Detailed Ophthalmologic Evaluation of 43 Individuals with PAX6 Mutations

Abstract

purpose. Heterozygous mutations of the PAX6 gene cause a variety of ocular malformations, the best known being aniridia (absence of the iris). Mutation analyses and detailed clinical evaluations were performed in 43 individuals with aniridia or closely related ocular anomalies, to investigate whether phenotype correlates with mutation type.

methods. Case notes and medical records were reviewed and patients were reexamined when necessary. Denaturing high-performance liquid chromatography (DHPLC) analysis and sequencing of the PAX6 coding region was performed in individuals whose mutation was unknown.

results. The most common PAX6 mutations identified were premature termination mutations, amino acid substitutions, and C-terminal extensions. Six novel mutations are reported. Mutations that inactivate one copy of the gene typically caused a severe phenotype including foveal hypoplasia, marked iris anomalies, and severe visual impairment. Missense mutations, all affecting invariant amino acids in the paired domain, caused milder phenotypes in this cohort, with a lower incidence of foveal hypoplasia and less severe visual loss. C-terminal extension mutations caused relatively severe anomalies and marked reduction in vision. Two C-terminal extension cases had a unilateral exudative retinopathy, resembling Coats’ disease, which has not previously been reported in association with PAX6 mutation.

conclusions. PAX6 mutations cause panocular malformations that vary considerably in pattern and severity. In our cohort, iris hypoplasia, nystagmus, and foveal hypoplasia were most common, with cataracts, corneal anomalies, and high refractive errors also frequently observed. In this cohort, loss-of-function and C-terminal extension mutations were found to cause more severe phenotypes than missense mutations.

The PAX6 gene encodes a key regulator of ocular and neural development. ^{1 2} The PAX6 protein is highly conserved in a wide variety of species and contains two DNA binding motifs, a paired domain and a homeodomain, and a C-terminal transcriptional activation domain. During eye development PAX6 controls the expression of other regulators, including PAX2, SOX2, and a series of retinal helix-loop-helix transcription factors, as well as structural proteins, such as the crystallins. ^{2 3 4 5}  

In humans, PAX6 mutations are associated with a variety of congenital ocular anomalies. The classic phenotype is aniridia (absence of the iris), a dominantly inherited condition caused by mutations in one copy of the PAX6 gene. ¹ Consistent with the expression pattern of PAX6 in the embryonic eye and brain, aniridia is often accompanied by anomalies of the cornea, lens, retina, optic nerve, pineal gland, and anterior commissure. ^{6 7 8}  

Understanding of the phenotypic consequences of different types of PAX6 mutation has improved through increased knowledge of the molecular mechanisms associated with different kinds of mutant allele. ^{9 10 11 12 13 14} The first mutations to be associated with aniridia were whole gene deletions, leading to the hypothesis that aniridia is caused by inactivation of one allele of PAX6. ¹⁵ It soon became clear that aniridia is also caused by mutations that result in the introduction of a premature termination codon (PTC) into the PAX6 coding region (i.e., nonsense mutations, frame-shift insertions and deletions, and most splice mutations). A recent survey found that more than 99% of all PTC mutations cause classic aniridia. ¹¹ The similarity between the phenotypes associated with whole gene deletions and PTC mutations strongly suggest that the latter create null alleles. Until recently, it was believed that PTC mutations result in truncated proteins, some of which may have partial function or dominant negative activity, and it was therefore unclear how PTC mutations in different parts of the coding region could be functionally equivalent. ^{16 17} It is now known that mRNAs containing a PTC are generally detected and degraded by the nonsense mediated decay (NMD) pathway, rendering most PTC-containing mRNAs from multiexon genes nonfunctional and preventing the accumulation of truncated proteins in the cell. ¹³  

Missense mutations, which cause one amino acid to be replaced by another, are associated with a variety of phenotypes including aniridia, corectopia, isolated foveal hypoplasia, Peters’ anomaly, microphthalmia, and optic nerve defects. ^{11 18 19 20 21 22} There is evidence from Pax6 mutant mice that missense alleles are indeed translated to give full-length proteins. ^{9 10 14} Depending on the location and nature of the amino acid change, the function of the mutant protein may be partially reduced, abolished, or enhanced. ^{10 14 21 23}  

C-terminal extension (CTE) mutations cause the open reading frame to continue into the 3′ untranslated region, where a termination codon is eventually encountered. NMD of the mutant RNA is not predicted, because the NMD surveillance zone ends approximately 50 bp before the penultimate splice junction. ¹³ As previously discussed, ¹¹ no late-truncating mutations have been observed for PAX6, but examples of extension beyond the usual stop codon have been reported. The properties of such CTE mutant proteins are largely unknown. 

We present a detailed ophthalmic evaluation of 43 individuals with a diagnosed aniridia-like phenotype. PAX6 mutations have been identified for all cases and classified according to type: probable null alleles (gross deletions and PTC mutations), missense mutations, CTE mutations, and mutations with other consequences. 

