Paired Box Mutations in Familial and Sporadic Aniridia ... - Europe PMC

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Dec 23, 1993 - Ophthalmology and Pediatrics, Baylor College of Medicine, Houston; and *Department of Human Genetics, Graduate ... The University of Texas M. D. Anderson Cancer Center, 1515 Hol- .... (antisense); end of exon 6, nucleotide positions 702- ...... Rose EA,Glaser T, Jones C, Smith CL, Lewis WH, Call KM,.
Am.J. Hum. Genet. 54:801-811, 1994

Paired Box Mutations in Familial and Sporadic Aniridia Predicts Truncated Aniridia Proteins Aruna Martha,* Robert E. Ferrell,* Helen Mintz-Hittner,t Leslie A. Lyons,* and Grady F. Saunders* *Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, and tDepartments of Ophthalmology and Pediatrics, Baylor College of Medicine, Houston; and *Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh

Summary Aniridia, an autosomal dominant ocular disorder characterized by iris hypoplasia, results from mutations in the PAX6 gene, which encodes paired box and homeobox motifs. In this report we describe five new mutations in the paired box region of the human PAX6 gene that are associated with aniridia. The paired box mutations that we detected were in both familial (three) and sporadic (two) cases. All five mutations predict truncated PAX6 proteins. Our study indicates that early premature translational termination mutations in the PAX6 gene result in haploinsufficiency and generate the aniridia phenotype.

Introduction Aniridia is a congenital, bilateral, panocular disorder that occurs with an incidence of 1/64,000-1/96,000 live births (Shaw et al. 1960). This disorder is characterized by the complete or partial absence of the iris and iris hypoplasia. Aniridia can appear in a highly penetrant form in association with a range of other ocular anomalies, e.g., poor vision (20/100 or worse), cata-

racts, glaucoma, corneal pannus, optic nerve hypoplasia, absence of macular reflex, ectopia lentis, nystagmus, and photophobia. All of these conditions generally result in poor vision. A less common form of aniridia occurs in which the iris defect predominates, but visual acuity remains relatively unimpaired. About two-thirds of children with aniridia have an affected parent, with the disorder being inherited as an autosomal dominant trait, which is expressed with high frequency in carriers (Shaw et al. 1960). One-third of cases occur in the sporadic form, and about one-third Received August 27, 1993; accepted for publication December 23, 1993.

Address for correspondence and reprints: Grady F. Saunders, Ph.D., Department of Biochemistry and Molecular Biology, Box 117, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. © 1994 by The American Society of Human Genetics. All rights reserved.

0002-9297/94/5405-0009$02.00

of these individuals also develop Wilms tumor. Those with both aniridia and Wilms tumor are usually mentally retarded and often suffer from genitourinary abnormalities (Fraumeni and Glass 1968). Patients with this constellation of systematic disorders, typical of the WAGR (Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation) contiguous-gene syndrome (Miller et al. 1974), are usually heterozygous for constitutional deletions of chromosomal band 11pl3 (Riccardi et al. 1978; Francke et al. 1979). Two families in which there was concordant transmission of aniridia with translocations involving llpi3 have been described by Simola et al. (1983) and Moore et al. (1986). Their research suggests that the rearrangements may either physically disrupt the gene or impair its function through positional effects. The aniridia gene has been localized to a small region on chromosomal band 1 lpl3 by a combination of genetic linkage, physical mapping, and cytogenetic analysis (Davis et al. 1989; Gessler et al. 1989; Rose et al. 1990; Lyons et al. 1992). The first candidate aniridia locus described was cloned from the cell line SIMO and lies at one of the breakpoints of the translocation t(4;11) (q23;pl3) (Gessler et al. 1989). Recently, a second candidate gene for an aniridia locus at 1ipl3 has been isolated by positional cloning (Ton et al. 1991), and this gene is located about 700 kb telomeric of the Wilms tumor predisposition gene (WT1). The gene at this locus, originally 801

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Genomic and cDNA structure of the human PAX6 gene. The PAX6 gene contains 12 introns flanked by 13 exons (alternative included). The thicker bar in PAX6 cDNA indicates the coding region (1,266 bp). Hatched, solid, and dotted areas represent paired box, homeobox, and pro-ser-thr-rich regions, respectively. Individual exons are numbered, and introns are designated as "A"-"L." The vertical lines in the PAX6 cDNA represent intron-exon boundaries. This representation is modified from that of Glaser et al. (1992).

