- Email: [email protected]
Correlation of novel PAX6 gene abnormalities in aniridia and clinical presentation
Correlation of novel PAX6 gene abnormalities in aniridia and clinical presentation Naif S. Sannan, BSc,* Cheryl Y. Gregory-Evans, PhD,* Christopher J. Lyons, MD,*,† Anna M. Lehman, MD,‡ Sylvie Langlois, MD,‡ Simon J. Warner, MD,* Helen Zakrzewski, BSc,§ Kevin Gregory-Evans, MD, PhD* ABSTRACT ● Objective: To describe the clinical presentation and genotype of subjects with aniridia with a particular focus on foveal hypoplasia. Design: Prospective cohort study. Participants: Thirty-three Canadian participants with aniridia and of various ethnic backgrounds residing in British Columbia. Methods: Full ophthalmic examinations and posterior segment spectral domain-optical coherence tomography (SD-OCT) imaging were performed. Foveal hypoplasia was graded independently by 2 staff ophthalmologists. PAX6 sequencing was performed and chromosomal 11p anomalies investigated. Candidate gene and single-nucleotide polymorphism sequencing in genes functionally related to PAX6 were also studied. Results: Best corrected visual acuities in the cohort ranged from 0.0 logMAR to no light perception. Total absence of iris tissue was seen in the majority (42 of 66 eyes). In those in whom SD-OCT was possible, foveal hypoplasia was seen in the majority (45 of 56 eyes, 80%). Molecular genetic defects involving PAX6 were identified in 30 participants (91%), including 4 novel PAX6 mutations (Gly18Val; Ser65ProfsX14; Met337ArgfsX18; Ser321CysfsX34) and 4 novel chromosome 11p deletions inclusive of PAX6 or a known PAX6 regulatory region. Conclusions: The number of PAX6 mutations associated with aniridia continues to increase. Variable foveal architecture despite nearly identical anterior segment disease in 4 participants with an Ex9 ELP4-Ex4 DCDC1 deletion suggested that molecular cues causing variation in disease in the posterior segment differ from those at play in the anterior segment. Results in 3 patients without identifiable PAX6 mutations and a review of the literature suggest that such cases be described as phenocopies rather than actual cases of the syndrome of aniridia.
Aniridia (OMIM 106210) is an autosomal dominant, congenital, bilateral, panocular condition.1 Populationbased studies in the USA and Europe have estimated the prevalence of the condition to be 1:64 000 to 1:96 000.1,2 Iris hypoplasia comprises minor iris anomalies (pigmentary defects, stromal defects, coloboma), remnant iris root tissue, or complete absence of the iris (total aniridia). 1,3–5 Other common ocular manifestations include nystagmus, cataract, glaucoma, corneal opacification, and foveal hypoplasia.6–9 Systemic abnormalities have also been observed, including interhemispheric brain anomalies, olfactory defects, obesity, and diabetes.10–12 Aniridia may also coexist with various systemic syndromes, such as Gillespie syndrome (partial aniridia, cerebellar-ataxia, and mental retardation),13 and WAGR syndrome (Wilms tumour, aniridia, genitourinary malformations, and mental retardation).14 Aniridia is caused by heterozygous mutations in the PAX6 gene,15 its regulatory elements, or, more rarely, by chromosomal defects16 leading to haploinsufficiency of the PAX6 transcription factor. In 8%–20% of cases, no defects in PAX6 have been identified.17–19 Variable expressivity in phenotype has been well documented and does not appear to correlate
well with genotype. In addition to its role in eye development, PAX6 is also important in brain, olfactory, and pancreas development, which may provide a rationale for the condition’s coexistence with various systemic syndromes.4,20 The literature investigating the foveal hypoplasia associated with aniridia remains scarce. Foveal development begins during midgestation and continues in postnatal life, reaching full cellular maturity by approximately 4 years of age.21 Foveal hypoplasia describes a foveal pit or depression that is partially or completely absent.22 Despite the implications of foveal hypoplasia on vision impairment, the process by which arrested development of the fovea occurs remains unknown. Spectral domain optical coherence tomography (SD-OCT) has improved evaluation of foveal hypoplasia.23,24 An SD-OCT–based grading system of foveal hypoplasia has been proposed,24 which suggests a relationship between the gradation of foveal hypoplasia and visual acuity. However, the gradation of foveal hypoplasia in most studies using this tool has been limited to that seen in albinism.25,26 We undertook an assessment of the ocular clinical presentation and genotype of individuals with aniridia living in British Columbia to
& 2017 Published by Elsevier Inc on behalf of the Canadian Ophthalmological Society. http://dx.doi.org/10.1016/j.jcjo.2017.04.006 ISSN 0008-4182/17 CAN J OPHTHALMOL — VOL. ], NO. ], ] 2017
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Aniridia and PAX6 mutation—Sannan et al. study the association of aniridia, grade of foveal hypoplasia, and genotype.
Biosystems), and results were assessed using CopyCaller software v2.1 (Life Technologies).
METHODS
Cytogenetic Analysis
Participants with aniridia were prospectively enrolled into a clinical and genetic study. Seven other affected family members were subsequently recruited, totaling 33 participants who were examined. Informed written consent for clinical investigations was obtained from those enrolled, in accordance with the University of British Columbia Clinical Research Ethics Board and the tenets of the Declaration of Helsinki.
In one family, standard fluorescence in situ hybridization was carried out on cultured lymphocytes using bacterial artificial chromosome probes: RP11-133E13, RP11-915F11, and CTD-2643K156. PCR-mediated amplification of microsatellite markers in the WAGR region were analyzed by gel electrophoresis as follows: tel-D11S914, PAX6, WT1, D11S4101, D11S672, D11S1301, and D11S907-cen. In a second family, chromosomal microarray analysis using the CytoScan HD array platform was used to identify the extent of a deletion on chromosome 11. Chromosome Analysis Suite version 1.15.1 (Affymetrix, Santa Clara, Calif.) was used in the data analysis.
Clinical Investigations and Imaging
Full ophthalmic examinations were performed. Posterior segment SD-OCT imaging was undertaken in vivo using a Spectralis SD-OCT platform (Heidelberg Engineering, Carlsbad, Calif.). In those with an absent foveal pit, scans of the posterior pole were centered approximately 3 mm directly temporal from the edge of the optic nerve head. Two ophthalmologists (KGE and CJL) with expertise in evaluation of posterior segment SD-OCT imaging independently graded foveal hypoplasia as per the grading system previously described.24 Briefly, images are graded on a scale of 0 to 4, where 0 is normal foveal architecture, grade 1 shows a shallow foveal pit, grade 2 features no foveal pit, grade 3 has no foveal pit plus loss of photoreceptor outer segment thickening at the fovea, and grade 4 is a complete absence of differentiation at the fovea.
