phenotypic variations in patients with a 1630 a>t point mutation in the pax6 gene

Phenotypic variations in patients with a 1630 A>T point mutation in the PAX6 gene

Inge De Becker, * MD, FReSe; Michael Walter, t PhD; Leon-Paul Noel,:j: MD, FReSe

ABSTRACT • RESUME

Background: Th~ extreme clinic.al case-tq-cas~ variability of aniridia, even within families, can cause difficulties in making the correct diagnosis, prognosis and treatment plan. We describe seven patients from two families demonstrating variable expression of ,this ,syndrome, all with the same single point mutation within the PAX6 gene.

Methods: Case presentations. The authors review the clinical ophthalmic findings of the aniridia-keratopathy syndrome from two families, one involving four generations, the other with two generations. Polymerase chain reaction amplificatiqn of all 14 exons of the PAX6 gene was performed for five patients.

Results: The iris findings varied from classic total absence to nearly normal iris appearance. Corneal changes were minimal in the younger patients but visionimpairing in the older. None of the patients had glaucoma. The five patients who underwent DNA analysis shared the same PAX6 point mutation defect (1630 A> T).

Interpretation: Bilateral corneal changes progressing from mild opacification at the limbus to vascularized central keratopathy, especially in the presence of nystagmus, are highly suggestive of a PAX6 mutation. Such patients may theoretically benefit from early limbal stem cell replacement therapy.

Contexte: L'extreme variabilite clinique de I'aniridie, meme au sein d'une famille, peut rendre Ie diagnostic, Ie pronostic et Ie plan de traitement difficiles a faire avec exactitude. La description de sept patients de deux families montre les diverses expressions du syndrome, toutes a partir d'une seule et meme mutation ponctuelle du gene PAX6.

Methodes: Presentation de cas. Les auteurs passent en revue les donnees ophtalmologiques cliniques du syndrome d'aniridie et de keratopathie dans deux families, I'une sur quatre generations et I'autre, sur deux. On a effectue I'amplification en chaine par polymerase des 14 exons du gene PAX6 chez cinq patients.

From the Departments of Ophthalmology, *Dalhousie University,

Halifax, NS, tUniversity of Alberta, Edmonton, Alta., and *State

University of New York Upstate Medical University, Syracuse, NY

Originally received Mar. 10, 2003

Accepted for publication Nov. 20,2003

272 PAX6 phenotypic variations-De Becker et a1

Correspondence to: Dr. Leon-Paul Noel, Department of Ophthalmology, State University of New York Upstate Medical University, 1101 Erie Blvd. E, Syracuse NY 13210, USA; fax (315) 422-4690, [email protected]

This article has been peer-reviewed.

Can J Ophthalmol 2004;39:272--8

PAX6 phenotypic variations-De Becker et al

Resultats : Les constatations sur I'iris ont varie entre la classique absence totale et I'apparence presque normale de I'iris. Les changements de la cornee etaient minimes chez les jeunes patients mais affaiblissaient profondement la vue chez les plus vieux.Aucun des patients ne souffraient de glaucome. Les cinq patients dont on a analyse I' ADN partageaient Ie me me defaut de mutation ponctuelle du gene PAX6 (1630 A> T).

Interpretation: Les changements bilateraux de la cornee, progressant d'une faible opacification du limbe jusqu'a une keratopathie centrale vascularisee, surtout en presence de nystagmus, semblent indiquer nettement une mutation du gene PAX.6. Ces patients pourraient en principe beneficier d'une greffe precoce de la cellule souche du systeme limbique.

Aniridia is an uncommon panocular hereditary disease that can affect development not only of

the iris but also of the cornea, anterior chamber angle, lens, retina and optic nerve.1 Profound vision loss due to a combination of ocular abnormalities may be present from birth. These include macular hypoplasia, abnormal retinal function,2 cataracts, nystagmus and iris aplasia. The residual subnormal vision may progressively worsen because of glaucoma, cataracts or corneal changes.