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called "AN," is now named "PAX6," because it is clearly the human homologue of the mouse Pax-6 gene (Hill et al. 1991; Walther and Gruss 1991; Ton et al. 1992). PAX6 belongs to the class of paired-like developmental protein-producing genes first described in Drosophila and contains two highly conserved motifs, the paired box and the homeobox regions. The mammalian PAX-gene family has become a focus of intense research since the discovery that mutations in some of these genes cause developmental defects (Balling et al. 1988; Epstein et al. 1991; Hill et al. 1991) and that genetic alterations in PAX3 and PAX6 that are responsible for Waardenburg syndrome type I and aniridia, respectively, indicate involvement of PAX genes in human diseases (Baldwin et al. 1992; Glaser et al. 1992; Jordan et al. 1992). In the present report we describe different mutations found in the paired-box region of the PAX6 gene in three familial cases and in two sporadic cases. Our results provide evidence that aniridia seems to arise only from apparent PAX6 null alleles or alleles that are likely to cause a severe loss of function. Patients and Methods

The Families A detailed description of the large aniridia pedigree VMR-1 used in the present study has been reported elsewhere (Ferrell et al. 1980; Hittner et al. 1980). Individuals are designated as clinically affected if either eye

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has assigned the aniridia gene to chromosome llpl3 (Lyons et al. 1992). Recently we showed that a polymorphic dinucleotide repeat region segregated with the aniridia phenotype in this family (Martha et al. 1993, in press-a). In the two smaller families-AN1W and ANS3-analyzed in this study, patient identification was obtained from the proband, who was found through the medical records of the Department of Ophthalmology at the University of Pittsburgh. The family was contacted through their primary physician; and clinical, demographic, and family-history data and blood samples were obtained with the informed consent of the individuals. The proband IV-1 in family ANIW was a 28-yearold white woman with aniridia, macular hypoplasia, amblyopia, glaucoma cataract, corneal scarring, pannus, and nystagmus. She was mentally normal, but her eye findings were at the more severe end of the aniridia spectrum. Clinical histories in affected family members of ANS3 were unremarkable except for the occurrence of aniridia, which varied in expression among family members. The sporadic aniridia case AN017 reported here is that of a spouse included in family ANSI, in which a mutation in exon 10 of the homeobox region has been transmitted for three generations (Martha et al., in press-b). The other sporadic aniridia case analyzed was

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Analysis of aniridia family AN1W. A, Four-generation a Figure 3 ANIW. The proband (IV-1) in this family is at the more severe end o spectrum. Blackened circles represent affected females, and blackened si sent affected males. B, SSCP analysis of PCR products. DNA was amplifie with primers MTP-2 and KM-6. The SSCP panel show altered mobility c ucts from affected individuals in lanes 1-7. C, Sequence analysis of PCR p lanes show sequencing of PCR products of affected (111-1) and unaffected ( uals. Oligonucleotide primer MTP-2 was used to prime the sequencing re tion of a single base (A) from codon AAC (Asn 124) gives rise to TAA (st codon CTA (Leu 146).

ANO15, and the clinical history of this patient was unremarkable except for the occurrence of aniridia.

Oligonucleotide Primers The following oligonucleotide primers were used for exons 5-7 in the present study, with the nucleotide positions in PAX6: MTP-la (5'-GTCACAG-CGGAGTGAATCA) (sense), start of exon 5, nucleotide positions 373-392; TMP-4 (5'-CTGCAGAATTCGGGAAATGT) (antisense), end of exon 5, nucleotide positions 484-503 in Ton et al. (1991); MTP-1 (5'-GCTTGGTATGTTATCGTT) (sense), start of exon 6, nucleotide positions 504-522; TMP-3 (5'-GCTTGGTATGTTATCGTT) (antisense); end of exon 6, nucleotide positions 702719; MTP-2 (CAGGAGACACTACCA7TTTGG) (sense), 3' end of intron, between exons 6 and 7 in Glaser et al. (1992); and KM-6 (5'-CTTGCG-TAGGTTGCCCTGGC) (antisense), end of exon 7, nucleotide positions 866-885 in Ton et al. (1991). PCR and SSCP Analysis To identify intron-exon boundaries in the PAX6 gene, DNA from cosmid clone (cHl-7) and two normal

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\A genomic DNA samples were amplified by PCR with different pairs of oligonucleotides selected randomly from the PAX6 cDNA sequence. To identify point mutations in the PAX6 gene, genomic DNA samples from aniridia patients and their relatives from one large aniridia pedigree, eight different small families, and 13 apparently sporadic aniridia cases were analyzed. DNA prepared from blood (Miller et al. 1988) was PCR amplified for exons 1-13 in the present study. PCR was performed on 100 ng of genomic DNA samples by using the forward and backward primer set for each exon. SSCP analysis of PCR products was performed according to the method of Orita et al. (1989), with slight modifications. The PCR mixture contained 10 pmol of each unlabeled primer, 2 nmol of each of the four dNTPs, 100 ng of genomic DNA, and 0.15 U of Taq DNA polymerase in 5 g1 of the 10 X PCR buffer supplied with the enzyme. All reactions were labeled with 5 iCi of [ca-32P] dCTP (ICN Biochemicals). The PCR parameters for exons 1-13 were 3 min at 950C, followed by 25 cycles of 1 min at 940C, 1 min at 550C (annealing), and 2 min at 720C, followed by a 3-min extension at 720C. No amplified products were found in the nega-

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3-4 h at 4VC. The gel was dried onto filter paper and was exposed to X-ray film at -80'C for 18 h.