Methods

Patients

The PAX6 mutation studies were performed on DNA samples provided from a single center (Moorfields Eye Hospital) over a significant period (16 years). All patients gave informed consent, and the study protocol was in keeping with the tenets of the Declaration of Helsinki. A retrospective review of hospital clinical records was performed and where there was incomplete clinical data, patients were re-examined. All patients were examined by ATM and MH. A full eye examination was performed with careful documentation of the morphology of anterior segment changes (including degree of iris absence and subtle changes in iris anatomy and structure), dilated funduscopy, refraction, and orthoptic examination. The phenotype of each individual was given a severity grading based on a combination of the extent of ocular anatomic changes, the occurrence of complications, and the degree of visual impairment. 

Molecular Analysis

The PAX6 mutation had been determined in 24 of the 43 individuals involved in the study. For the remaining individuals, genomic DNA was extracted from blood as previously described, ²⁴ and the coding exons of the PAX6 gene (4 to 13 and 5a) were individually amplified by PCR. Mutation screening was performed on the PCR products by DHPLC ²⁵ (Transgenomic Ltd., Cramlington, UK) followed by direct sequencing of shifted fragments. All sequences were bidirectional and confirmed by repeat PCR. Where individuals (usually children) were not available for screening, the mutation was inferred from the affected parent. In Tables 1 1 2 3 4the location of each mutation is reported in two formats: the new nomenclature system recommended by the Human Genome Variation Society ²⁶ and the traditional nomenclature using the original PAX6 cDNA numbering. ²⁷ Reference sequences for the PAX6 cDNA and protein are contained in GenBank entry NM_000280.3 (http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). In the reference cDNA sequence, the A of the initiating methionine codon is base 534. This A corresponds to base 1 in the new nomenclature system and base 363 in the traditional system. A database of known PAX6 mutations is maintained at http://pax6.hgu.mrc.ac.uk/ (using the Leiden Open Variation Database [LOVD] format, provided in the public domain by the Leiden University Medical Center, Leiden, The Netherlands). 

Results

Forty three individuals were evaluated in this study, 26 females and 17 males from 22 families (Tables 1 1 2 3 4) . Their ages ranged from <1 year to 57 years. There were 15 children (mean age 8.5 years) and 28 adults (mean age 37 years). Seven cases were sporadic, with no affected relatives; the remaining 36 were familial. Thirty-four cases were examined prospectively for this report; the remaining nine cases are described from detailed archival information, maintained as structured notes at each visit. Most of the patients described are under long-term follow-up by the Moorfields team. 

The PAX6 mutation was identified in each individual (Tables 1 1 2 3 4) . There were 24 independent mutations in total. Six mutations were novel, including three missense changes in the paired domain. For 12 of the mutations, two or more family members were examined, allowing the consequence of the same mutation to be assessed in different individuals. 

Every individual in our cohort had iris anomalies, although these varied considerably in severity from complete iris absence to relatively subtle architectural anomalies. 

The majority of patients either had a full iris with structural anomalies, or no iris, as observed on slit lamp inspection, although some of these cases may have had a small stump of iris tissue on gonioscopy. In the few cases described as having a partial iris, a significant and somewhat variable amount of tissue was evident on slit lamp examination. Most individuals studied had good concordance of iris phenotype between the two eyes; variability between fellow eyes was mostly observed in the small partial iris category. Details are described in Tables 1 1 2 3 4 . 

Nystagmus and foveal hypoplasia were extremely common, being present in 41 of 43 and 37 of 43 individuals, respectively. Thirty patients had cataracts, 16 had corneal abnormalities, 10 had optic nerve hypoplasia, 5 had glaucoma, and 4 had bilateral ptosis. The corneal abnormalities typically occurred in adult life and manifested as corneal epithelial instability, stromal opacity and vascularization occurring as a result of limbal stem cell deficiency. Nearly half the cohort (21/43) had a significant refractive error, with moderate to high myopia present in five. A novel finding was a unilateral exudative retinopathy, resembling Coats’ disease, in two patients. Progressive features, such as cataracts, glaucoma and keratopathy may be underrepresented as some cases are infants or young children who may develop further complications later on. 

Loss-of-Function Mutations

Eighteen individuals had mutations that are predicted to cause loss of function of one copy of PAX6 (Table 1) 1 . One case had a whole gene deletion as described previously. ⁷ The other 17 had mutations predicted to introduce a PTC, leading to nonsense-mediated decay of the mutant mRNA and failure of translation. These included two novel frame-shifting deletions, c.1035_1048del14 and c.1061_1070del10. 

Every single individual in this category had foveal hypoplasia and nystagmus. The iris anomalies were relatively severe with complete iris absence in 13 cases, partial iris absence in 1 case, and full irides with abnormal architecture in 4 cases. Cataract and corneal changes were very common. There was a relatively high incidence of glaucoma and dysplasia or developmental anomaly of the optic nerve. Visual acuities in the better eye ranged from perception of light to 20/80 with a median of 20/400. Phenotypes in 10 individuals were classified as severe and only two cases were considered mildly affected. 