Aniridia occurs in the general population at a frequency of 1164 000 to 1196 000 live births.3 About two-thirds of cases are inherited in an autosomal dominant manner with almost complete penetrance but with variable expressivity even within families. The remaining one-third of cases are sporadic.

In sporadic cases of aniridia there is a 30% risk of development of Wilms' tumour, a high-grade malignant disease. A small number of sporadic cases are associated with WAGR syndrome (Wilms' tumour, aniridia, genitourinary anomalies and mental retardation), which results from an interstitial deletion of chromosome 11 p 13 . Positional cloning has led to the isolation of complementary DNA clones of the gene predisposing to Wilms' tumour (WTl) and the aniridia candidate gene, PAX6.4,5 The WTl and PAX6 genes are about 750 kb apart, the former being centromeric to the latter. Early diagnosis of Wilms' tumour in children with sporadic aniridia requires sequential renal ultrasound examinations until the age of 5 to 7 years. A rapid test bas~d on the polymerase chain reaction (peR) is now available to rule out a chromosome llp13 deletion covering the PAX6 to WTl region.6

Aniridia is caused by mutations affecting the PAX6 gene located on chromosome llp13.7,8 At the molecular level, aniridia is a haploinsufficiency disorder

caused by the loss of function of one genome copy of the PAX6 gene. This may occur through a point mutation or a chromosomal deletion. Inactivation of both copies of the PAX6 gene is lethal.9,10

The extreme clinical case-to-case variability of aniridia, even within families, can cause difficulties in making the correct diagnosis, prognosis and treatment plan. We describe seven patients from two families demonstrating variable expression of this syndrome, all with the same single point mutation within the PAX6 gene.

CASE REPORTS

Family I

Patient 1

The propositus of this family, IV-l (Fig. 1), an only child, was seen at age 1 year by a pediatric ophthalmologist, who noted classic absence of iris, foveal hypoplasia, anterior polar cataracts, clear corneas and

II

III

IV

Fig. I-Pedigree of family I. Circles = females; squares = males; black figures = affected; diagonal line = deceased. Arrow indicates index case.

CAN J OPHTHALMOL-VOL. 39, NO.3, 2004 273


horizontal nystagmus. The boy's father was reported to have a history of retinitis pigmentosa and to have been blind since infancy, but this could not be confirmed as the father was estranged from the immediate family. Ophthalmic follow-up was erratic, but the boy was reassessed at age 9 years at the request of pediatric immunology, who were trying to link the child's eye findings to his repeated pulmonary infections. His visual acuity was 20/100 in both eyes, and at near he saw 20/70 in the right eye and 20/100 in the left eye. With both eyes open he read 20/100 at distance and 20/70 at near. Krimsky's light reflex showed exotropia of 10 prism dioptres. The extraocular movements were full, but there was constant searching horizontal nystagmus. The lids and conjunctiva appeared normal. The central corneas were clear, although there was fine peripheral vascular pannus measuring 1.5 mm involving the entire limbus. The anterior chambers were clear. The right iris was totally absent, and the left iris had only a small, thin iris stump from the 1 to 5 0' clock position in the left eye. A dense anterior polar opacity was present in both lenses (Fig. 2). The cycloplegic refraction was +0.75 +2.00 x 90° in the right eye and +0.50 +1.75 x 90° in the left. On fundus examination the discs, vessels and periphery appeared normal, but there was foveal hypoplasia bilaterally. No change was noted over 2 years of follow-up.

Patient 2

The mother (III-2) of the index case was originally seen in the residents' clinic at the age of 27 years with a best-corrected visual acuity of 20/400 (refraction -1.25 + 1.00 x 85°) in the right eye and 20/200 (refraction -2.00 +2.75 x 101°) in the left eye. At near with a +4.75 add she read 20/30 binocularly. There was large constant roving horizontal nystagmus. There was no conjunctival injection, but significant vascular corneal pannus was present inferiorly in both eyes. The intraocular pressure was 18 mm Hg in the right eye and 19 mm Hg in the left. A persistent pupillary membrane was noted on the left as well as foveal hypoplasia bilaterally.