Candidate Gene and Single Nucleotide Polymorphisms Sequencing in PAX6-Negative Subjects
Gene-specific PCR-amplified products were generated and screened by direct sequencing using primers and conditions, as previously described, or were redesigned in-house (Table S3) using the Primer3 Program (http:// bioinfo.ut.ee/primer3-0.4.0/). These candidates have previously been associated with aniridia (FOXC1, PITX2),30 anterior segment dysgenesis (BMP4),31 or isolated foveal hypoplasia (SCL38A8)32 or were previously found to be downstream targets of PAX6 in the iris (BMP4, TGFâ2).33
PAX6 Sequencing Analysis
Genomic DNA was extracted either from buccal swabs (DNA Isolation Kit, Isohelix, Harrietsham, UK) or from 10 mL EDTA blood samples using a Nucleon BACC Genomic DNA Extraction Kit (GE Healthcare, Baie d’Urfe, Que.). PAX6 sequencing was performed using primers (Table S1) as previously described.27,28 Multiplex Ligation-Dependent Probe Amplification
A commercially available P219 multiplex ligationdependent probe amplification (MLPA) protocol (MCR Holland, Amsterdam, Netherlands) was used to detect chromosomal duplications or deletions in the PAX6 genomic region when sequencing of PAX6 failed to detect abnormalities as previously described.29 Each MLPA analysis included 3 control DNA samples from healthy individuals. Quantitative Polymerase Chain Reaction
Deletions detected with MLPA were confirmed using a quantitative real-time polymerase chain reaction (PCR) assay with predesigned TaqMan probes to target multiple loci of the suspected deleted region (Table S2). Reactions were analysed on a ViiA7 real-time PCR system (Applied
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RESULTS Subject Characteristics and Clinical Examination
Thirty-three participants of various ethnic backgrounds living in British Columbia with a diagnosis of aniridia were recruited over a 2-year period. The average age of the cohort was 25 years, and 50% of the cohort was male. There was family history of disease in 56% of cases. Clinical details of ocular disease in each participants are presented in Table 1. Nystagmus was found in 19 participants (58%), and keratopathy was seen in 31 eyes (41% of eyes). This keratopathy was a peripheral corneal neovascular pannus in all 29 eyes. In addition, mild stromal opacities were seen in 7 of these 29 eyes. A further 4 of the 29 exhibited advanced or severe stromal scarring (patients A3, A16, A27, and A33; Table 1); all consequently underwent keratoplasty (using Boston Keratoprosthesis in the cases of A16 and A27). Total absence of iris tissue was noted in 42 eyes (64%). In those with partial loss of iris tissue (Fig. 1), a remnant of iris root tissue was seen in 16 eyes (24%), iris coloboma was evident in 6 eyes (9%), and milder iris stroma defects only were seen in 2 eyes (3%). Cataract or intraocular lens in situ was evident in 48 eyes (72%), and a history of
Aniridia and PAX6 mutation—Sannan et al. Table 1.—Patient characteristics and clinical findings Patient Characteristics
Ocular Findings and Imaging
No. Sex Age
BCVA logMAR
A1 A2 A3 A4
F F M F
33 17 6 41
0.4/HM 0.4/0.1 1.0/0.5 1.0 / 1.0
-
-/-/þ/þ þ/þ
remnant/aniridia aniridia/aniridia remnant/remnant aniridia/aniridia
þ/þ þ/þ þ/þ þ/þ
þ/þ þ/þ þ/þ -/-
4/4 1/1 Scar/normal 4/4
A5 A6
M F
16 13
1.0/CF 0.4/0.3
þ þ
-/-/-
þ/þ -/-
-/-/-
4/4 normal/normal
A7
M
17
CF/0.65
þ
þ/þ
þ/þ
þ/þ
4/3
c.406C4T; p.Gln136X
A8 A9 A10 A11
M M M M
8 6 57 52
1.0/1.0 1.0/1.0 0.7/1.0 0.6/0.4
þ þ -
-/þ/þ þ/þ -/-
þ/þ þ/þ þ/þ þ/þ
þ/þ þ/þ þ/þ -/-
4/4 4/4 4/4 3/3
PAX6 del - D11S914 Ex4 PAX6 - Ex9 ELP4 Ex4 PAX6 - Ex9 ELP4 c.112delC; p.Arg38GlyfsX16
A12 A13 A14 A15 A16 A17 A18
F M F F F M M
13 80 8 46 34 14 20
NLP/1.3 0.3/0.3 0.3/0.7 0.2/0.2 LP/CF 0.5/0.0 0.9/0.6
þ þ þ
þ/þ þ/þ -/-/þ/þ -/-/-
þ/þ þ/þ -/þ/þ þ/þ þ/þ -/-
þ/þ -/-/-/þ/þ þ/þ -/-
-/4 normal/normal 1/1 3/3 scar/scar 3/4 4/4
c.112delC; p.Arg38GlyfsX16 c.1197T4A; p.Pro339Pro Ex9 ELP4 - Ex4 DCDC1 Ex9 ELP4 - Ex4 DCDC1 Ex9 ELP4 - PAX6 c.53G4T; p.Gly18Val c.1058C4A; p.Ser353X
A19 A20 A21 A22
F F M F
9 33 10 4
1.0/1.0 1.0/1.3 1.0/1.0 0.5/0.6
þ þ þ
-/þ/þ -/-/-
remnant/remnant coloboma/ coloboma aniridia/stromal strands aniridia/aniridia aniridia/aniridia aniridia/aniridia coloboma/stroma defects aniridia/remnant remnant/remnant remnant/remnant aniridia/aniridia aniridia/aniridia remnant/aniridia coloboma/ coloboma remnant/remnant aniridia/aniridia aniridia/aniridia aniridia/aniridia
-/þ/þ þ/þ -/-
-/þ/þ þ/þ -/-
4/4 4/4 4/3 4/4
A23 A24 A25 A26 A27 A28 A29 A30 A31 A32 A33
F M M M M F F M F M M
77 4/12 48 9/12 43 3/12 54 9 29 3/12 26
0.9/CF FL/FL HM/HM FL/FL 1.0/1.3 FL/FL 1.0/1.2 1.0/0.9 CF/CF FL/FL CF/1.0
þ þ þ þ þ þ þ þ
þ/þ þ/þ/þ -/þ/þ -/þ/þ -/þ/þ -/þ/þ
coloboma/remnant remnant/remnant aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia
þ/þ -/þ/þ -/þ/þ -/þ/þ þ/þ þ/þ -/þ/þ
-/þ -/-/-/þ/þ -/þ/þ þ/þ -/-/þ/þ
normal/scar -/-/3/3 3/3 -/4/scar 4/4 3/4 NR -/4
Nystagmus Keratopathy
Iris defect
Cataract Glaucoma
Foveal hypoplasia, grade
PAX6 genetic abnormality Ex9 ELP4 - Ex4 DCDC1 Ex9 ELP4 - Ex4 DCDC1 Not found c.375_376delAG; p. Arg125SerfsX7 c.193delT; p.Ser65ProfsX14 Not found
Ex6 PAX6 - Ex4 DCDC1 Ex6 PAX6 - Ex4 DCDC1 c.532C4T; p.Gln178X c.40_41delGT; p. Val14LeufsX41 Not found 11p14.2 - p12 del c.718C4T; p.Arg240X c.781C4T; p.Arg261X c.781C4T; p.Arg261X c.781C4T; p.Arg261X c.1010delT; p.Met337ArgfsX28 c.270T4A; p.Tyr90X c.781C4T; p.Arg261X c.781C4T; p.Arg261X c. 961_962insGTTT;p. Ser321CysfsX34
Grayed entries indicate novels mutations identified in this study. BCVA, best-corrected visual acuity; NR, not recorded, unable to obtain imaging; FL, follows light; CF, counting fingers; HM, hand motion; LP, light perception; NLP, no light perception.
glaucoma was elicited in 48%. No cases with optic nerve anomalies (e.g., hypoplasia, coloboma) were seen. Glaucomatous optic nerve changes, however, were present in all eyes with a history of glaucoma (48%).
Figs. 2 and 3). Agreement between raters for gradation of foveal hypoplasia was good (κ ¼ 0.60).
