5. 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.
Acknowledgements and disclosures
None.
Footnotes
Supplementary data related to this article can be found at https://doi.org/10.1016/j.ajoc.2018.02.021.
Appendix A. Supplementary data
The following is the supplementary data related to this article:
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