The patient was lost to follow-up for 6 years. She then presented because of an intermittent foreign-body sensation for the previous 3 years. Her corrected visual acuity was 20/400 in the right eye and 20/200 in the left eye. At near she read 20/70 in the right eye and 20/400 in the left. With both eyes open there was no improvement in reading vision. Krimsky's light reflex

274 CAN J OPHTHALMOL-VOL. 39, NO.3, 2004

Fig. 2-Patient I: Left eye: Total aniridia with central anterior polar cataract. Note iris remnant from I to 5 o'clock.

showed exotropia of 20 prism dioptres, and constant roving horizontal nystagmus was noted. On slit-lamp examination peripheral pannus for 360° of the right cornea was noted as well as an anterior stromal opacity extending toward the centre of the visual axis. The left cornea had anterior and midstromal opacification into which multiple vessels were directed from the 3 to 6 o'clock position. The anterior chamber was clear bilaterally. The right iris appeared normal, but the left iris had an area of loss of anterior stroma extending from the pupillary margin to the angle at the 5 to 6 o'clock position. Gonioscopy was not possible. Retinoscopy was no longer possible in the left eye because of the corneal scar. The cycloplegic refraction in the right eye was -1.50 +3.00 x 10°. The lens appeared normal in both eyes. Fundus examination, although difficult, showed a poor macular reflex bilaterally. No retinal pigment epithelial clumping was identified. Electroretinography (ERG) was attempted, without success because of poor patient compliance.

Patient 3

The maternal grandmother (II-3) of the index case was originally seen in the residents' clinic at the age of 47 years. She stated that she had had good vision until the age of 40. An optometrist had last seen her 9 years previously. Her eyeglass correction at the time of presentation was +9.00 in the right eye and +8.00 in the left eye. Her mother, the only one of 12 children with vision problems, had had poor vision since about the age of 5 years because of "measles." Her father is described below (patient 5). The patient has three sisters, all in the United Kingdom, two with normal

vision and one (patient 4) with heterochromia irides (one blue iris and the other green) who is legally blind because of corneal problems. Other than the daughter described above, patient 3 has a son, who has normal vision and works as a chauffeur.

The residents' clinic records indicate a visual acuity of counting fingers at 4 feet (1.2 m) in both eyes. The intraocular pressure was 10 mm Hg in the right eye and 12 mm Hg in the left eye. There was roving horizontal nystagmus as well as large exotropia. Corneal pannus for 360° was noted in both eyes, with vessels extending into the central cornea, and anterior and midstromal opacification. The anterior chamber was still visible and appeared clear. An iris coloboma was noted from the 5 to 6:30 position in the right eye. The left iris appeared normal. Fundus examination was not possible, but B-scan Ultrasonography gave normal results.

At age 53, visual acuity was limited to hand movements in both eyes. There was constant searching, roving nystagmus. Lid xanthelasma was present on all four lids, and there was mild conjunctival injection of both eyes. The corneal scarring was quite marked, with prominent vascularization of both corneas. A partial coloboma of the iris from the 5 to 6:30 position was still visible in the right eye. The left pupil was miotic, but the iris itself appeared normal. Dense nuclear sclerotic cataracts were present in both eyes, which, along with the corneal abnormalities, obstructed any view of the fundus.

Patient 4

The younger sister (II-5) of patient 3 resides in the United Kingdom and was not examined by the authors. She was originally seen at the age of 3 months for suspicion of blindness. Findings were poor fixation in both eyes, poorly reactive pupils and bilateral anterior polar cataracts. There is no mention of corneal scarring until the patient was seen again, at age 17 years. The ophthalmologist in England who has personally followed her since the age of 37 describes counting-finger vision in either eye and nystagmus as well as a congenital coloboma of the right iris, a left anterior polar cataract and bilateral corneal opacification with vascularization. No retinal details are available. She has no offspring.

Patient 5

Patient 5 (1-1) is the deceased father of patients 3


and 4. Patient 4's chart contains a note by a consultant: "I knew this patient's father. He had congenital coloboma, congenital cataracts, nystagmus and vascularized corneas." Patient 5 had a seeing-eye dog when he died, at age 54.