Molecular Genetic Analysis Assessment of Foveal Hypoplasia
SD-OCT imaging was obtained in 56 eyes. Anterior segment disease, which limited the view of the posterior pole; young age of the participant; or severe nystagmus precluded imaging in others. SD-OCT imaging revealed that foveal hypoplasia was present in 45 eyes (80%). In the other 11 eyes, 6 fovea were considered normal, and scar tissue (unrelated to aniridia) was recorded in the remainder. In this latter group, macular scarring was attributed to previous retinal vein occlusions (A3 and A23), bilateral retinal detachments (A16), and wet macular degeneration (A29). Most eyes with foveal hypoplasia were observed to exhibit severe hypoplasia (grade 4, 28 of 44 eyes;
PAX6 genetic defects were identified in 91% (30 of 33) of subjects within the cohort (Table 1). Three novel PAX6 sequence mutations were identified (Gly18Val; Ser65ProfsX14; Met337ArgfsX18), as were one novel insertion (Ser321CysfsX34) and one synonymous PAX6 sequence change (Pro339Pro). MLPA analysis uncovered 4 different deletions in 9 participants that were either within or downstream of the PAX6 genomic region (Fig. 4).29,34 This included 2 parent/sibling families with novel deletions (A9 and A10; A19 and A20). Cytogenetic analysis yielded novel positive results in a further 2 unrelated individuals (A8; A24) (Table 1, Fig. 4). A total of 6 families were enrolled, allowing comparison of phenotypes among family members with the same CAN J OPHTHALMOL — VOL. ], NO. ], ] 2017
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Aniridia and PAX6 mutation—Sannan et al.
Fig. 1 — Iris phenotype variability observed in the study cohort. A, Iris phenotype observed in patient A5 (p.Ser65ProfsX14). B, Remnant iris observed in patient A6 (no PAX6-related mutation identified). C, Ring iris defect observed in patient A13 (p.Pro339Pro). D, Remnant iris observed in patient A14 (Ex9 ELP4 - Ex4 DCDC1 deletion).
PAX6 mutation. A1 and A2 were mother and daughter with a DCDC1-ELP4 deletion. Interestingly, the daughter (A2) had mild foveal hypoplasia only (grade 1) whereas the mother had severe foveal hypoplasia (grade 4). There were no other differences on clinical examination. This same deletion was also seen in 2 other unrelated individuals (A14 and A15), in whom quite variable grading of foveal hypoplasia again was seen (grade 1 and grade 3, respectively). In all 4, complete aniridia was seen without keratopathy or glaucoma (despite 2 of the 4 being over 30 years of age). That no cataract was visible in A14 is probably attributable to her young age. In another deletion family (A11 and A12), significant differences in clinical appearance were again seen both in the anterior segment and posterior segment (milder iris abnormalities and milder foveal hypoplasia in older family member A11). Contrastingly, in the other 3 families (A19:A20, A26:A27: A28, and A31:A32) clinical features among family members were either nearly identical or could be attributed to age differences. No PAX6 abnormality could be identified in 3 unrelated participants (A3, A6, and A23). In addition, a screen for molecular abnormalities in genes with a functional relationship to PAX6 (PITX2, FOXC1, and SCL38A8)30–32
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also failed to identify genetic mutations. It was noticeable that in each case, milder iris defects were seen in comparison to PAX6-mutant aniridia cases and that none of the 3 exhibited foveal hypoplasia. A Val152Ala polymorphism in BMP431 was identified in A3, A6, and A23; however, this polymorphism was also seen in 80% of the rest of the cohort and is a known nonpathogenic variant. We also took this opportunity to screen PITX2, FOXC1, and SCL38A8 to see whether sequence variants might explain the extreme phenotypic heterogeneity seen in foveal hypoplasia in aniridia. A heterozygous p.Val64Asp substitution in SLC38A8 was identified in A2, who had partial foveal hypoplasia (grade 1) with good visual acuity (0.4 logMAR OD, 0.1 LogMAR OS). The participant’s affected mother (A1) had a normal SLC38A8 genotype and significant foveal hypoplasia with poorer vision (0.4 logMAR OD; hand motion OS). Paternal DNA was not available for testing so it could not be determined if the Val64Asp mutation was a spontaneous mutation or of paternal origin. We could not identify sequence variants in SLC38A8 in the other participant in the study (A14) with grade 1 foveal hypoplasia who coincidentally had the same PAX6 deletion as A2 (Ex9 ELP4-Ex4 DCDC1). All participants within the cohort
Aniridia and PAX6 mutation—Sannan et al.
Fig. 2 — Posterior segment spectral domain-optical coherence tomography (SD-OCT) imaging of patients with novel PAX6 mutations identified. A, SD-OCT imaging right eye of patient A5 (p.Ser65ProfsX14) demonstrating significant foveal hypoplasia (grade 4). B, SD-OCT imaging right eye of patient A17 (p.Gly18Val) demonstrating significant foveal hypoplasia (grade 4). White arrow denotes thickening of the outer nuclear layer. C, SD-OCT imaging of the right eye of patient A29 (p.Met337ArgfsX28) demonstrating significant foveal hypoplasia (grade 4).
were identified as having the alternate major G alleles and not the minor effect alleles for both SLC38A8 single nucleotide polymorphisms.
DISCUSSION With an incidence of aniridia of 1:64 000–1:96 000, the 33 cases presented here represent approximately 46%– 69% of the estimated cases in British Columbia (population 4.6 million), so they are reasonably representative of aniridia cases across the province. One recent Mexican35 study identified PAX6 genetic defects in as little as 30% of
cases, whereas Saudi Arabian36 and Korean37 studies have reported PAX6 genetic abnormalities in as much as 88% of cases. We report PAX6 genetic abnormalities in the majority of cases (91%), higher than in previous reports, and describe 4 novel PAX6 mutations (1 missense, 2 nonsense, and 1 insertion) and 4 novel chromosomal deletions inclusive of PAX6 or a known PAX6 regulatory region. One structural feature detected in posterior segment SD-OCT imaging used to grade foveal hypoplasia is outer segment (OS) lengthening.24 In that study, no cases were reported in which a foveal pit was present without OS lengthening, suggesting these 2 features are mutually CAN J OPHTHALMOL — VOL. ], NO. ], ] 2017
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Aniridia and PAX6 mutation—Sannan et al.
Fig. 3 — Foveal hypoplasia phenotypic variability observed in the study cohort. Each bar denotes the number of eyes observed with each grade of foveal hypoplasia. Two staff ophthalmologists with expertise in evaluation of posterior spectral domain-optical coherence tomography imaging independently graded observed foveal hypoplasia as per the grading system previously described. Anterior segment disease, which limited the view of the posterior pole, or age of the patient precluded the imaging of 9 eyes.