Missense Mutations

Nine individuals had missense mutations (Table 2) , sharing four different mutations between them: p.Gly64Val has been described previously ²⁰ but the other mutations (p.Gly36Arg, p.Lys55Arg, and p.Gly72Ser) are novel. All are predicted to result in an amino acid substitution in the paired domain. The affected residues (Gly36, Lys55, Gly64, and Gly72) are absolutely invariant in all PAX6 paired domains sequenced to date, including those from invertebrates such a Drosophila and squid. In the crystal structure of the PAX6 paired domain, Lys55 and Gly72 both make direct contact with the DNA target sequence. ²⁹  

This group contains the only two individuals in the entire cohort without nystagmus (mother 2/1A and daughter 2/1B, Gly36Arg). Despite this similarity, they had different iris phenotypes: 2/1B had partial iris absence, whereas 2/1A, in common with all other members of the group, had full irides with abnormal architecture (Fig. 1a 1b 1c 1d) . Only three missense individuals had foveal hypoplasia. Preserved foveal morphology in case 2/2B (Lys55Arg) is illustrated in Figures 1e and 1f . Cataracts were common in this group (6/9) but only one individual had corneal abnormalities. 

The phenotypes observed in missense mutation carriers clearly overlap those associated with loss of function mutations, but they were significantly milder. Five of the nine missense individuals were mildly or very mildly affected, and none was severely affected. The low incidence of foveal hypoplasia was associated with good preservation of visual function; visual acuities in the better eye ranged from 20/30 to 20/250 with a median of 20/60. In addition, there were no severe optic nerve malformations and no glaucoma, both of which can have highly deleterious effects on vision. 

C-Terminal Extension Mutations

Ten individuals had mutations predicted to result in a protein with a C-terminal extension (Table 3) . One patient (3/1) had a late frame-shifting deletion of nucleotide T1239 that is predicted to replace the last 10 amino acids of the PAX6 protein with a peptide of 45 amino acids. The other individuals all had a duplication of nucleotide T1267 (c.1267dupT) which converts the stop codon (TAA) to a leucine codon (TTA). This mutation is predicted to result in the addition of 107 residues to the end of the PAX6 protein, increasing its length by 25% from 422 amino acids to 529. To date, c.1267dupT has been independently observed 19 times and is one of the most common mutations in the PAX6 Allelic Variant Database (http://pax6.hgu.mrc.ac.uk/). Nucleotide T1267 immediately precedes a stretch of 23 A’s that may increase the chance of mispairing and therefore of errors during DNA replication. 

Exudative vascular retinopathy resembling that observed in Coats’ disease ³⁰ was observed in two CTE cases, 3/2C with the 1267dupT mutation and 3/1 with the 1239delT mutation (Figs. 1g 1h) . Exudative vascular retinopathy has not, to our knowledge, been described previously in association with a PAX6 mutation. Retinal vascular changes were absent in other CTE cases, including the two brothers of 3/2C. 

The severity of the anomalies in the CTE group is comparable to that seen with the loss-of-function mutations (Table 1) 1but the iris anomalies were generally milder. Foveal hypoplasia (10/10), keratopathy (6/10), cataracts (7/10) and myopia (5/10) were all common in this group, but glaucoma was unusual. Visual acuities in the better eye ranged from 20/80 to counting fingers, with a median of 6/200. An example of severe bilateral keratopathy associated with the c.1629dupT mutation is shown in Figures 1i 1j . 

Other Mutations

Six individuals had PAX6 sequence changes that do not fit into the other categories or are of uncertain consequence (Table 4) . These include four patients with a mutation in the splice donor of intron 6. Patient 4/1 had the mutation c.357+1G>A (IVS6+1G>A) and 4/2 had c.357+2insT (IVS6+2insT), both of which lead to utilization of a cryptic splice donor within exon 6 leading to an in-frame deletion of 36 amino acids from the paired domain, as previously described. ³¹ The deletion encompasses two of the three α helices in the C-terminal subdomain and would be predicted to impair DNA binding substantially. ²⁹ Cryptic splice donor activation was confirmed by RT-PCR for individuals 4/2 ³¹ and 4/4 ⁷ but the intron 6 splice donor was normal in individual 4/4, and so the underlying genomic mutation, which may lie deep within intron 6, remains to be found. 

Two patients (4/3A and 4/3B) had the novel intron 6 splice donor mutation c.357+5G>A (IVS6+5G>A). Unlike the first and second positions of the splice donor sequence, the fifth is not invariant and the consequence of this change is difficult to predict in the absence of RNA analysis. 

The mutation c.1044+2T>A (IVS8+2T>A) in case 4/5 affects the invariant T at position 2 of the intron 8 splice donor, making it highly probable that splicing is disrupted, but the effect on RNA and protein structure is unknown. Consistent with the idea that this is a causative mutation, the same change has been reported in a family with a similar phenotype of cataracts, foveal hypoplasia, and iris defects. ³²  

The patients in this group were all moderately affected, but it is difficult to correlate genotype with phenotype, because the molecular outcome of three of the mutations (4/3A, 4/3B, and 4/5) is unclear. All patients in this group had foveal hypoplasia and three had complete absence of the iris. Corneal changes and optic nerve defects were rare; there were no cases of glaucoma, although two cases had ocular hypertension. One individual 4/3A had a rhegmatogenous retinal detachment. Visual acuities in the better eye ranged from 28/80 to 20/400 with a median of 20/200. 

