Missense Mutation at the C Terminus of the PAX6 Gene in Ocular Anterior Segment Anomalies Noriyuki Azuma} and Masao Yamada2

Home , Ectropion

PURPOSE. TO report a rare case of ocular anterior segment anomalies including uveal ectropion of the iris, invasion of the conjunctival epithelia into the cornea, and posterior embryotoxon with a missense mutation of the PAX6 gene. METHODS. The authors performed polymerase chain reaction-single-strand conformation polymorphism analysis and sequencing of the PAX6 gene using genomic DNA of family members and more than 100 control subjects. RESULTS. The A to G transition at nucleotide 1682 in exon 13 in the patient was identified in an allele that resulted in a Gin to Arg substitution (Q422R) at the C terminus of the protein. The mutation was not found in the parents, a sibling, or control subjects. CONCLUSIONS. The mutation indicates that the proline- serine-threonine-rich domain at the C terminus of the PAX6 protein plays a role in ocular anterior segment morphogenesis. (Invest Ophthalmol Vis Set. 1998;39: 828-830)

t is well known that aniridia is caused by mutations of the IPAX6 gene, a transcription factor gene in ocular development.1"3 Because most of these mutations result in premature translational termination on one of the alleles, haploinsuffi- ciency of the gene has been suggested to cause the aniridia 3 FIGURE 1. Photographs of the anterior segments of the right phenotype. Other mutations in the PAX6 gene have been (A) and left (B) eyes of the patient. The corneoscleral limbus is detected in patients with Peters' anomaly,'' cornea! dystrophy,5 obscure because of the invasion of the conjunctival epithelia. and isolated foveal hypoplasia.6 Thus, the PAX6 gene acts in Uveal ectropion is seen at the pupillary margin. A pigment the formation of the anterior segments and the neural retina. In clump is on the lens surface in the right eye, and the cornea is mutants of the Small eye (Sey) locus, in which the murine partially opacified in the left eye. homologue of the PAX6 gene is located,7 the inactivation of both alleles causes the eyes not to develop and severe cranio- facial disorders to occur; inactivation of one allele results in the phenotypes, including Peters' anomaly, aniridia, and foveal development of small eyes and mild central nervous system hypoplasia, provides evidence in support of position-depen- disorders, the latter of which are detectable only at an embry- dent effects of PAX6 mutations in normal ocular develop- onic stage. In humans, compound heterozygotes have no eyes ment.6'8 7 and central nervous system disorders. Thus, the abnormalities We report a missense mutation at the C-terminal end of depend on the severity of the mutation of the PAX6 gene. Few the open reading frame of the PAX6 gene in a patient with missense mutations have been identified in the PAX6 ocular anterior segment anomalies. This mutation suggests the 2 1 6 8 gene " ' * however; the correlation between genotypes and possibility of another noteworthy function of the PAX6 gene.

From the 'Department of Ophthalmology, National Children's CASE REPORT Hospital, 2National Children's Medical Research Center, Tokyo Japan. Submitted for publication July 10, 1997; revised November 3, A 10-year-old girl was diagnosed at a clinic with an abnormality 1997 and December 5, 1997; accepted January 7, 1998. of the iris and was referred to our hospital. Ocular examination Proprietary interest category: N. revealed corneal opacities without iridocorneal adhesion in the Reprint requests: Noriyuki Azuma, Department of Ophthalmol- ogy, National Children's Hospital, 3-35-31, Taishido, Setagaya-ku, To- left eye, bilateral invasion of the conjunctival epithelia into the kyo 154, Japan. cornea, and uveal ectropion at the pupillary margin (Fig. 1).

Downloaded from iovs.arvojournals.org on 10/02/2021 IOVS, April 1998, Vol. 39, No. 5 Reports 829

digested into 2 fragments with Hindlll before being subjected to electrophoresis. Single-strand conformation polymorphism analyses also were performed after radiolabeling with [a-32P]dATP in the PCR reaction, using conventionally sized ill gels of 5% polyacrylamide under at least three different condi- tions in concentrations of glycerol and at running temperature. Nucleotide sequences were determined directly or after clon- ing on pUC18 using a Sequenase version 2 kit (Amersham, Cleveland, OH) with PCR primers or universal primers in pUC18.