coincident. However, we observed a partial foveal pit but no OS lengthening in one participant (A2), and accumulation of photoreceptors was present in the region of the expected foveal pit in 2 others (A11, A17). These observations suggest that OS lengthening and foveal pit excavation are 2 distinct events. This is supported by the known timeline of foveal development21,22: Foveal pit excavation begins at 28 weeks gestation and is complete by birth, whereas outer segments of cone photoreceptors start to form at 36 weeks but do not reach their full length until approximately 3–4 years of age. The mechanism by which PAX6 regulates various phases of foveal pit formation remains to be determined, but multiple target genes are likely involved in the different phases. It might also be expected that foveal development would be arrested earlier in gestation in those with significant foveal hypoplasia. A broad range of best-corrected visual acuity was found in this study. This is a feature of many ocular syndromes in which acuity may be reduced due to a number of ocular abnormalities; corneal scarring, nystagmus, amblyopia, cataract, advanced glaucoma, and foveal hypoplasia are common causes of reduced acuity in aniridia patients. In such a multifactorial condition, it can be challenging to apportion percentages of vision loss to each specific abnormality. In the context of foveal hypoplasia, though, it would be expected that the proportion of vision loss that can be ascribed to this abnormality will be relatively small in the context of significant corneal scarring, for example. Furthermore, previous studies have suggested that advanced stages of foveal hypoplasia can be associated with relatively good vision.38 We studied 6 independent families, and although none were large families and despite previous reports on single, individual families,6,39,40 this study is one of the few to concurrently study intrafamilial variation in a number of families. Of particular note was the marked variation in
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foveal hypoplasia seen with the ELP4-DCDC1 deletion (family A1/A2 and unrelated individuals A14 and A15). This was seen in the context of total aniridia, but otherwise mild anterior segment changes were observed in each subject. We therefore hypothesize that the factors (either genetic or environmental) causing variation in the posterior segment are not an influence in anterior segment pathogenesis. Of particular note were the 3 unrelated cases in which no PAX6 abnormality could be identified (A3, A6, and A23), although we did not undertake an extensive study of PAX6 3’-cis regulatory abnormalities that can map hundreds of kilobases from the PAX6 gene locus.16,41–44 It was noted that these cases differed clinically from others in this study in that iris anomalies were milder, and none exhibited fovea hypoplasia. It should be noted, however, that some PAX6 mutant aniridia cases were seen with partial aniridia (A11 and A18) and normal foveal architecture (A13), although not with both. Previous reports have suggested non-PAX6 aniridia-like phenotypes associated with FOXC1 and PITX2.30 However, our screen failed to identify abnormalities of these genes or in the functionally related SCL38A8 gene. A review of these past cases noted that none have reported classical ocular aniridia (complete absence of iris tissue, juvenile onset glaucoma, epithelial keratopathy, cataract, and frequent foveal hypoplasia). FOXC1 mutation was associated with partial aniridia, aniridia with corneal disease (Peter’s anomaly), or aniridia with congenital glaucoma (not normally present in classic aniridia). Furthermore, foveal hypoplasia was not reported in these cases.19,45–47 Similarly, in the 2 PITX2 mutation patients, the phenotypic descriptions were limited with no report on foveal architecture.17,48 It is striking to note that in our study all aniridia cases with PAX mutation had abnormal
Fig. 4 — Chromosome deletions in patients without PAX6 coding mutations identified in this study. The gene organization of chromosome 11p is represented (not to scale) with horizontal arrows denoting the direction of gene transcription. DRR (vertical arrows) is the DNA regulatory region containing hypersensitivity sites. D11S914 denotes microsatellite marker. Gray bars indicate the extent of deletion in each patient as detected by multiplex ligation-dependent probe amplification analysis. White bars indicate deletions in patients as determined by cytogenetics analysis. Patient ID number is denoted inside each bar.
Aniridia and PAX6 mutation—Sannan et al. foveal architecture. This calls into doubt the existence of aniridia without PAX6 mutation and suggests that these other cases might more correctly be labelled as anterior segment dysgenesis, in which the aniridia is only a physical sign of a lack of iris tissue and not indicative of the genetic syndrome of aniridia. This genetic syndrome of aniridia could therefore be defined as a condition with a PAX6 defect, anterior segment dysgenesis, and foveal hypoplasia (with additional high risk of keratopathy, cataract, glaucoma, and systemic defects). A larger survey of aniridia patients needs to be undertaken to verify this. Although PAX6 genetic abnormalities were first identified more than 2 decades ago, only now is the value of their identification coming to the fore. Screening for 11p13 chromosomal deletions is now routine in patients diagnosed with aniridia because it identifies those at risk for Wilms tumor, prompting regular kidney ultrasound surveillance. Sequencing of the PAX6 gene is often limited, however, because it is seen as having a lesser impact on medical management. This may be about to change as new concepts in therapeutics emerge. We recently reported a small-molecule, nonsense-suppression therapy in the Pax6Sey mouse model of aniridia49 that has now progressed to clinical trials (ClinicalTrials.gov identifier: NCT02647359). Approximately 40% of those diagnosed with aniridia are identified as having PAX6 nonsense mutations50; this makes this therapy potentially useful to a significant proportion of individuals with the disease. Such mutation-based therapeutic options mandate more widespread molecular genetic testing and new natural history studies to more closely examine the clinical variability seen in this and other studies.
APPENDIX A. SUPPORTING
INFORMATION
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j. jcjo.2017.04.006.
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9. Park KA, Oh SY. Clinical characteristics of high grade foveal hypoplasia. Int Ophthalmol. 2013;33:9-14. 10. Sisodiya SM, Free SL, Williamson KA, et al. PAX6 haploinsufficiency cause cerebral malformation and olfactory dysfunction in humans. Nat Genet. 2001;28:214-6. 11. Peter NM, Leyland M, Mudhas HS, Lowndes J, Owens KR, Stewart H. PAX6 mutation with ptosis, cataract, iris hypoplasia, corneal opacification and diabetes: a new variant of familial aniridia. Clin Exp Ophthalmol. 2013;41:835-41. 12. Netland PA, Scott ML, Boyle JW, Lauderdale JD. Ocular and systemic findings in a survey of aniridia subjects. J AAPOS. 2011;15:562-6. 13. Glaser T, Ton CC, Mueller R, et al. Absence of PAX6 gene mutations in Gillespie syndrome (partial aniridia, cerebellar ataxia, and mental retardation). Genomics. 1994;19:145-8. 14. Riccardi VM, Sujansky E, Smith AC, Francke U. Chromosomal imbalance in the Aniridia-Wilms' tumor association: 11p interstitial deletion. Pediatrics. 1978;61:604-10. 15. Jordan T, Hanson I, Zaletayev D, et al. The human PAX6 gene is mutated in two subjects with aniridia. Nat Genet. 1992;1:328-32. 16. Kleinjan DA, Seawright A, Schedl A, Quinlan RA, Danes S, van Heyningen V. Aniridia-associated translocations, DNase hypersensitivity, sequence comparison and transgenic analysis redefine the functional domain of PAX6. Hum Mol Genet. 2001;10:2049-59. 17. Traboulsi EI, Ellison J, Sears J, Maumenee IH, Avallone J, Mohney BG. Aniridia with preserved visual function: a report of four cases with no mutations in PAX6. Am J Ophthalmol. 2008;145:760-4. 18. Bobilev AM, McDougal ME, Taylor WL, Geisert EE, Netland PA, Lauderdale JD. Assessment of PAX6 alleles in 66 families with aniridia. Clin Genet. 2016;89:669-77. 19. Ansari M, Rainger J, Hanson IM, et al. Genetic analysis of ‘PAX6Negative’ individuals with aniridia or Gillespie syndrome. PLoS One. 2016;11:e0153757. 20. Hittner HM, Riccardi VM, Ferrell RE, Borda RR, Justice J. Variable expressivity in autosomal dominant aniridia by clinical, electrophysiologic, and angiographic criteria. Am J Ophthalmol. 1980;89: 531-539. 21. Yuodelis C, Hendrickson A. A qualitative and quantitative analysis of the human fovea during development. Vision Res. 1986;26:847-55. 22. Gregory-Evans CY, Gregory-Evans K. Foveal hypoplasia: the case for arrested development. Expert Rev Ophthalmol. 