Additional Phenotype Data

The variability of iris and corneal phenotypes and also the spectrum of fundus changes in some of the cases described in Tables 1123are shown in Supplementary Figures S1 and S2. 

Discussion

We set out to determine whether there was a correlation between the PAX6 mutation type and the severity and pattern of ocular malformation in a large panel of individuals with aniridia-like phenotypes. Based on our current understanding of the effect of different kinds of mutation at the RNA and protein level, we were able to categorize 37 of the 43 mutant alleles in our cohort into three main classes: predicted loss of protein expression, amino acid substitution, and predicted C-terminal extension. Within each mutation class there was phenotypic variability; however, individuals with loss-of-function mutations (Table 1) 1and CTE mutations (Table 3)were generally the most severely affected, whereas individuals with missense mutations (Table 2)had the mildest phenotypes. The main ocular features associated with PAX6 mutations in our cohort were iris anomalies (100%), nystagmus (95%), foveal hypoplasia (84%), cataracts (68%) and ametropia (48%). Missense mutations were associated with better preserved iris structure and a lower incidence of foveal hypoplasia (33%) and nystagmus (78%). In accordance with the emerging pattern, all intragenic PAX6 mutations recently reported from a certified service laboratory, where it may be expected that only classic aniridia cases are sent, were PTC, CTE or splice site changes. ³³ It should also be noted that our mutation detection methods did not routinely include deletion analysis, so that this molecularly defined cohort is deficient in the relatively frequent submicroscopic deletion changes that have been observed among isolated aniridia cases in the broader service laboratory study. ³³  

Of five subjects described as having ptosis, two (1/6C, 3/1) appeared to have reactive ptosis in response to keratopathy. The other three (1/5A, 1/5B, 4/2) had no reported corneal surface changes and appeared to have true ptosis. Subject 1/5B also had blepharophimosis. The etiology of ptosis and phimosis in terms of the known PAX6 expression pattern is currently unclear. Fourteen cases of ptosis have been reported in the PAX6 mutation database in 480 entries. 

Two patients, both with CTE mutations, had a unilateral exudative retinopathy, which to our knowledge has not been previously described in association with PAX6 mutation. It is uncertain how frequently this occurs in aniridic individuals and whether it is confined to those with CTE mutations. If the fundal view is compromised by nystagmus, corneal changes, and cataracts, subtle retinovascular changes may be difficult to assess and may be underreported. 

Retinal vascularization initially occurs along an astrocyte framework derived from PAX2-expressing cells in the developing optic nerve. ^{34 35} Since PAX6 represses PAX2, ² it is possible that PAX6 CTE proteins have a specific effect on PAX2 expression, causing abnormalities of the astrocyte network and consequently of the retinal vasculature. Furthermore, PAX6 suppresses corneal vascularization by promoting the expression of a soluble receptor for vascular endothelial growth factor (VEGF) which neutralizes the angiogenic activity of VEGF itself. ³⁶ Retinal astrocytes strongly express VEGF, ³⁴ and although it is not yet known whether PAX6 controls retinal VEGF receptors, this could be another mechanism by which specific PAX6 mutations lead to abnormal retinal angiogenesis. 

To assess the phenotypic variability associated with a particular mutation, we examined more than one affected family member when possible. Some aspects of the phenotype were strikingly similar, such as the absence of nystagmus in mother and daughter 2/1A and 2/1B (Gly36Arg, Table 2 ) and the consistently severe effect on visual function caused by the c.763C>T and c.775dupT mutations (Table 1) 1 . Other anomalies were highly variable within families, such as the variable iris hypoplasia in 2/1A and 2/1B and the Coats’-like retinopathy in 3/2C but not in her brothers. During eye development, PAX6 interacts with many other genes and proteins which may themselves have functional variants that differ from person to person. ^{2 3 4 5} Although there may be chance fluctuations in PAX6 levels in the tissues that affect the phenotypic outcome, it is notable that the iris phenotypes are very similar in the two eyes of a given individual. 

It seems clear from this study and the existing literature, ¹¹ however, that both null mutations (gene deletions and PTCs) and CTE mutations tend to generate relatively severe phenotypes. Presumably, this occurs through different mechanisms because gene deletions and PTC mutations are predicted to generate no protein, whereas CTE mutations are predicted to generate proteins with an abnormally elongated C terminus. At present, the effect on protein function and turnover, if any, of these C-terminal mutations is unknown. In our cohort the CTE group demonstrated qualitatively different phenotypes compared with the null group, supporting the hypothesis that CTE alleles impair ocular development by a different mechanism from null alleles. 

Missense phenotypes are particularly difficult to predict because the outcome depends on the activity of the mutant protein, and this will vary from mutation to mutation depending on the location and function of the substituted residue. All the missense changes in our cohort affected invariant amino acids in the paired domain. These residues, which have been conserved through hundreds of millions of years of evolutionary time, are likely to be absolutely essential for normal PAX6 function. ^{20 29} Reduced DNA binding and altered transcriptional activation function has been demonstrated for paired domain missense mutant proteins. ^{21 23} The two individuals with the Gly36Arg change had neither foveal hypoplasia nor nystagmus and were particularly mildly affected, suggesting that the function of this mutant protein is only moderately impaired. Although the missense individuals in this study were relatively mildly affected, missense mutations can cause visually debilitating conditions like Peters anomaly and optic nerve defects, ^{18 21} and the function of the mutant protein may be more severely compromised in these cases. 

