Human Aniridia Research Articles

PAX6 mutations reviewed

Mutations inPAX6 are responsible for human aniridia and have also been found in patients with Peter's anomaly, with congenital cataracts, with autosomal dominant keratitis, and with isolated foveal hypoplasia. No locus other than chromosome 11p13 has been implicated in aniridia, and PAX6 is clearly the major, if not only, gene responsible. Twenty-eight percent of identified PAX6 mutations are C–T changes at CpG dinucleotides, 20% are splicing errors, and more than 30% are deletion or insertion events. There is a noticeably elevated level of mutation in the paired domain compared with the rest of the gene. Increased mutation in the homeodomain is accounted for by the hypermutable CpG dinucleotide in codon 240. Very nearly all mutations appear to cause loss of function of the mutant allele, and more than 80% of exonic substitutions result in nonsense codons. In a gene with such extraordinarily high sequence conservation throughout evolution, there are presumed undiscovered missense mutations, these are hypothesized to exist in as-yet unidentified phenotypes. Hum Mutat 11:93–108, 1998. © 1998 Wiley-Liss, Inc.

The Rx homeobox gene is essential for vertebrate eye development.

Development of the vertebrate eye requires a series of steps including specification of the anterior neural plate, evagination of the optic vesicles from the ventral forebrain, and the cellular differentiation of the lens and retina. Homeobox-containing genes, especially the transcription regulator Pax6, play a critical role in vertebrate and invertebrate eye formation. Mutations in Pax6 function result in eye malformations known as Aniridia in humans and Small eye syndrome in mice. The Drosophila homologue of Pax6, eyeless, is also necessary for correct invertebrate eye development, and its misexpression leads to formation of ectopic eyes in Drosophila. Here we show that a conserved vertebrate homeobox gene, Rx, is essential for normal eye development, and that its misexpression has profound effects on eye morphology. Xenopus embryos injected with synthetic Rx RNA develop ectopic retinal tissue and display hyperproliferation in the neuroretina. Mouse embryos carrying a null allele of this gene do not form optic cups and so do not develop eyes. The Rx gene family plays an important role in the establishment and/or proliferation of retinal progenitor cells.

Transcriptional Regulation of the Human PAX6 Gene Promoter

PAX6, a member of the highly conserved paired-type homeobox gene family, is expressed in a spatially and temporally restricted pattern during early embryogenesis, and its mutation is responsible for human aniridia. Here we examined the transcriptional regulation of the PAX6 gene by transient transfection assays and identified multiple cis-regulatory elements that function differently in different cell lines. The transcriptional initiation site was identified by RNase protection and primer extension assay. Examination of the genomic DNA sequence indicated that the PAX6 promoter has a TATA like-box (ATATTTT) at -26 base pairs (bp), and two CCAAT boxes are positioned at -70 and -100 bp. A 38-bp poly(CA) sequence was located 992 bp upstream from the initiation site. Transient transfection assays in glioblastoma cells and leukemia cells indicate that a 92-bp region was required for basal level PAX6 promoter activity. A negative transcriptional element, silencer (bases -1518 to -1268), functioned differently in different cell lines. The activation of the promoter is positively correlated with the expression of PAX6 transcripts in all cells tested. These results indicate that a cis-regulatory element or elements is responsible for selective activation of the PAX6 promoter in cells that can express PAX6 mRNA.

The master control gene for morphogenesis and evolution of the eye.

The human Aniridia, the murine Small eye, and the eyeless mutations of Drosophila affect homologous (Pax-6) genes that contain both a paired- and a homeobox. By ectopic expression of these genes, functional eyes can be induced on the legs, wings, and antennae of the fly, indicating that eyeless (Pax-6) is the master control gene for eye morphogenesis. The finding of Pax-6 from flatworms to humans suggests that eyeless is a universal master control gene and that the various types of eyes in the various animal phyla may have evolved from a single prototype.