2011;6:565-74. 23. Recchia FM, Carvalho-Recchia CA, Trese MT. Optical coherence tomography in the diagnosis of foveal hypoplasia. Arch Ophthalmol. 2002;120:1587-8. 24. Thomas MG, Kumar A, Mohammad S, et al. Structural grading of foveal hypoplasia using spectral-domain optical coherence tomography: a predictor of visual acuity. Ophthalmology. 2011;118: 1653-1660. 25. Harvey PS, King RA, Summers CG. Spectrum of foveal development in albinism detected with optical coherence tomography. J AAPOS. 2006;10:237-42. 26. Chong GT, Farsiu S, Freedman SF, et al. Abnormal foveal morphology in ocular albinism imaged with spectral-domain optical coherence tomography. Arch Ophthalmol. 2009;127:37-44. 27. Love J, Axton R, Churchill A, van Heyningen, Hanson I. A new set of primers for mutation analysis of the human PAX6 gene. Hum Mutat. 1998;12:128-34. 28. Robinson DO, Howarth RJ, Williamson KA, van Heyningen V, Beal SJ, Crolla JA. Genetic analysis of chromosome 11p13 and the PAX6 gene in a series of 125 cases referred with aniridia. Am J Med Genet A. 2008;146A:558-69. 29. Redeker EJ, de Visser AS, Bergen AA, Mannens MM. Multiplex ligation-dependent probe amplification (MPLA) enhances the molecular diagnosis of aniridia and related disorders. Mol Vis. 2008;14:836-40. 30. Weisschuh N, Dressler P, Schuettauf F, Wolf C, Wissinger B, Gramer E. Novel mutations of FOXC1 and PITX2 in subjects with Axenfeld-Rieger malformations. Invest Ophthalmol Vis Sci. 2006;47:3846-52. 31. Mangino M, Torrente I, de Luca A, Sanchez O, Dallapiccola B, Novelli G. A single-nucleotide polymorphism in the human bone morphogenetic protein-4 (BMP4) gene. J Hum Genet. 1999;44: 76-77. CAN J OPHTHALMOL — VOL. ], NO. ], ] 2017
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Aniridia and PAX6 mutation—Sannan et al. 32. Poultier JA, Al-Araimi M, Conte I, et al. Recessive mutations in SLC38A8 cause foveal hypoplasia and optic nerve misrouting without albinism. Am J Hum Genet. 2013;93:1143-50. 33. Wang X, Shan X, Gregory-Evans CY. A mouse model of aniridia reveals the in vivo downstream targets of Pax6 driving iris and ciliary body development in the eye. Biochem Biophys Acta. 2017;1863: 66-67. 34. D’Elia AV, Pellizzari L, Fabbro D, et al. A deletion 3’ to the PAX6 gene in familial aniridia cases. Mol Vis. 2007;13:1245-50. 35. Villarroel CE, Villanueva-Mendoza C, Orozco L, et al. Molecular analysis of the PAX6 gene in Mexican patients with congenital aniridia: report of four novel mutations. Mol Vis. 2008;14:1650-8. 36. Khan AO, Aldahmesh MA, Alkuraya FS. Genetic and genomic analysis of classic aniridia in Saudi Arabia. Mol Vis. 2011;17: 708-714. 37. Park SH, Kim MS, Chae H, Kim Y, Kim M. Molecular analysis of the PAX6 gene for congenital aniridia in the Korean population: identification of four novel mutations. Mol Vis. 2012;18:488-94. 38. Marmor MF, Choi SS, Zawadzki RJ, Werner JS. Visual insignificance of the foveal pit: reassessment of foveal hypoplasia as fovea plana. Arch Ophthalmol. 2008;126:907-13. 39. Davis A, Cowell JK. Mutations in the PAX6 gene in patients with hereditary aniridia. Hum Mol Genet. 1993;12:2093-7. 40. Gregory-Evans K, Cheong-Leen R, George S, Xie J, Moosajee M, Gregory-Evans CY. Non-invasive anterior segment and posterior segment optical coherence tomography and phenotypic characterization of aniridia. Can J Ophthalmol. 2011;46:337-44. 41. Kawano T, Wang C, Hotta Y, et al. Three novel mutations of the PAX6 gene in Japanese patients. J Hum Genet. 2007;52:571-4. 42. Chen P, Zang X, Sun D, et al. Mutation analysis of paired box 6 gene in inherited aniridia in northern China. Mol Vis. 2013;30:1169-77. 43. Kammandel B, Chowdhury K, Stoykova A, Aparicio S, Brenner S, Gruss P. Distinct cis-essential modules direct the time-space pattern of the Pax6 gene activity. Dev Biol. 1999;205:79-97. 44. McBride DJ, Buckle A, van Heyningen V, Kleinjan DA. DNaseI hypersensitivity and ultraconservation reveal novel, interdependent long-range enhancers at the complex Pax6 cis-regulatory region. PLoS One. 2011;6:e28616. 45. Khan AO, Aldahmesh MA, Al-Amri A. Heterozygous FOXC1 mutation (M161K) associated with congenital glaucoma and aniridia in an infant and a milder phenotype in her mother. Ophthal Genet. 2008;29:67-71.
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46. Ito YA, Footz TK, Berry FB, et al. Severe molecular defects of a novel FOXC1 W152G mutation result in aniridia. Invest Ophthalmol Vis Sci. 2009;50:3573-9. 47. Sadagopan KA, Liu GT, Capasso JE, Wuthisiri W, Keep RB, Levin AV. Aniridia-like phenotype caused by 6p25 dosage aberrations. Am J Med Genet A. 2015;167A:524-8. 48. Perveen R, Lloyd IC, Clayton-Smith J, et al. Phenotypic variability and asymmetry of Rieger syndrome associated with PITX2 mutations. Invest Ophthalmol Vis Sci. 2000;41:2456-60. 49. Gregory-Evans CY, Wang X, Wasan KM, Zhao J, Metcalfe AL, Gregory-Evans K. Postnatal manipulation of Pax6 dosage reverses congenital tissue malformation defects. J Clin Invest. 2014;124:111-6. 50. Tzoulaki I, White IMS, Hanson IM. PAX6 mutations: genotypephenotype correlations. BMC Genet. 2005;6:e27.
Footnotes and Disclosure: The authors have no proprietary or commercial interest in any materials discussed in this article. This work was supported by the Sharon Stewart Trust and a scholarship from the Saudi Cultural Bureau. The authors thank Laura Hall for optical coherence tomography imaging support. From the nDepartment of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, B.C; †Department of Ophthalmology, BC Children’s Hospital, Vancouver, B.C; ‡Department of Medical Genetics, University of British Columbia, Vancouver, B.C; §Cumming School of Medicine, University of Calgary, Calgary, Alta. Originally received Oct. 27, 2016. Final revision Mar. 24, 2017. Accepted Apr. 5, 2017. Correspondence to Kevin Gregory-Evans MD, PhD, Department of Ophthalmology and Visual Sciences, University of British Columbia, 2550 Willow Street, Vancouver BC, V5Z 3N9, Canada; [email protected].
Aniridia (OMIM 106210) is an autosomal dominant, congenital, bilateral, panocular condition.1 Populationbased studies in the USA and Europe have estimated the prevalence of the condition to be 1:64 000 to 1:96 000.1,2 Iris hypoplasia comprises minor iris anomalies (pigmentary defects, stromal defects, coloboma), remnant iris root tissue, or complete absence of the iris (total aniridia). 1,3–5 Other common ocular manifestations include nystagmus, cataract, glaucoma, corneal opacification, and foveal hypoplasia.6–9 Systemic abnormalities have also been observed, including interhemispheric brain anomalies, olfactory defects, obesity, and diabetes.10–12 Aniridia may also coexist with various systemic syndromes, such as Gillespie syndrome (partial aniridia, cerebellar-ataxia, and mental retardation),13 and WAGR syndrome (Wilms tumour, aniridia, genitourinary malformations, and mental retardation).14 Aniridia is caused by heterozygous mutations in the PAX6 gene,15 its regulatory elements, or, more rarely, by chromosomal defects16 leading to haploinsufficiency of the PAX6 transcription factor. In 8%–20% of cases, no defects in PAX6 have been identified.17–19 Variable expressivity in phenotype has been well documented and does not appear to correlate
well with genotype. In addition to its role in eye development, PAX6 is also important in brain, olfactory, and pancreas development, which may provide a rationale for the condition’s coexistence with various systemic syndromes.4,20 The literature investigating the foveal hypoplasia associated with aniridia remains scarce. Foveal development begins during midgestation and continues in postnatal life, reaching full cellular maturity by approximately 4 years of age.21 Foveal hypoplasia describes a foveal pit or depression that is partially or completely absent.22 Despite the implications of foveal hypoplasia on vision impairment, the process by which arrested development of the fovea occurs remains unknown. Spectral domain optical coherence tomography (SD-OCT) has improved evaluation of foveal hypoplasia.23,24 An SD-OCT–based grading system of foveal hypoplasia has been proposed,24 which suggests a relationship between the gradation of foveal hypoplasia and visual acuity. However, the gradation of foveal hypoplasia in most studies using this tool has been limited to that seen in albinism.25,26 We undertook an assessment of the ocular clinical presentation and genotype of individuals with aniridia living in British Columbia to
& 2017 Published by Elsevier Inc on behalf of the Canadian Ophthalmological Society. http://dx.doi.org/10.1016/j.jcjo.2017.04.006 ISSN 0008-4182/17 CAN J OPHTHALMOL — VOL. ], NO. ], ] 2017
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Aniridia and PAX6 mutation—Sannan et al. study the association of aniridia, grade of foveal hypoplasia, and genotype.