In this article we have concentrated solely on ophthalmic phenotypes, but it is important to remember that PAX6 is also expressed outside the eye. Many of the individuals in this study have also been evaluated for changes in olfaction and brain structure and function (as referenced in Tables 1 1 2 3 4 ), thus building up a comprehensive picture of the effects of different PAX6 mutations. ^{7 8 20 28} Careful clinical evaluation continues to reveal novel consequences of PAX6 alteration and to extend the range of phenotypes with which PAX6 is associated. 

²  Present affiliation: Hinchingbrooke Hospital, Huntingdon, United Kingdom.

 Supported by funding from the Medical Research Council and from the National Institute for Health Research UK (Moorfields Eye Hospital Biomedical Research Centre).

 Submitted for publication September 3, 2008; revised December 2, 2008; accepted March 30, 2009.

 Disclosure: M. Hingorani, None; K.A. Williamson, None; A.T. Moore, None; V. van Heyningen, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Summary of Phenotypes Associated with Loss-of-Function Mutations

ID	Sex/Age	Inheritance	Mutation*	Exon (Domain)	Protein Change	Mutation Type	Iris Anomaly
1/1	F/41	S	Gross deletion of one copy of PAX6	n/a	n/a	Gross deletion	No iris
1/2A	F/32	F, sister of 1/2B	115_116dupCC [c.477_478dupCC]	Ex5 (PD)	p.Cys40ArgfsX15	Frameshift, PTC in Ex6	No iris
1/2B	F/37	F, sister of 1/2A	115_116dupCC [c.477_478dupCC]	Ex5 (PD)	p.Cys40ArgfsX15	Frameshift, PTC in Ex6	Full iris, abnormal structure
1/2C	M/12	F, son of 1/2B	115_116dupCC [c.477_478dupCC] Inferred	Ex 5 (PD)	p.Cys40ArgfsX15	Frameshift, PTC in Ex6	Full iris, abnormal structure
1/3	F/child	S	c.607C>T [c.969C>T]	Ex8 (LNK)	p.Arg203X	Nonsense, immediate PTC	No iris
1/4A	F/34	F, mother of 1/4B and 1/4C; wife of 3/2B	c.718C>T [c.1080C>T]	Ex9 (HD)	p.Arg240X	Nonsense, immediate PTC	No iris
1/4B	M/12	F, son of 1/4A and 3/2B	c.718C>T [c.1080C>T]	Ex9 (HD)	p.Arg240X	Nonsense, immediate PTC	No iris
1/4C	F/7	F, daughter of 1/4A and 3/2B	c.718C>T [c.1080C>T]	Ex9 (HD)	p.Arg240X	Nonsense, immediate PTC	No iris
1/5A	M/55	F, father of 1/5B and 1/5C	c.763C>T [c.1125C>T]	Ex9 (HD)	p.Gln255X	Nonsense, immediate PTC	No iris
1/5B	M/34	F, son of 1/5A	c.763C>T [c.1125C>T]	Ex9 (HD)	p.Gln255X	Nonsense, immediate PTC	No iris
1/5C	F/32	F, daughter of 1/5A	c.763C>T [c.1125C>T]	Ex9 (HD)	p.Gln255X	Nonsense, immediate PTC	No iris
1/6A	F/57	F, mother of 1/6B and 1/6C	c.775dupT [c.1137dupT]	Ex10 (HD)	p.Ser259PhefsX2	Frameshift; PTC in Ex10	No iris
1/6B	M/27	F, son of 1/6A	c.775dupT [c.1137dupT]	Ex10 (HD)	p.Ser259PhefsX2	Frameshift; PTC in ex10	No iris
1/6C	M/24	F, son of 1/6A	c.775dupT [c.1137dupT]	Ex10 (HD)	p.Ser259PhefsX2	Frameshift; PTC in Ex10	No iris
1/6D	F/2	F, daughter of 1/6C	c.775dupT [c.1137dupT] Inferred	Ex10 (HD)	p.Ser259PhefsX2	Frameshift; PTC in Ex10	No iris
1/7	F/<1	S	c.949C>T [c.1131C>T]	Ex11 (PST)	p.Arg317X	Nonsense, immediate PTC	Thin rim of iris, ∼20%
1/8	M/19	F	c.1035_1048del14 [c.1397_1410del14]	Ex12 (PST)	p.Pro346AspfsX20	Frameshift; PTC in Ex12	Full iris abnormal structure
1/9	M/20	S	c.1061_1070del10 [c.1423_1432del10]	Ex12 (PST)	p.Tyr354CysfsX8	Frameshift; PTC in Ex12	Full iris abnormal structure

 S, sporadic; F, familial; Ex, exon; PD, paired domain; LNK, linker region; HD, homeodomain; PST, proline-threonine-serine-rich domain; PTC, premature termination codon; CF, counting fingers; PL, perception of light; HM, hand movements.