RESULTS By single-strand conformation polymorphism analysis, an ab- normal pattern indicating a heterozygous mutation was detected in the exon 13 product of the patient, but it was not in the unaffected members of the immediate family or in more FIGURE 2. Polymerase chain reaction-single-strand conforma- than 100 normal persons (Fig. 2). Sequencing analysis demon- tion polymorphism analysis of exon 13 reveals a bandshift in strated that one of the patient's alleles had a single A-to-G the patient, but not in the parents, a sibling, or more than 100 normal control subjects. transition at nucleotide 1682 (Fig. 3), which caused a Gln-to- Arg substitution (Q422R) at the endpoint of the protein (in this study, the number of the nucleotide and amino acid was based on the sequence of accession no. M93650). No other changes The lens, vitreous, and fundus were normal by slit lamp biomi- in nucleotide sequences were detected in PCR products from croscopy and ophthalmoscopy. Her visual acuity was 1.0 with the patient's genomic DNA. slight bilateral myopia. The ocular position was orthophoric. The electroenceph- alogram was normal bilaterally; computed tomography failed DISCUSSION to show any orbital or cranial abnormality. The patient was normal in size for her age, and she had a normal intelligence We identified a missense mutation at the C-terminal end of the level and karyotype (46XX). No ocular abnormality was found PAX6 protein that causes congenital anterior segment anoma- in her parents and a sibling. lies. The corneal opacity in the left eye of our patient was similar to that in Peters' anomaly, although obvious iridocorneal adhesion was not seen. Epithelial abnormalities at the METHODS corneoscleral limbus and corneal opacities are often seen in eyes with aniridia. It has been reported that the PAX6 gene DNA mutation results in varying degrees of aniridia, which range These studies were conducted in accordance with the tenets of from an iris of normal size to the absence of one.5* The present the Declaration of Helsinki. Our use of human subjects was case with minute abnormalities of the iris and limbus may be conducted with the patient's informed consent, was approved representative of mild aniridia or a related condition. by the National Children's Hospital Experimental Review Most patients with aniridia have either a deletion of the Board, and was deemed exempt from human subject regula- whole gene or mutations of nonsense, a frameshift, or a defect tions. After informed consent, blood samples of the patient and at the splicing junction, that result in premature translational family members were collected from the peripheral veins into lithium heparin tubes. Genomic DNA was prepared from isolated leukocytes using a standard phenol/chloroform proce- Normal Mutant 9 dure. DNA samples from a normal control subject were also 31 GATCGATC obtained. A Polymerase Chain Reaction-Single-Strand Stop A Stop Conformation Polymorphism Assay and T Sequencing G Gin A Arg Polymerase chain reaction (PCR) primers used for amplifica- II C tion of 14 exons of PAX6 were synthesized using a DNA-RNA A synthesizer (model 392; Applied Biosystems, Urayasu, Japan), Leu T Leu following a previous report.] The annealing temperatures were T adjusted to 55°C for exons 5a and 13, and to 60°C for all others, and Mg2+ concentration was 1.5 mM. Single-strand conforma- 5' tion polymorphism analyses were performed using automated FIGURE 3. Sequencing of the normal and mutant alleles iden- minigel electrophoresis coupled with silver staining (Phastsys- 9 tifies the heterozygous A-to-G transition at the nucleotide 1682 tem, Pharmacia, Little Chalfont, UK). Because DNA fragments in exon 13, resulting in a Gln-to-Arg substitution (Q422R) in generated by PCR for exon 13 were large, the products were the protein.

Downloaded from iovs.arvojournals.org on 10/02/2021 830 Reports IOVS, April 1998, Vol. 39, No. 5