Biosystems), and results were assessed using CopyCaller software v2.1 (Life Technologies).
METHODS
Cytogenetic Analysis
Participants with aniridia were prospectively enrolled into a clinical and genetic study. Seven other affected family members were subsequently recruited, totaling 33 participants who were examined. Informed written consent for clinical investigations was obtained from those enrolled, in accordance with the University of British Columbia Clinical Research Ethics Board and the tenets of the Declaration of Helsinki.
In one family, standard fluorescence in situ hybridization was carried out on cultured lymphocytes using bacterial artificial chromosome probes: RP11-133E13, RP11-915F11, and CTD-2643K156. PCR-mediated amplification of microsatellite markers in the WAGR region were analyzed by gel electrophoresis as follows: tel-D11S914, PAX6, WT1, D11S4101, D11S672, D11S1301, and D11S907-cen. In a second family, chromosomal microarray analysis using the CytoScan HD array platform was used to identify the extent of a deletion on chromosome 11. Chromosome Analysis Suite version 1.15.1 (Affymetrix, Santa Clara, Calif.) was used in the data analysis.
Clinical Investigations and Imaging
Full ophthalmic examinations were performed. Posterior segment SD-OCT imaging was undertaken in vivo using a Spectralis SD-OCT platform (Heidelberg Engineering, Carlsbad, Calif.). In those with an absent foveal pit, scans of the posterior pole were centered approximately 3 mm directly temporal from the edge of the optic nerve head. Two ophthalmologists (KGE and CJL) with expertise in evaluation of posterior segment SD-OCT imaging independently graded foveal hypoplasia as per the grading system previously described.24 Briefly, images are graded on a scale of 0 to 4, where 0 is normal foveal architecture, grade 1 shows a shallow foveal pit, grade 2 features no foveal pit, grade 3 has no foveal pit plus loss of photoreceptor outer segment thickening at the fovea, and grade 4 is a complete absence of differentiation at the fovea.
Candidate Gene and Single Nucleotide Polymorphisms Sequencing in PAX6-Negative Subjects
Gene-specific PCR-amplified products were generated and screened by direct sequencing using primers and conditions, as previously described, or were redesigned in-house (Table S3) using the Primer3 Program (http:// bioinfo.ut.ee/primer3-0.4.0/). These candidates have previously been associated with aniridia (FOXC1, PITX2),30 anterior segment dysgenesis (BMP4),31 or isolated foveal hypoplasia (SCL38A8)32 or were previously found to be downstream targets of PAX6 in the iris (BMP4, TGFâ2).33
PAX6 Sequencing Analysis
Genomic DNA was extracted either from buccal swabs (DNA Isolation Kit, Isohelix, Harrietsham, UK) or from 10 mL EDTA blood samples using a Nucleon BACC Genomic DNA Extraction Kit (GE Healthcare, Baie d’Urfe, Que.). PAX6 sequencing was performed using primers (Table S1) as previously described.27,28 Multiplex Ligation-Dependent Probe Amplification
A commercially available P219 multiplex ligationdependent probe amplification (MLPA) protocol (MCR Holland, Amsterdam, Netherlands) was used to detect chromosomal duplications or deletions in the PAX6 genomic region when sequencing of PAX6 failed to detect abnormalities as previously described.29 Each MLPA analysis included 3 control DNA samples from healthy individuals. Quantitative Polymerase Chain Reaction
Deletions detected with MLPA were confirmed using a quantitative real-time polymerase chain reaction (PCR) assay with predesigned TaqMan probes to target multiple loci of the suspected deleted region (Table S2). Reactions were analysed on a ViiA7 real-time PCR system (Applied
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RESULTS Subject Characteristics and Clinical Examination
Thirty-three participants of various ethnic backgrounds living in British Columbia with a diagnosis of aniridia were recruited over a 2-year period. The average age of the cohort was 25 years, and 50% of the cohort was male. There was family history of disease in 56% of cases. Clinical details of ocular disease in each participants are presented in Table 1. Nystagmus was found in 19 participants (58%), and keratopathy was seen in 31 eyes (41% of eyes). This keratopathy was a peripheral corneal neovascular pannus in all 29 eyes. In addition, mild stromal opacities were seen in 7 of these 29 eyes. A further 4 of the 29 exhibited advanced or severe stromal scarring (patients A3, A16, A27, and A33; Table 1); all consequently underwent keratoplasty (using Boston Keratoprosthesis in the cases of A16 and A27). Total absence of iris tissue was noted in 42 eyes (64%). In those with partial loss of iris tissue (Fig. 1), a remnant of iris root tissue was seen in 16 eyes (24%), iris coloboma was evident in 6 eyes (9%), and milder iris stroma defects only were seen in 2 eyes (3%). Cataract or intraocular lens in situ was evident in 48 eyes (72%), and a history of
Aniridia and PAX6 mutation—Sannan et al. Table 1.—Patient characteristics and clinical findings Patient Characteristics
Ocular Findings and Imaging
No. Sex Age
BCVA logMAR
A1 A2 A3 A4
F F M F
33 17 6 41
0.4/HM 0.4/0.1 1.0/0.5 1.0 / 1.0
-
-/-/þ/þ þ/þ
remnant/aniridia aniridia/aniridia remnant/remnant aniridia/aniridia
þ/þ þ/þ þ/þ þ/þ
þ/þ þ/þ þ/þ -/-
4/4 1/1 Scar/normal 4/4
A5 A6
M F
16 13
1.0/CF 0.4/0.3
þ þ
-/-/-
þ/þ -/-
-/-/-
4/4 normal/normal
A7
M
17
CF/0.65
þ
þ/þ
þ/þ
þ/þ
4/3
c.406C4T; p.Gln136X
A8 A9 A10 A11
M M M M
8 6 57 52
1.0/1.0 1.0/1.0 0.7/1.0 0.6/0.4
þ þ -
-/þ/þ þ/þ -/-
þ/þ þ/þ þ/þ þ/þ
þ/þ þ/þ þ/þ -/-
4/4 4/4 4/4 3/3
PAX6 del - D11S914 Ex4 PAX6 - Ex9 ELP4 Ex4 PAX6 - Ex9 ELP4 c.112delC; p.Arg38GlyfsX16
A12 A13 A14 A15 A16 A17 A18
F M F F F M M
13 80 8 46 34 14 20
NLP/1.3 0.3/0.3 0.3/0.7 0.2/0.2 LP/CF 0.5/0.0 0.9/0.6
þ þ þ
þ/þ þ/þ -/-/þ/þ -/-/-
þ/þ þ/þ -/þ/þ þ/þ þ/þ -/-
þ/þ -/-/-/þ/þ þ/þ -/-
-/4 normal/normal 1/1 3/3 scar/scar 3/4 4/4
c.112delC; p.Arg38GlyfsX16 c.1197T4A; p.Pro339Pro Ex9 ELP4 - Ex4 DCDC1 Ex9 ELP4 - Ex4 DCDC1 Ex9 ELP4 - PAX6 c.53G4T; p.Gly18Val c.1058C4A; p.Ser353X
A19 A20 A21 A22
F F M F
9 33 10 4
1.0/1.0 1.0/1.3 1.0/1.0 0.5/0.6
þ þ þ
-/þ/þ -/-/-
remnant/remnant coloboma/ coloboma aniridia/stromal strands aniridia/aniridia aniridia/aniridia aniridia/aniridia coloboma/stroma defects aniridia/remnant remnant/remnant remnant/remnant aniridia/aniridia aniridia/aniridia remnant/aniridia coloboma/ coloboma remnant/remnant aniridia/aniridia aniridia/aniridia aniridia/aniridia
-/þ/þ þ/þ -/-
-/þ/þ þ/þ -/-
4/4 4/4 4/3 4/4
A23 A24 A25 A26 A27 A28 A29 A30 A31 A32 A33
F M M M M F F M F M M
77 4/12 48 9/12 43 3/12 54 9 29 3/12 26
0.9/CF FL/FL HM/HM FL/FL 1.0/1.3 FL/FL 1.0/1.2 1.0/0.9 CF/CF FL/FL CF/1.0