*  Mutations are numbered according to the Human Genome Variation Society system (no brackets) and the traditional PAX6 system (square brackets). Inferred mutations were deduced from an affected relative.

Table 1A.

 

Summary of Phenotypes Associated with Loss-of-Function Mutations

Foveal Hypoplasia	Other Features	Refraction	Binocular Vision (Corrected)	Severity	Reference
Yes	Severe keratopathy, cataracts, glaucoma, nystagmus	−3.5 OD; −3.0 OS	CF	Severe	7 8
Yes	Cataracts, nystagmus	+1.5/−1.5×25 OD; +13.0/−1.0 OS (aphakia)	20/200	Moderate	7 8 28
Yes	Nystagmus	+0.25/−0.5×90 OD; +0.75 OS	20/80	Mild	7 8
Yes	Moderate hypermetropia, nystagmus	+6.25/−1.0×180 OD; +6.0/−1.0×180 OS	20/120	Mild	28
Yes	Mild keratopathy, cataracts, glaucoma, nystagmus	ND	20/200	Severe	Current
Yes	Severe keratopathy, cataracts, severe glaucoma, nystagmus	+16.0 OU (aphakia)	PL	Severe	7 8 28
Yes	Mild keratopathy, cataracts, nystagmus, epilepsy	ND	20/600	Moderate-severe	28
Yes	Cataracts, nystagmus, epilepsy	ND	20/300	Moderate	28
Yes	Ptosis, cataracts, nystagmus	0/−1.5×5 OD; −1.5/−1.5×10 OS	20/120	Severe	7 8
Yes	Blepharophimosis with ptosis, cataracts, ocular hypertension, nystagmus	+1.5 OD; +1.0 OS	20/120	Severe	7 8
Yes	Cataracts, nystagmus	+2.5/−3.0×180 OD; +2.0 OS	HM	Severe	7 8
Yes	Severe keratopathy, cataracts, glaucoma, mild optic nerve hypoplasia, moderate myopia, nystagmus	−3.0/−3.5×180 OD; −4.5/−3.5×180 OS	20/120	Severe	7 8
Yes	Keratopathy, cataracts, mild optic nerve hypoplasia, moderate myopia, nystagmus	−2.0/−2.0×20 OD; −3.5/−2.0×40 OS	20/400	Severe	7 8
Yes	Mild ptosis, keratopathy, cataracts, mild optic nerve hypoplasia, moderate myopia, nystagmus	−5.5 OD; −3.5 OS	20/400	Severe	7 8
Yes	Mild optic nerve hypoplasia, nystagmus	0 OU	20/400	Severe	Current
Yes	Cataracts, hypermetropia, nystagmus	+6.0 OU	?(baby)	Moderate	Current
Yes	Cataracts, high myopia, nystagmus	−9.75/−1.5×175 OD; −10.25/−1.5×20 OS	20/200	Moderate	Current
Yes	Minor keratopathy, mild optic nerve hypoplasia, myopia, nystagmus	−5.5/−1.5×180 OD; −4.5/−1.0×15 OS	20/120	Moderate	Current

Table 2.

 

Summary of Phenotypes Associated with Missense Mutations

ID	Sex/Age	Inheritance	Mutation	Exon (Domain)	Codon Change	Protein Change	Iris Anomaly	Foveal Hypoplasia	Other Features	Refraction	Binocular Vision (Corrected)	Severity	Reference
2/1A	F/53	F, mother of 2/1B	c.106G>A [c.468G>A]	Ex5 (PD)	GGG>AGG	p.Gly36Arg	Full iris, abnormal structure	No	Cataracts	−1.0/−0.25×5 OD; +2.0/−1.75×165 OS	20/30	Very mild	28
2/1B	F/16	F, daughter of 2/1A	c.106G>A [c.468G>A]	Ex5 (PD)	GGG>AGG	p.Gly36Arg	Thin rim of iris, ∼20%	No	Mild cataracts	+1.5/−1.5×145 OD; +2.0/−1.25×20 OS	20/60	Mild	28
2/2A	M/48	F, father of 2/2B and 2/2C	c.164A>G [c.526A>G]	Ex6 (PD)	AAA>AGA	p.Lys55Arg	Full iris, abnormal structure	No	Cataracts, nystagmus	+3.5/−1.5×105 OD; +1.5/−1.5×35 OS	20/80	Mild	28
2/2B	M/13	F, son of 2/2A	c.164A>G [c.526A>G] Inferred	Ex6 (PD)	AAA>AGA	p.Lys55Arg	Full iris, abnormal structure	No	Blonde fundus, nystagmus	−2.5/−1.25×5 OD; +2.0/−2.0×180 OS	20/60	Mild	28
2/2C	F/11	F, daughter of 2/2A	c.164A>G [c.526A>G] Inferred	Ex6 (PD)	AAA>AGA	p.Lys55Arg	Full iris, abnormal structure	No	Blonde fundus, nystagmus	−2.0/−2.0×125 OD; −2.25/−3.25×25 OS	20/60	Mild	28
2/3A	M/40	F, brother of 2/3B	c.191G>T [c.553G>T]	Ex6 (PD)	GGC>GTC	p.Gly64Val	Full iris, abnormal structure	No	Cataracts, ocular hypertension, nystagmus	ND	20/40	Mild-moderate	20
2/3B	F/30	F, sister of 2/3A	c.191G>T [c.553G>T]	Ex6 (PD)	GGC>GTC	p.Gly64Val	Full iris, abnormal structure	Yes	Cataracts, keratopathy, mild optic nerve hypoplasia, nystagmus	ND	20/60	Moderate	8 20
2/4A	F/adult	F, mother of 2/4B	c.214G>A [c.576G>A]	Ex6 (PD)	GGT>AGT	p.Gly72Ser	Full iris, abnormal structure	Yes	Cataracts, high myopia, nystagmus	−6.0/−2.0×170 OD; −5.5/−2.0×20 OS	20/200	Moderate	Current
2/4B	F/6	F, daughter of 2/4A	c.214G>A [c.576G>A]	Ex6 (PD)	GGT>AGT	p.Gly72Ser	Full iris, abnormal structure	Yes	Nystagmus	−3.25/+2.0×80 OD; −3.75/+0.75×90 OS	20/250	Moderate	Current