Family 2

Patient 6

The propositus of the second family was first seen at the age of 7 weeks. He was referred by a pediatric neurologist for assessment of large-amplitude pendular horizontal nystagmus. The infant would follow and fixate with difficulty. The pupils were round and slightly irregular at 6 o'clock but well centred. They did not constrict well and measured 2.5 mm when tested with the indirect ophthalmoscope. The pupils did dilate well with 1 % cyclopentolate. The corneas were clear, and slit-lamp examination of the irides, which were blue, showed subtle stromal hypoplasia as well as Brushfield's spots. Fundus examination with the pupils dilated showed the optic nerve to be small but not hypoplastic; macular hypoplasia was also present. ERG showed a rod and cone response of low amplitude, giving the ERG a nearly flat appearance.

At the age of 9 years the boy's distance best-corrected visual acuity was 20/200 in both eyes, and the near visual acuity was 20/150 in the right eye and 20/200 in the left eye. With both eyes open the visual acuity was 20/150 at distance and 20/60 at near. The horizontal nystagmus was variable, sometimes beating right and sometimes beating left. The child would assume a variable head tum to decrease the amplitude of the nystagmus. The extraocular motility was normal. The cycloplegic refraction was plano +3.00 x 85° in the right eye and plano +2.50 x 99° in the left. The lenses were clear. Peripheral keratopathy was present at the inferior limbus that had progressed to 1.5 mm from less than 1.0 mm at age 7. The intraocular pressure was normal in both eyes (16 mm Hg). Fundus examination showed definite macular hypoplasia bilaterally. Compared to the ERG at age 7 weeks, that at age 9 was no longer flat but still showed decreased rod and cone amplitudes.

Patient 7

Patient 7, the mother of patient 6, is adopted but has been followed since early childhood for congenital



nystagmus and bilateral corneal changes. When she first presented with her infant son she had blepharospasm, photophobia and fine bilateral nystagmus. The best-corrected visual acuity was counting fingers at 0.5 m in the right eye and 20/400 in the left. The intraocular pressure was normal bilaterally, at 17 mm Hg. Slit-lamp examination of the right eye showed keratopathy involving the entire corneal surface, including the visual axis. The left cornea had milder keratopathy involving the limbal area superiorly and inferiorly, but the central visual axis remained clear (Fig. 3). The pupils were round and the irides, which were brown, appeared normal. There were moderate bilateral cortical lens opacities. ERG showed decreased rod and cone amplitudes.

At the age of 39 years the patient underwent right corneal grafting, but the corneal button scarred over, and the patient was again left with poor vision. Fundus examination was attempted in the left eye, but a definite diagnosis of macular hypoplasia could not be made.

METHODS

After obtaining patient or parental consent for genetic analysis, we took blood samples from both members of family 2 in 1993 and from patients 1, 2 and 3 in family 1 in 1999. Blood samples were collected in ethylenediamine tetraacetic acid tubes, and DNA was prepared from isolated leukocytes by standard organic solvent extraction procedures. ll PCR amplification of all 14 exons of the PAX6 gene was done as described by Mirzayans and colleagues. 11

PCR products were purified with QIAquick PCR Purification Kit columns (QIAGEN Inc. - Canada, Mississauga, Ont.), then directly sequenced by means of 33p cycle sequencing (Thermo Sequenase 33p

Terminator Cycle Sequencing Kit, Amersham Biosciences, Inc., Baie d'Urfe, Que.).

RESULTS

Visual acuity was best in the two children (patients 1 and 6) and worse in the adults. Corneal changes also showed worsening with increasing age, and in patient 6 definite progression of the pannus was documented over 2 years. Nystagmus was common to all. No patient had glaucoma.

Iris involvement was extremely variable, ranging from classic aniridia (patient 1) with almost total


Fig. 3-Patient 7: Left eye:Vascularized stromal opacity involving inferior third of cornea. Note normal-appearing iris.

absence of the iris to iris coloboma only (patients 3, 4 and 5) to a normal-appearing iris (patient 7). Lens appearance ranged from clear to congenital anterior polar opacities to dense total opacification.