þ þ þ þ þ þ þ þ
þ/þ þ/þ/þ -/þ/þ -/þ/þ -/þ/þ -/þ/þ
coloboma/remnant remnant/remnant aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia aniridia/aniridia
þ/þ -/þ/þ -/þ/þ -/þ/þ þ/þ þ/þ -/þ/þ
-/þ -/-/-/þ/þ -/þ/þ þ/þ -/-/þ/þ
normal/scar -/-/3/3 3/3 -/4/scar 4/4 3/4 NR -/4
Nystagmus Keratopathy
Iris defect
Cataract Glaucoma
Foveal hypoplasia, grade
PAX6 genetic abnormality Ex9 ELP4 - Ex4 DCDC1 Ex9 ELP4 - Ex4 DCDC1 Not found c.375_376delAG; p. Arg125SerfsX7 c.193delT; p.Ser65ProfsX14 Not found
Ex6 PAX6 - Ex4 DCDC1 Ex6 PAX6 - Ex4 DCDC1 c.532C4T; p.Gln178X c.40_41delGT; p. Val14LeufsX41 Not found 11p14.2 - p12 del c.718C4T; p.Arg240X c.781C4T; p.Arg261X c.781C4T; p.Arg261X c.781C4T; p.Arg261X c.1010delT; p.Met337ArgfsX28 c.270T4A; p.Tyr90X c.781C4T; p.Arg261X c.781C4T; p.Arg261X c. 961_962insGTTT;p. Ser321CysfsX34
Grayed entries indicate novels mutations identified in this study. BCVA, best-corrected visual acuity; NR, not recorded, unable to obtain imaging; FL, follows light; CF, counting fingers; HM, hand motion; LP, light perception; NLP, no light perception.
glaucoma was elicited in 48%. No cases with optic nerve anomalies (e.g., hypoplasia, coloboma) were seen. Glaucomatous optic nerve changes, however, were present in all eyes with a history of glaucoma (48%).
Figs. 2 and 3). Agreement between raters for gradation of foveal hypoplasia was good (κ ¼ 0.60).
Molecular Genetic Analysis Assessment of Foveal Hypoplasia
SD-OCT imaging was obtained in 56 eyes. Anterior segment disease, which limited the view of the posterior pole; young age of the participant; or severe nystagmus precluded imaging in others. SD-OCT imaging revealed that foveal hypoplasia was present in 45 eyes (80%). In the other 11 eyes, 6 fovea were considered normal, and scar tissue (unrelated to aniridia) was recorded in the remainder. In this latter group, macular scarring was attributed to previous retinal vein occlusions (A3 and A23), bilateral retinal detachments (A16), and wet macular degeneration (A29). Most eyes with foveal hypoplasia were observed to exhibit severe hypoplasia (grade 4, 28 of 44 eyes;
PAX6 genetic defects were identified in 91% (30 of 33) of subjects within the cohort (Table 1). Three novel PAX6 sequence mutations were identified (Gly18Val; Ser65ProfsX14; Met337ArgfsX18), as were one novel insertion (Ser321CysfsX34) and one synonymous PAX6 sequence change (Pro339Pro). MLPA analysis uncovered 4 different deletions in 9 participants that were either within or downstream of the PAX6 genomic region (Fig. 4).29,34 This included 2 parent/sibling families with novel deletions (A9 and A10; A19 and A20). Cytogenetic analysis yielded novel positive results in a further 2 unrelated individuals (A8; A24) (Table 1, Fig. 4). A total of 6 families were enrolled, allowing comparison of phenotypes among family members with the same CAN J OPHTHALMOL — VOL. ], NO. ], ] 2017
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Aniridia and PAX6 mutation—Sannan et al.
Fig. 1 — Iris phenotype variability observed in the study cohort. A, Iris phenotype observed in patient A5 (p.Ser65ProfsX14). B, Remnant iris observed in patient A6 (no PAX6-related mutation identified). C, Ring iris defect observed in patient A13 (p.Pro339Pro). D, Remnant iris observed in patient A14 (Ex9 ELP4 - Ex4 DCDC1 deletion).
PAX6 mutation. A1 and A2 were mother and daughter with a DCDC1-ELP4 deletion. Interestingly, the daughter (A2) had mild foveal hypoplasia only (grade 1) whereas the mother had severe foveal hypoplasia (grade 4). There were no other differences on clinical examination. This same deletion was also seen in 2 other unrelated individuals (A14 and A15), in whom quite variable grading of foveal hypoplasia again was seen (grade 1 and grade 3, respectively). In all 4, complete aniridia was seen without keratopathy or glaucoma (despite 2 of the 4 being over 30 years of age). That no cataract was visible in A14 is probably attributable to her young age. In another deletion family (A11 and A12), significant differences in clinical appearance were again seen both in the anterior segment and posterior segment (milder iris abnormalities and milder foveal hypoplasia in older family member A11). Contrastingly, in the other 3 families (A19:A20, A26:A27: A28, and A31:A32) clinical features among family members were either nearly identical or could be attributed to age differences. No PAX6 abnormality could be identified in 3 unrelated participants (A3, A6, and A23). In addition, a screen for molecular abnormalities in genes with a functional relationship to PAX6 (PITX2, FOXC1, and SCL38A8)30–32
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also failed to identify genetic mutations. It was noticeable that in each case, milder iris defects were seen in comparison to PAX6-mutant aniridia cases and that none of the 3 exhibited foveal hypoplasia. A Val152Ala polymorphism in BMP431 was identified in A3, A6, and A23; however, this polymorphism was also seen in 80% of the rest of the cohort and is a known nonpathogenic variant. We also took this opportunity to screen PITX2, FOXC1, and SCL38A8 to see whether sequence variants might explain the extreme phenotypic heterogeneity seen in foveal hypoplasia in aniridia. A heterozygous p.Val64Asp substitution in SLC38A8 was identified in A2, who had partial foveal hypoplasia (grade 1) with good visual acuity (0.4 logMAR OD, 0.1 LogMAR OS). The participant’s affected mother (A1) had a normal SLC38A8 genotype and significant foveal hypoplasia with poorer vision (0.4 logMAR OD; hand motion OS). Paternal DNA was not available for testing so it could not be determined if the Val64Asp mutation was a spontaneous mutation or of paternal origin. We could not identify sequence variants in SLC38A8 in the other participant in the study (A14) with grade 1 foveal hypoplasia who coincidentally had the same PAX6 deletion as A2 (Ex9 ELP4-Ex4 DCDC1). All participants within the cohort
Aniridia and PAX6 mutation—Sannan et al.
Fig. 2 — Posterior segment spectral domain-optical coherence tomography (SD-OCT) imaging of patients with novel PAX6 mutations identified. A, SD-OCT imaging right eye of patient A5 (p.Ser65ProfsX14) demonstrating significant foveal hypoplasia (grade 4). B, SD-OCT imaging right eye of patient A17 (p.Gly18Val) demonstrating significant foveal hypoplasia (grade 4). White arrow denotes thickening of the outer nuclear layer. C, SD-OCT imaging of the right eye of patient A29 (p.Met337ArgfsX28) demonstrating significant foveal hypoplasia (grade 4).
were identified as having the alternate major G alleles and not the minor effect alleles for both SLC38A8 single nucleotide polymorphisms.
