Mutations of the PAX6 gene detected in patients with a variety of optic-nerve malformations

Abstract

The PAX6 gene is involved in ocular morphogenesis and is expressed in the developing central nervous system and numerous ocular tissues during development. PAX6 mutations have been detected in various ocular anomalies, including aniridia, Peters anomaly, corneal dystrophy, congenital cataracts, and foveal hypoplasia. However, it has not been identified in patients with optic-nerve malformations. Here, we identified novel mutations in eight pedigrees with optic-nerve malformations, including coloboma, morning glory disc anomaly, optic-nerve hypoplasia/aplasia, and persistent hyperplastic primary vitreous. A functional assay demonstrated that each mutation decreased the transcriptional activation potential of PAX6 through the paired DNA-binding domain. PAX6 and PAX2 are each thought to downregulate the expression of the other. Four of the detected mutations affected PAX6-mediated transcriptional repression of the PAX2 promoter in a reporter assay. Because PAX2 gene mutations were detected in papillorenal syndrome, alternation of PAX2 function by PAX6 mutations may affect phenotypic manifestations of optic-nerve malformations.

Figure 1

a, Fundus photography (panel 1) and fluorescein angiography (panel 2) of the right eye of patient 1, showing a morning glory disc anomaly. The optic-nerve region is excavated, and white tissue is present at the bottom. Panel 3, Mutation analysis identified 619C→T in exon 6 (P68S). b, Fundus photography (panel 1) and fluorescein angiography (panel 2) of the right eye of patient 2, showing a small optic-nerve head surrounded by a hypopigmented ring. Panel 3, Mutation detection of 1030C→T in exon 8 (Q205X). c, Photographs showing the anterior segments (panel 1 and panel 4) and fundus (panel 2, panel 3, and panel 5) of patient 3. The iris was dysplastic and the posterior fundus showed large coloboma of the optic nerve, retina, and choroid. Hyaloid vessel proliferation is seen in the vitreous cavity. Panel 6, Mutation detection of 1190T→C in exon 10 (F258S). d, Fundus photography (panel 1) and fluorescein angiography (panel 2) of the right eye of patient 4, showing ONH. Panel 3, Mutation detection of 1292G→T in exon 10 (S292I). e, The right eye of patient 5 has a slight corneal opacity and iridocorneal adhesion (panel 1) and deep excavation of the optic-nerve head (3). The left eye has substantial corneal opacity (panel 2). Panel 4, Mutation detection of 1504T→C in exon 12 (S363P). f, The anterior segment (panel 1), fundus photography (panel 2), and fluorescein angiography (panel 3) of the left eye, with optic-nerve aplasia, of patient 6. The iris was dysplastic, and the optic-nerve head and retinal vessels were absent, with a remnant of the hyaloid vessels. Panel 4, Mutation detection of 1550A→G in exon 12 (Q378R). g, Fundus photography (panel 1) and fluorescein angiography (panel 2) of the left eye, with ONH, of patient 7. Panel 3, Mutation analysis identified 1558A→G in exon 12 (M381V). h, Fundus photography (panel 1) and fluorescein angiography (2) of the left eye, with optic-nerve aplasia, of patient 8. c, Mutation analysis identified 1588A→G in exon 12 (T391A).

Figure 2

Effects of PAX6 on PAX2 (a) and of PAX2 on PAX6 (c). CAT activities were measured in P19 cells after cotransfection of effecter and reporter constructs. Total volume of DNA was adjusted with an empty vector, pBluescript. Cell extracts were prepared after 48 h and assayed for CAT activities by use of a FAST CAT Green Reagent (Molecular Probes). The CAT activity was quantified by measurement with a phospho-fluor-imager (Molecular Dynamics) and illustrated in a fold-activation, compared with the condition with the vector alone. The levels of PAX2 were suppressed with increasing amounts of wild type of PAX6, and vice versa. b, Transactivating potential of PAX6 mutants. 0.1 μg of effecter construct and 1 μg of reporter constructs were cotransfected in P19 cells. Transcription level from P6CON was disturbed significantly by P68S, Q205X, F258S, and S292I mutants and slightly by S363P, Q378R, M381V, and T391A mutants. d, Effects of PAX6 mutants on PAX2 expression. One μg of effecter construct and 1 μg of reporter constructs were cotransfected in P19 cells. The decreasing level of PAX6 was significantly disturbed with the P68S, Q205X, S292I, and M381V mutants. Each photograph of CAT assay under the bar graph is representative of at least three independent experiments.