Fundus examination was possible in four patients. Foveal hypoplasia was identified in three, and none showed classic optic nerve hypoplasia with the double-ring sign (Table 1). The ERG was abnormal in the two patients who were successfully tested.

In all five patients tested, direct DNA sequence analysis of the PAX6 gene revealed a mutation in exon 13, with a transversion from an adenine to a thymine at position 1630 (1630 A>T).

INTERPRETATION

Patients 4 and 5 were not examined by us and did not undergo DNA analysis. The clinical description provided by their ophthalmic records indicated that they most likely had the same condition as their direct relatives who have the PAX6 (1630 A>T) point mutation.

Visual acuity is very limited in all seven patients, showing progressive deterioration with increasing age. Ultimately the vision is hand motion because of the lens changes but, more significantly, because of the corneal opacification and vascularization. These findings underline the need to suspect aniridia or, more specifically, a PAX6 abnormality in a patient with bilateral progressive keratopathy of unknown type. The iris changes may be very subtle, as in our


Table I-Clinical findings in seven patients from two families with a 1630 A>T point mutation in the PAX6 gene*

Age at last Keratopathy

examination, Acuity Iris Macular Patient no. yr < 201100 Peripheral Central changes Nystagmus hypoplasia

Family I I 9 + + - + + + 2 34 + + + + + + 3 53 + + + + + UN 4 50 + + + + + UN 5 54 +t + + + + UN Family 2 6 9 + + - + + + 7 41 + + + - + ?

*UN = unable to visualize fundus; ? = not confirmed. tPatient deceased, but vision presumed to be less than 20/200 because of use of seeing-eye dog.

patients 6 and 7, or may present as a coloboma, as in patients 3, 4 and 5.

The PAX6 gene is one of nine members of the PAX gene family and is found in most or all members of the animal kingdom. PAX6 is transcribed as a 2.07-kb messenger RNA and encodes 422 amino acids. The gene product acts as a transcriptional regulator. In mice, PAX6 is expressed in the developing neuroectoderm and subsequently in the developing eye and nasal tissue as well as in the forebrain, hindbrain and neural tube.10,12,13 The PAX6 gene is thus considered a master control gene for eye development. More than 200 different mutations in the PAX6 gene have been reported as an underlying cause of aniridia; most are truncating mutations. Missense mutations occur less frequently and demonstrate a variant form of aniridia, such as autosomal dominant keratopathy (as noted in our families), Peters' anomaly, foveal hypoplasia and nystagmus. 14

Although our patients carry the same single base mutation (1630 A>T), phenotypic variations run the gamut, from classic aniridia with nearly total iris absence to a predominantly corneal problem with minimal iris involvement. This phenotypic variability may be the result of modifying effects of other genes but may also be explained solely by the PAX6 mutation.

PAX6 mutations have been classified into amorphic (i.e., nonfunctioning), hypomorphic (weaker loss of function) and neomorphic (gain of function or dominant-negative) alleles. 15,16 If this (1630 A>T) point

mutation resulted in an amorphic nonfunctioning allele, the phenotypic differences in our patients would be due solely to variations in the expression of the wild type (more normal) PAX6 allele.

However, the worsening corneal changes noted with increasing age in our patients support a neomorphic or a hypomorphic allele alteration. The (1630 A> T) point mutation replaces a stop codon with an amino-acid-generating PAX6 protein that is substantially longer than normal. The buildup of this abnormal protein in the corneal limbal stem cells could result in abnormallimbal stem cells that fail to differentiate throughout the patient's lifetime into normal transparent corneal epithelium. The cornea slowly becomes covered by a conjunctival opaque epithelium. This limbal stem cell abnormality may also explain the poor response to penetrating keratoplasty in our patient 7 and other patients,17 as in these cases defective epithelial cells derived from the abnormal limbal stem cells again cover the engrafted tissue. This has important theoretical implications for treatment. Remedial or prophylactic limbal stem cell allograft therapy may be indicated in the younger members of these families before irreversible corneal changes compromise the residual vision.