 See Table 1 1for abbreviations and explanation of mutation nomenclature.

Figure 1.

 

(a–d) Ocular anomalies in a mother and daughter with the PAX6 missense mutation c.106G>A (Gly36Arg). Full iris with abnormal architecture in the left (a) and right (b) eyes of patient 2/1A. Partial iris loss (c) and normal fovea (d) in the left eye of her daughter 2/1B. (e, f) Preserved foveal morphology in patient 2/2B with the missense mutation c.164A>G (Lys55Arg). (e) Retinal OCT, showing a normal foveal pit (arrow). (f) Autofluorescence image showing normal macular region. (g, h) Phenotype in case 3/1 with the C-terminal extension mutation c.1239delT: (g) partial iris defect, opaque lens capsule remnants, and surgical aphakia; and (h) exudative retinal detachment. (i, j) Severe keratopathy due to limbal stem cell failure in both eyes of case 3/4A with the C-terminal extension mutation c.1267dupT. Note that the iris is completely formed (the pupil has been dilated pharmacologically).

Table 3.

 

Summary of Phenotypes Associated with C-terminal Extension Mutations

ID	Sex/Age	Inheritance	Mutation	Exon (Domain)	Protein Change	Iris Anomaly	Foveal Hypoplasia	Other Features	Refraction	Binocular Vision (Corrected)	Severity	Reference
3/1	F/54	S	c.1239delT [c.1601delT]	Ex13 (PST)	p.Asp413GlufsX*46	Scattered peripheral iris remnant ∼30% LE; thin rim, ∼40% RE	Yes	Mild ptosis, severe keratopathy, cataracts, glaucoma, uni-ocular exudative retinopathy	+1.0/−0.5×70; OD; +1.0 OS	20/200	Severe	8
3/2A	M/46	F, brother of 3/2B and 3/2C	c.1267dupT [c.1629dupT]	Ex13 (PST)	p.X423LeuextX*108	Full iris, abnormal structure	Yes	High myopia	−7.0 OU	20/200	Moderate	8
3/2B	M/43	F, husband of 1/3A	c.1267dupT [c.1629dupT]	Ex13 (PST)	p.X423LeuextX*108	Full iris, abnormal structure	Yes	Cataracts, high myopia	−8.0 OD; −9.0 OS	20/600	Severe	8
3/2C	F/40	F, sister of 3/2A and 3/2B	c.1267dupT [c.1629dupT]	Ex13 (PST)	p.X423LeuextX*108	Full iris, abnormal structure; slightly enlarged pupil RE	Yes	Severe keratopathy, cataracts, exudative retinopathy, high myopia	−18.0 OD; −16.0 OS	20/600	Severe	8
3/3A	F/44	F, mother of 3/3B	c.1267dupT [c.1629dupT]	Ex13 (PST)	p.X423LeuextX*108	Full iris, abnormal structure	Yes	Severe keratopathy, cataracts, optic nerve hypoplasia, moderate myopia	−3.0/−1.0×70 OD; −4.0/−0.5×90 OS	20/600	Severe	8 28
3/3B	F/16	F, daughter of 3/3A	c.1267dupT [c.1629dupT]	Ex13 (PST)	p.X423LeuextX*108	Full iris, abnormal structure	Yes	Optic disc hypoplasia, blonde fundus, high myopia	−7.0/−1.0×180 OU	20/200	Severe	28
3/4A	F/adult	F, mother of 3/4B	c.1267dupT [c.1629dupT]	Ex13 (PST)	p.X423LeuextX*108	Full iris, abnormal structure	Yes	Severe keratopathy, mild cataracts	ND	CF	Moderate-severe	Current
3/4B	F/23	F, mother of 3/4C and 3/4D	c.1267dupT [c.1629dupT] Inferred	Ex13 (PST)	p.X423LeuextX*108	Full iris, abnormal structure	Yes	Very mild keratopathy, cataracts	0/+1.0×90 OD; −0.5 OS	20/80	Mild-moderate	Current
3/4C	M/3	F, son of 3/4B	c.1267dupT [c.1629dupT] Inferred	Ex13 (PST)	p.X423LeuextX*108	Full iris, abnormal structure	Yes	Very mild keratopathy, mild cataract LE	−1.75/−0.25×90 OD; −2.0/−0.25×90 OS	20/160	Mild-moderate	Current
3/4D	F/<1	F, daughter of 3/4B	c.1267dupT [c.1629dupT] Inferred	Ex13 (PST)	p.X423LeuextX*108	Full iris, abnormal structure	Yes	Blonde fundus	ND	20/250	Mild-moderate	Current