termination on one of the alleles.1"3 Only two pedigrees with pared with previously reported cases, including aniridia, aniridia have been reported to have missense mutations, re- caused by PST-rich domain mutations. spectively, in the paired domain and homeodomain,2'8 the Although the PST-rich domain shows a great variation of DNA-binding motifs of the transcriptional regulators. A single amino acid levels among animal species, the C-terminal amino amino acid substitution in an important motif may result in acid, glutamine, is conserved in all known vertebrate Pax6 significant protein dysfunction, the same as that in the trun- proteins and some more distant ones, including those of hu- cated protein usually seen in aniridia. Other missense muta- mans (accession no. M93650), mouse (P32117), rat (U69644), tions found in Peters' anomaly and foveal hypoplasia were also chick (P47237), quail (X82151), zebrafish (X63183), Xenopus in the paired domain. The paired domain consists of a highly (U64513), Phallusia (Y09975), squid (U59830), and Caeno- conserved N-terminal subdomain and a more variable C-termi- rhabditis elegans (U29184). The Q422R mutation together nal subdomain. Two subdomains that possess different DNA- with previous studies strongly suggests that a full-length PAX6 binding activities work independently to activate transcrip- protein is necessary for eye morphogenesis. tion,10 and each negatively regulates the function of the other.'' Because a missense mutation in the N-terminal subdomain of the paired domain was found in Peters' anomaly4 and References because those in the C-terminal subdomain were identified in 1. Glaser T, Walton DS, Maas RL. Genomic structure, evolutionary foveal hypoplasia6 and aniridia,7 the two subdomains play conservation and aniridia mutation in the human PAX6 gene. Nat significant roles in ocular anterior and posterior segment for- Genet. 1992; 1:232-238. mation through development. 2. Hanson IM, Seawright A, Hardman K, et al. PAX6 mutations in aniridia. Hum Mol Genet. 1993;2:915-920. The present study provides additional evidence to show 3. Martha A, Ferrell RE, Mintz-Hittner H, Jyons LA, Saunders GF. the importance of a C-terminal motif of the PAX6 protein for Paired box mutations in familial and sporadic aniridia predicts ocular anterior segment formation. The region (152 amino truncated aniridia proteins. Am J Hum Genet. 1994;54:801- acids) enriched with proline, serine, and threonine residues is 811. similar to that region in CTF-112 and Oct-lli genes, which has 4. Hanson I, Fletcher JM, Jordan T, et al. Mutations at the PAX6 locus been indicated to be an activation motif of the transcription are found in heterogeneous anterior segment malformations in- 7 cluding Peters' anomaly. Nat Genet. 1994;6:l68-173- factor. In this region, 17 mutations have been identified so far 5. Myrzayans F, Pearce WG, MacDonald IM, Walter MA. Mutation of (they are summarized in the database at http://www.hgu.mr- the PAX6gene in patients with autosomal dominant keratitis. Am] cac.uk/Softdata/PAX6), most of which manifested various Hum Genet. 1995;57:539-548. compound anomalies through the anterior ocular segment to 6. Azuma N, Nishina S, Okuyama T, Yanagisawa H, Yamada M. PAX6 the posterior segment including aniridia, congenital cataracts, missense mutation in isolated foveal hypoplasia. Nat Genet. 1996; 13:141-142. autosomal dominant keratitis, and foveal hypoplasia. All muta- 7. Glaser T, Jepeal L, Edwards JG, et al. Pax6 gene dosage effect in tions but one were nonsense or frameshift, which were pre- a family with congenital cataracts, aniridia, anophthalmia and dicted to cause premature translational termination or exten- central nervous system defects. Nat Genet. 1994;7:463- sion out of the frame of the protein. One missense mutation of 471. a single G-to-C transition at nucleotide 1545 that causes G395R 8. Tang HK, Chao L, Saunders GF. Functional analysis of paired box in association with —1 out-of-frame deletion of exon 12 has missense mutations in the PAX6 gene. Hum Mol Genet. 1997;6: 381-386. been identified in familial aniridia. However, the missense 9. Yanagisawa H, Fujii K, Nagafuchi S, et al. A unique origin and mutation probably is unimportant, and a mutation at —1 of multistep process for the generation of expanded DRPLA triplet exon 12 results in an out-of-frame excision of exon 12, remov- repeats. Hum Mol Genet. 1996;5:373-379- ing 50 additional amino acids of the PST-rich domain and 10. Epstein JA. Glaser T, Cai J, et al. Two independent and interactive resulting in an out-of-frame extension of exon 13- Thus, the DNA-binding subdomains of the Pax6 paired domain are regulated Q422R is the first missense mutation in the PST-rich domain by alternative splicing. Genes Dev. 1994;8:2022-2034. 11. Yamaguch Y, Sawada J, Yamada M, Handa H, Azuma N. Autoreg- that may cause ocular anomaly. ulation of Pax6 transcriptional activation by two distinct DNA- An in vitro study using nonsense mutations in aniridia and binding subdomains of the paired domain. Genes Cells. 1997;2: SeylNc" mouse demonstrated that the mutants in the C termi- 255-261. nus residues of the PAX6 PST-rich domain decrease their au- 12. Mermod N, O'Neill EA, Kelly TJ, Tijan R. The proline-rich tran- 7 scriptional activator of CNF/NF-1 is distinct from the replication tonomous activation. The present study also confirms the and DNA binding domain. Cell. 1989;58:74l-753. importance of the C terminus PST-rich domain. The Q422R 13- Tanaka M, Herr W. Differential transcriptional activation by Oct-1 mutation may alter PAX6 protein function in a relatively subtle and Oct-2: interdependent activation domains induce Oct-2 phos- way, and the mutation may manifest a mild phenotype com- phorylation. Cell. 1990;60:375-386.

Downloaded from iovs.arvojournals.org on 10/02/2021