Although glaucoma is an important cause of progressive visual loss in aniridia, none of our patients had increased intraocular pressure.

All our patients had nystagmus. Nystagmus in patients with aniridia has implicitly been considered secondary to poor development of central vision due



to foveal hypoplasia or abnormal retinal function.2

The presence of nystagmus before age 7 weeks in our patient 6 lends support to the theory that nystagmus in aniridia is a developmental defect in the neural control of eye movements rather than having a sensory cause. 14

Fundus examination is a challenge in patients with nystagmus, photophobia, keratopathy and cataracts. In our four patients in whom the fundi could be examined, no optic nerve hypoplasia was found, but macular hypoplasia was noted in three of the four patients.

In conclusion, typical bilateral corneal changes manifesting subtly at the limbus and progressing over time are just as suggestive of a PAX6 mutation as are the iris anomalies classically described in aniridia, particularly if these corneal changes are associated with nystagmus. Once properly identified, such patients may theoretically be prime candidates for early limbal stem cell replacement therapy before central corneal opacification occurs.

We thank Jody Marshall, Ocular Genetics Laboratory, University of Alberta, for technical assistance with this project.

REFERENCES

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2. Tremblay F, Gupta SK, De Becker I, Guernsey DL, Neumann PE. Effect of PAX6 mutations on retinal function: an electroretinographic study. Am J Ophthalmol 1998;126:211-8.

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a paired box- and homeobox-containing gene from the aniridia region. Cell 1991;67:1059-74.

6. Gupta SK, De Becker I, Guernsey DL, Neumann PE. Polymerase chain reaction-based risk assessment for Wilms tumor in sporadic aniridia. Am J Ophthalmoll998; 125:687-92.

7. Glaser T, Walton DS, Maas RL. Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene. Nat Genet 1992;2:232-9.

8. Davis A, Cowell JK. Mutations in the PAX6 gene in patients with hereditary aniridia. Hum Mol Genet 1993;2: 2093-7.

9. Hill RE, Favor J, Hogan BL, Ton CCT, Saunders GF, Hanson 1M, et al. Mouse small eye results from mutations in a paired-like homeobox-containing gene. Nature 1991;354:522-5.

10. Glaser T, Jepeal L, Edwards JG, Young SR, Favor J, Maas RL. PAX 6 gene dosage effect in a family with congenital cataracts, aniridia, anophthalmia and CNS defects. Nat Genet 1994;7:463-71.

11. Mirzayans F, Pearce WG, MacDonald 1M, Walter MA. Mutation of the PAX6 gene in patients with autosomal dominant keratitis. Am J Hum Genet 1995;57:539--48.

12. Walther C, Gruss P. PAX6, a murine paired box gene, is expressed in the developing CNS. Development 1991; 113:1435--49.

13. Halder G, Callaerts P, Gehring WJ. Introduction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 1995;267: 1788-92.

14. Sonoda S, Isashiki Y, Tabata Y, Kimura K, Kakiuchi T, Ohba N. A novel PAX6 gene mutation (P118R) in a family with congenital nystagmus associated with a variant form of aniridia. Graefes Arch Clin Exp Ophthalmol 2000;238:552-8.

15. Glaser T, Walton DS, Cai J, Epstein JA, Jepeal L, Mass RL. PAX6 gene mutations in aniridia. In: Wiggs JL, editor. Molecular genetics of ocular disease. New York: Wiley-Liss; 1995. p. 51-82.

16. Gupta SK, De Becker I, Tremblay F, Guersney DL, Neuman PE. Genotype/phenotype correlations in aniridia. Am J OphthalmolI998;126:203-1O.

17. Kremer I, Rajpal RK, Rapuano CJ, Cohen EJ, Laibson PRo Results of penetrating keratoplasty in aniridia. Am J OphthalmolI993;115:317-20.

Key words: aniridia, iris coloboma, keratopathy, nystagmus, PAX6 gene, phenotypic variations

phenotypic variations in patients with a 1630 a>t point mutation in the pax6 gene

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