 See Table 1 1for other abbreviations and explanation of mutation nomenclature.

Table 4.

 

Summary of Phenotypes Associated with Other Types of Mutation

ID	Sex/Age	Inheritance	Mutation	Intron (Domain)	Protein Change	Mutation type	Iris Anomaly	Foveal Hypoplasia	Other Features	Refraction	Binocular Vision (Corrected)	Severity	Reference
4/1	M/7	S	c.357+1G>A [IVS6+1G>A]	Intron 6 (PD)	Predicted 36-aa deletion in Ex6	Probable splice defect (unconfirmed)	?	Yes	OHT, moderate hypermetropia	+4.0/−1.0×180 OD; +4.0/−1.0×180 OS	20/400	Moderate	Current
4/2	F/40	S	c.357+2insT [IVS6+2 insT]	Intron 6 (PD)	Predicted 36-aa deletion in Ex6	Splice defect confirmed by RT-PCR	Iris tissue only present temporally. ∼50% RE; nearly full iris, ∼90% LE	Yes	Mild ptosis, cataracts, mild optic nerve hypoplasia	0/−2.0×180 OD; 0/−2.0×180 OS	20/200	Moderate	8
4/3A	M/adult	F, father of 4/4B	c.357+5G>A [IVS6+5G>A]	Intron 6 (PD)	Unknown	Possible splice defect (unconfirmed)	No iris	Yes	Mild keratopathy, OHT, retinal detachment RE	ND	20/120	Moderate	28
4/3B	F/12	F, daughter of 4/4A	c.357+5G>A [IVS6+5G>A]	Intron 6 (PD)	Unknown	Possible splice defect (unconfirmed)	No iris	Yes	Moderate hypermetropia	+5.0 OD; +4.0/−1.0×180 OS	20/200	Moderate	28
4/4	F/37	F	Splice defect confirmed, but genomic mutation not yet identified	Unknown	Predicted 36-aa deletion in Ex6	Splice defect confirmed by RT-PCR	No iris	Yes	Mild cataracts, subluxed lenses	−1.0 OU	20/200	Moderate	7 8
4/5	M/28	F	c.1044+2T>A [IVS8+2T>A]	Intron 8 (HD)	Unknown	Probable splice defect (unconfirmed)	Coloboma-like defect LE; normal RE	No	Cataracts	+12.0 (aphakia) OD; 0/−0.5×100 OS	20/80	Moderate	Current

 See Table 1 1for abbreviations and explanation of mutation nomenclature. aa, amino acid.

Supplementary Materials

Supplementary Figure S1 - 3.3 MB (PDF) 

Supplementary Figure S1. Fundus details of additional cases. 

Left eye in left column and right eye on the right. Magnification may be variable. (a, b) 1/2B Bilateral foveal hypoplasia with no foveal pit or pigmentation, and crossing of fovea by retinal blood vessels. (c, d) 1/9 Mild optic nerve hypoplasia right eye and bilateral foveal hypoplasia with absence of normal foveal landmarks and retinal blood vessels passing across fovea. (e, f) 2/2B Well-preserved foveal morphology as shown in Figure 1 (e, f). (g, h) 2/2C Mildly hypopigmented (blonde) fundus with well preserved foveal structure. (i, j) 3/2B Myopic chorioretinal degeneration, peripapillary atrophy and foveal hypoplasia in the left and right eyes. 

Supplementary Figure S2 - 3.7 MB (PDF) 

Supplementary Figure S2. Iris features of additional cases. 

Left eye in left column and right eye on the right. Magnification may be variable. (a, b) 1/1 No visible iris tissue and severe keratopathy in left eye with neovascularisation extending into corneal graft; the graft sutures are visible. No visible iris tissue and severe keratopathy with central scarring and neovascularisation in right eye. (c, d) 1/4A Peripheral scarring and vascularisation of cornea in left eye. Right phthisical eye has endstage corneal scarring and vascularisation following failed penetrating keratoplasty and multiple glaucoma operations. (e, f) 1/6B No visible iris tissue and moderate to severe keratopathy with neovascularisation and conjunctivalisation of superficial cornea in left eye and marked opacification and neovascularisation of the peripheral cornea and posterior cataract in the right eye. (g, h) 1/9 Full iris with mild structural abnormality in both eyes. (i, j) 2/3B Full iris with abnormal structure in both eyes. 

The authors thank the patients and their families for participating in the study.