Specification of sense-organ identity by a Caenorhabditis elegans Pax-6 homologue.

The Pax-6 transcription-factor gene, containing a paired domain and a paired-type homeodomain, is conserved in structure and ubiquitously present among Metazoa. It is required for development of the central nervous system, and is mutated in human aniridia, mouse and rat small eye and Drosophila eyeless. We identified the Pax-6 gene of the nematode Caenorhabditis elegans in genetic studies of male tail morphology. C. elegans Pax-6 encodes at least two independent genetic functions. One, like other Pax-6 genes, contains paired and homeodomains; this constitutes the genetic locus vab-3. The other, described here, is expressed from an internal promoter and contains only the homeodomain portion; this constitutes the genetic locus mab-18. The mab-18 form of the gene is expressed in a peripheral sense organ and is necessary for specification of sense-organ identity. Its function in this context could be to regulate the expression of cell recognition and adhesion proteins required for sense-organ assembly.

Three novel aniridia mutations in the human PAX6 gene

Aniridia (iris hypoplasia) is an autosomal dominant congenital disorder of the eye. Mutations in the human aniridia (PAX6) gene have now been identified in many patients from various ethnic groups. In the study reported here we describe PAX6 mutations in one sporadic and five familial cases with aniridia. Of the four different mutations identified, one was identical to a previously reported mutation (C-->T transition at codon 240), and three were novel: two in the glycine-rich region and one in the proline/serine/threonine-rich (PST) region. One PAX6 mutation found in the PST region was associated with cataracts in an aniridia family. Another splice mutation in the PST domain occurred in an aniridia patient with anosmia (inability to smell). The six new aniridia cases reported here have mutations predicted to generate incomplete PAX6 proteins. These results support the theory that human aniridia is caused by haploinsufficiency of PAX6.

Molecular analysis of aniridia patients for deletions involving the Wilms' tumor gene.

A human aniridia candidate (AN) gene on chromosome 11p13 has been cloned and characterized. The AN gene is the second cloned gene of the contiguous genes syndrome WAGR (Wilms' tumor, aniridia, genitourinary malformations, mental retardation) on chromosome 11p13, WT1 being the first gene cloned. Knowledge about the position of the AN and WT1 genes on the map of 11p13 makes the risk assessment for Wilms' tumor development in AN patients possible. In this study, we analyzed familial and sporadic aniridia patients for deletions in 11p13 by cytogenetic analyses, in situ hybridization, and pulsed field gel electrophoresis (PFGE). Cytogenetically visible deletions were found in 3/11 sporadic AN cases and in one AN/WT patient, and submicroscopic deletions were identified in two sporadic AN/WT patients and in 1/9 AN families. The exact extent of the deletions was determined with PFGE and, as a result, we could delineate the risk for Wilms' tumor development. Future analyses of specific deletion endpoints in individual AN cases with the 11p13 deletion should result in a more precise risk assessment for these patients.

Two independent and interactive DNA-binding subdomains of the Pax6 paired domain are regulated by alternative splicing.

Vertebrate Pax proteins share a conserved 128-amino-acid DNA-binding motif, the paired domain. The PAX6 gene, which is mutated in the murine Small eye and human aniridia developmental defects, also encodes a second protein with a 14-amino-acid insertion in the paired domain. This protein, which arises by alternative mRNA splicing, exhibits unique DNA-binding properties. Unlike other paired domains, which bind DNA predominantly by their amino termini, the extended Pax6 paired domain interacts with DNA exclusively through its carboxyl terminus. This property can be stimulated by deletion of 30 amino-terminal residues from the Pax6 or Pax2 paired domains. Thus, the insertion acts as a molecular toggle to unmask the DNA-binding potential of the carboxyl terminus. The functional nonequivalence of the two Pax6 proteins is underscored by a T-->C mutation at position -3 of the alternative splice acceptor site that changes the ratio of the two isoforms and causes a distinct human ocular syndrome.

Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans.

A Drosophila gene that contains both a paired box and a homeobox and has extensive sequence homology to the mouse Pax-6 (Small eye) gene was isolated and mapped to chromosome IV in a region close to the eyeless locus. Two spontaneous mutations, ey2 and eyR, contain transposable element insertions into the cloned gene and affect gene expression, particularly in the eye primordia. This indicates that the cloned gene encodes ey. The finding that ey of Drosophila, Small eye of the mouse, and human Aniridia are encoded by homologous genes suggests that eye morphogenesis is under similar genetic control in both vertebrates and insects, in spite of the large differences in eye morphology and mode of development.

Characterization of quail Pax-6 (Pax-QNR) proteins expressed in the neuroretina.

After differential screening of a cDNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a cDNA clone, Pax-QNR, homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye of mice and in the disorder aniridia in humans. Here we report the characterization of the Pax-QNR proteins expressed in the avian neuroretina. From bacterially expressed Pax-QNR peptides, we obtained rabbit antisera directed against different domains of the protein: paired domain (serum 11), domain between the paired domain and homeodomain (serum 12), homeodomain (serum 13), and carboxyl-terminal part (serum 14). Sera 12, 13, and 14 were able to specifically recognize five proteins (48, 46, 43, 33, and 32 kDa) in the neuroretina. In contrast to proteins of 48, 46, and 43 kDa, proteins of 33 and 32 kDa were not recognized by the paired antiserum (serum 11). Paired-less and paired-containing proteins exhibited the same half-life (6 h) and were phosphorylated mostly on serine residues. Immunoprecipitations performed with subcellular fractions of neuroretinas showed that the paired-containing proteins were located in the nucleus, whereas the 33- and 32-kDa proteins were found essentially in the cytoplasmic compartment. However, immunofluorescence experiments performed after transient transfections showed that p46 and p33/32 were also located in vivo into the nucleus. Thus, the Pax-QNR/Pax-6 gene can produce proteins with two DNA-binding domains as well as proteins containing only the DNA-binding homeodomain.

Characterization of quail Pax-6 (Pax-QNR) proteins expressed in the neuroretina.

After differential screening of a cDNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a cDNA clone, Pax-QNR, homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye of mice and in the disorder aniridia in humans. Here we report the characterization of the Pax-QNR proteins expressed in the avian neuroretina. From bacterially expressed Pax-QNR peptides, we obtained rabbit antisera directed against different domains of the protein: paired domain (serum 11), domain between the paired domain and homeodomain (serum 12), homeodomain (serum 13), and carboxyl-terminal part (serum 14). Sera 12, 13, and 14 were able to specifically recognize five proteins (48, 46, 43, 33, and 32 kDa) in the neuroretina. In contrast to proteins of 48, 46, and 43 kDa, proteins of 33 and 32 kDa were not recognized by the paired antiserum (serum 11). Paired-less and paired-containing proteins exhibited the same half-life (6 h) and were phosphorylated mostly on serine residues. Immunoprecipitations performed with subcellular fractions of neuroretinas showed that the paired-containing proteins were located in the nucleus, whereas the 33- and 32-kDa proteins were found essentially in the cytoplasmic compartment. However, immunofluorescence experiments performed after transient transfections showed that p46 and p33/32 were also located in vivo into the nucleus. Thus, the Pax-QNR/Pax-6 gene can produce proteins with two DNA-binding domains as well as proteins containing only the DNA-binding homeodomain.

PAX6 mutations in aniridia.

Aniridia is a congenital malformation of the eye, chiefly characterised by iris hypoplasia, which can cause blindness. The PAX6 gene was isolated as a candidate aniridia gene by positional cloning from the smallest region of overlap of aniridia-associated deletions. Subsequently PAX6 intragenic mutations were demonstrated in Smalleye, a mouse mutant which is an animal model for aniridia, and six human aniridia patients. In this paper we describe four additional PAX6 point mutations in aniridia patients, both sporadic and familial. These mutations highlight regions of the gene which are essential for normal PAX6 function. In addition, the frequency at which we have found PAX6 mutations suggests that lesions in PAX6 will account for most cases of aniridia.

Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene.

Aniridia is a semidominant disorder in which development of the iris, lens, cornea and retina is disturbed. The mouse mutation Small eye (Sey), which has been proposed as a model for aniridia, results from defects in Pax-6, a gene containing paired-box and homeobox motifs that is specifically expressed in the developing eye and brain. To test the role of PAX6 in aniridia, we isolated human cDNA clones and determined the intron-exon structure of this gene. PAX6 spans 22 kilobases and is divided into 14 exons. Analysis of DNA from 10 unrelated aniridia patients revealed intragenic mutations in three familial and one sporadic case. These findings indicate that the human aniridia and murine Small eye phenotypes arise from homologous defects in PAX6.

Small eye (Sey): Cloning and characterization of the murine homolog of the human aniridia gene

Phenotypic parallels and genetic evidence from comparative mapping suggest that the murine Small eye (Sey) and human aniridia (AN) disorders are homologous. This report describes the isolation of a murine embryonic cDNA that is structurally homologous to the AN cDNA we recently cloned. The murine cDNA detects a 2.7-kb transcript in the adult mouse eye and cerebellum and in human glioblastomas, suggesting a neuroectodermal involvement in the etiology of Sey/AN. Sequence comparison between the murine and the human cDNAs revealed extensive homology in nucleotide sequence (>92%) and virtual identity at the amino acid level. None of the differing amino acids was located within the paired box and homeobox DNA-binding domains. These results provide evidence for a common molecular basis underlying the two genetic disorders and suggest that the Sey system would be an authentic model for human AN.

A Mouse Model of the Aniridia-Wilms Tumor Deletion Syndrome

Deletion of chromosome 11p13 in humans produces the WAGR syndrome, consisting of aniridia (an absence or malformation of the iris), Wilms tumor (nephroblastoma), genitourinary malformations, and mental retardation. An interspecies backcross between Mus musculus/domesticus and Mus spretus was made in order to map the homologous chromosomal region in the mouse genome and to define an animal model of this syndrome. Nine evolutionarily conserved DNA clones from proximal human 11p were localized on mouse chromosome 2 near Small-eyes (Sey), a semidominant mutation that is phenotypically similar to aniridia. Analysis of Dickie's Small-eye (SeyDey), a poorly viable allele that has pleiotropic effects, revealed the deletion of three clones, f3, f8, and k13, which encompass the aniridia (AN2) and Wilms tumor susceptibility genes in man. Unlike their human counterparts, SeyDey/+ mice do not develop nephroblastomas. These findings suggest that the Small-eye defect is genetically equivalent to human aniridia, but that loss of the murine homolog of the Wilms tumor gene is not sufficient for tumor initiation. A comparison among Sey alleles suggests that the AN2 gene product is required for induction of the lens and nasal placodes.

Location of the gene involving the Small eye mutation on mouse chromosome 2 suggests homology with human aniridia 2 (AN2)

Using and interspecific backcross, we have mapped the gene involved in the mouse Small eye mutation ( Sey MH) relative to six cloned markers on chromosome 2 ( Hox-5.1, Cas-1, Fshb, Bmp-2a, and ld) and the agouti locus. The results suggest that the Sey gene maps between Fshb and Cas-1. Human mapping studies have shown that the aniridia ( AN2) gene, which is part of the Wilms tumor susceptibility, aniridia, genitourinary abnormalities, and mental retardation (WARG) complex, is also between FSHB and CAT on human chromosome 11. The conserved linkage of the cloned markers and the similarity of the Sey/+ and AN2/+ phenotypes suggest that the gene involved in the Sey mutation is the mouse homolog of the human AN2 gene.