DISCUSSION With an incidence of aniridia of 1:64 000–1:96 000, the 33 cases presented here represent approximately 46%– 69% of the estimated cases in British Columbia (population 4.6 million), so they are reasonably representative of aniridia cases across the province. One recent Mexican35 study identified PAX6 genetic defects in as little as 30% of
cases, whereas Saudi Arabian36 and Korean37 studies have reported PAX6 genetic abnormalities in as much as 88% of cases. We report PAX6 genetic abnormalities in the majority of cases (91%), higher than in previous reports, and describe 4 novel PAX6 mutations (1 missense, 2 nonsense, and 1 insertion) and 4 novel chromosomal deletions inclusive of PAX6 or a known PAX6 regulatory region. One structural feature detected in posterior segment SD-OCT imaging used to grade foveal hypoplasia is outer segment (OS) lengthening.24 In that study, no cases were reported in which a foveal pit was present without OS lengthening, suggesting these 2 features are mutually CAN J OPHTHALMOL — VOL. ], NO. ], ] 2017
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Aniridia and PAX6 mutation—Sannan et al.
Fig. 3 — Foveal hypoplasia phenotypic variability observed in the study cohort. Each bar denotes the number of eyes observed with each grade of foveal hypoplasia. Two staff ophthalmologists with expertise in evaluation of posterior spectral domain-optical coherence tomography imaging independently graded observed foveal hypoplasia as per the grading system previously described. Anterior segment disease, which limited the view of the posterior pole, or age of the patient precluded the imaging of 9 eyes.
coincident. However, we observed a partial foveal pit but no OS lengthening in one participant (A2), and accumulation of photoreceptors was present in the region of the expected foveal pit in 2 others (A11, A17). These observations suggest that OS lengthening and foveal pit excavation are 2 distinct events. This is supported by the known timeline of foveal development21,22: Foveal pit excavation begins at 28 weeks gestation and is complete by birth, whereas outer segments of cone photoreceptors start to form at 36 weeks but do not reach their full length until approximately 3–4 years of age. The mechanism by which PAX6 regulates various phases of foveal pit formation remains to be determined, but multiple target genes are likely involved in the different phases. It might also be expected that foveal development would be arrested earlier in gestation in those with significant foveal hypoplasia. A broad range of best-corrected visual acuity was found in this study. This is a feature of many ocular syndromes in which acuity may be reduced due to a number of ocular abnormalities; corneal scarring, nystagmus, amblyopia, cataract, advanced glaucoma, and foveal hypoplasia are common causes of reduced acuity in aniridia patients. In such a multifactorial condition, it can be challenging to apportion percentages of vision loss to each specific abnormality. In the context of foveal hypoplasia, though, it would be expected that the proportion of vision loss that can be ascribed to this abnormality will be relatively small in the context of significant corneal scarring, for example. Furthermore, previous studies have suggested that advanced stages of foveal hypoplasia can be associated with relatively good vision.38 We studied 6 independent families, and although none were large families and despite previous reports on single, individual families,6,39,40 this study is one of the few to concurrently study intrafamilial variation in a number of families. Of particular note was the marked variation in
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foveal hypoplasia seen with the ELP4-DCDC1 deletion (family A1/A2 and unrelated individuals A14 and A15). This was seen in the context of total aniridia, but otherwise mild anterior segment changes were observed in each subject. We therefore hypothesize that the factors (either genetic or environmental) causing variation in the posterior segment are not an influence in anterior segment pathogenesis. Of particular note were the 3 unrelated cases in which no PAX6 abnormality could be identified (A3, A6, and A23), although we did not undertake an extensive study of PAX6 3’-cis regulatory abnormalities that can map hundreds of kilobases from the PAX6 gene locus.16,41–44 It was noted that these cases differed clinically from others in this study in that iris anomalies were milder, and none exhibited fovea hypoplasia. It should be noted, however, that some PAX6 mutant aniridia cases were seen with partial aniridia (A11 and A18) and normal foveal architecture (A13), although not with both. Previous reports have suggested non-PAX6 aniridia-like phenotypes associated with FOXC1 and PITX2.30 However, our screen failed to identify abnormalities of these genes or in the functionally related SCL38A8 gene. A review of these past cases noted that none have reported classical ocular aniridia (complete absence of iris tissue, juvenile onset glaucoma, epithelial keratopathy, cataract, and frequent foveal hypoplasia). FOXC1 mutation was associated with partial aniridia, aniridia with corneal disease (Peter’s anomaly), or aniridia with congenital glaucoma (not normally present in classic aniridia). Furthermore, foveal hypoplasia was not reported in these cases.19,45–47 Similarly, in the 2 PITX2 mutation patients, the phenotypic descriptions were limited with no report on foveal architecture.17,48 It is striking to note that in our study all aniridia cases with PAX mutation had abnormal
Fig. 4 — Chromosome deletions in patients without PAX6 coding mutations identified in this study. The gene organization of chromosome 11p is represented (not to scale) with horizontal arrows denoting the direction of gene transcription. DRR (vertical arrows) is the DNA regulatory region containing hypersensitivity sites. D11S914 denotes microsatellite marker. Gray bars indicate the extent of deletion in each patient as detected by multiplex ligation-dependent probe amplification analysis. White bars indicate deletions in patients as determined by cytogenetics analysis. Patient ID number is denoted inside each bar.
Aniridia and PAX6 mutation—Sannan et al. foveal architecture. This calls into doubt the existence of aniridia without PAX6 mutation and suggests that these other cases might more correctly be labelled as anterior segment dysgenesis, in which the aniridia is only a physical sign of a lack of iris tissue and not indicative of the genetic syndrome of aniridia. This genetic syndrome of aniridia could therefore be defined as a condition with a PAX6 defect, anterior segment dysgenesis, and foveal hypoplasia (with additional high risk of keratopathy, cataract, glaucoma, and systemic defects). A larger survey of aniridia patients needs to be undertaken to verify this. Although PAX6 genetic abnormalities were first identified more than 2 decades ago, only now is the value of their identification coming to the fore. Screening for 11p13 chromosomal deletions is now routine in patients diagnosed with aniridia because it identifies those at risk for Wilms tumor, prompting regular kidney ultrasound surveillance. Sequencing of the PAX6 gene is often limited, however, because it is seen as having a lesser impact on medical management. This may be about to change as new concepts in therapeutics emerge. We recently reported a small-molecule, nonsense-suppression therapy in the Pax6Sey mouse model of aniridia49 that has now progressed to clinical trials (ClinicalTrials.gov identifier: NCT02647359). Approximately 40% of those diagnosed with aniridia are identified as having PAX6 nonsense mutations50; this makes this therapy potentially useful to a significant proportion of individuals with the disease. Such mutation-based therapeutic options mandate more widespread molecular genetic testing and new natural history studies to more closely examine the clinical variability seen in this and other studies.
APPENDIX A. SUPPORTING
INFORMATION
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j. jcjo.2017.04.006.
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Footnotes and Disclosure: The authors have no proprietary or commercial interest in any materials discussed in this article. This work was supported by the Sharon Stewart Trust and a scholarship from the Saudi Cultural Bureau. The authors thank Laura Hall for optical coherence tomography imaging support. From the nDepartment of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, B.C; †Department of Ophthalmology, BC Children’s Hospital, Vancouver, B.C; ‡Department of Medical Genetics, University of British Columbia, Vancouver, B.C; §Cumming School of Medicine, University of Calgary, Calgary, Alta. Originally received Oct. 27, 2016. Final revision Mar. 24, 2017. Accepted Apr. 5, 2017. Correspondence to Kevin Gregory-Evans MD, PhD, Department of Ophthalmology and Visual Sciences, University of British Columbia, 2550 Willow Street, Vancouver BC, V5Z 3N9, Canada; [email protected].