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Sequencing Analysis To detect mutations in exons 5-7 of the paired box region of the PAX6 gene, the PCR product was purified by electrophoresis in a 1% low-melting-point agarose gel in TAE buffer (50 mM Tris-acetate and 2 mM EDTA). A band of the correct size was excised, wrapped in parafilm, and stored overnight at -200C. DNA was extracted from the frozen gel by squeezing. Seven microliters of the gel-purified PCR product was sequenced using Sequenase (United States Biochemicals) according to the recommended protocol. Four microliters of each sequence reaction were run on a 6% polyacrylamide urea gel at 1,500 V for 2 h. The gel was soaked for 30 min in fixative, dried for 1 h, and exposed to X-Omat film (Kodak) for 48 h.

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Figure 4 Analysis of aniridia family ANS3. A, Three family members, two of whom are affected. B, PCR-SSCP analysis of exon 6 in affected individuals 1-2 and II-1, which revealed an additional band with altered mobility on SSCP gel. Amplification of PCR products was performed with primers MTP-1 and TMP-3. C, Sequence analysis of PCR products. Sequence panel of patient I-2 (right lane) showed a base substitution, G-IA, at codon TGG (Trp 100) to TAG (stop codon). Identical results were observed in patient 11-2. To prime sequencing reactions, the antisense primer TMP3 was used.

tive control reactions lacking the template. A portion of the reaction mixture (2 pl) was withdrawn and mixed with 50 pl of SSCP dilution buffer containing 0.1% SDS and 10 mM EDTA. Then 2 pl of this solution was mixed with an equal volume of dye (95% formamide, 20 mM EDTA, 0.05% brom-phenol blue, and 0.05% xylene cyanol) and heated at 850C for 5 min; 2 gl of sample was loaded onto a 7% nondenaturing polyacrylamide gel. Electrophoresis was performed at 30 W for

Human PAX6 Gene and Intron-Exon Structures of PAX6 Gene The human PAX6 cDNA sequence (Ton et al. 1991) was used in analysis of intron-exon structures in PAX6 cosmid clone cHl-7, as well as in the genomic DNA of normal individuals. Initially, oligonucleotide pairs were randomly selected. These randomly selected primer sets could detect two introns in the paired domain, two introns located in the homeodomain, and one intron in the pro-ser-thr-rich (PST) domain. Introns B, D, and G were not detected in these experiments, suggesting that these introns were too long to amplify efficiently under the PCR conditions used. A subsequent report by Glaser et al. (1992) established the larger size of these introns. Our PAX6 intron-exon structure (fig. 1) is in agreement with that reported by Glaser et al. (1992), except for intron K at codon 344 in the PST domain, which is more than three times larger than that reported by Glaser et al. (1992).

Mutations in the Human PAX6 Gene That Are Associated with Aniridia To identify mutations in the PAX6-coding region, exons 1-13 from related and unrelated patients were amplified by PCR and were analyzed by the SSCP technique. Cytogenetic analysis in the large aniridia pedigree (VMR-I) and in the families analyzed in the present

study showed no detectable deletions or translocations in this region. The work presented here discusses exons

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Analysis of sporadic aniridia patients. A, Aniridia family ANS1 segregating for homeodomain mutation (Martha et al., in Figure 5 press-b). A spouse, AN017 (arrow), with sporadic aniridia showed mutation in exon 6 of the paired domain. B, SSCP analysis of the PCR products. The two sporadic aniridia cases, AN015 and AN017, analyzed for mutations in exon 6 of the paired domain depicted altered mobility of single strands, which indicates mutations in these patients. Primers used for PCR were MTP-1 and TMP3. C, Sequence analysis of PCR products of a normal individual and AN015. The lanes show sequencing gels corresponding to a normal individual and to AN015. Patient AN015 shows a single base (C) insertion at TGC (cys 94), causing a frameshift mutation that results in a stop codon (TAA) at codon GAT (ASP115). D, Sequence analysis of PCR products of a normal individual and AN017. Patient AN017 showed a single base deletion at codon ATT (ile 56), causing a frameshift and altering codon GTA (val 78) into a stop codon (TAG). To prime sequencing reactions, the oligonucleotide prime TMP-3 was used for patient AN015. To sequence PCR product of AN017, an intronic primer PXI 1 (5'-GTGGTTTTCTGTCCACTTCC; in Glaser et al. 1992) was used.

5-7 of the human PAX6 gene, as no other exons were found to be abnormal. The first mutation presented here was found in the VMR-I family, which includes 27 affected family members (fig. 2A). RFLP studies in this large, four-generation family detected complete cosegregation of a GT-repeat allele of 36 repeats in the intronic region of the homeobox with the aniridia phenotype (Martha et al. 1993). The exon 5 mutation originally detected by SSCP (fig. 2B) is in exon 5 of the highly conserved paired domain of PAX6 and was further analyzed by sequencing the PCR product of one affected individual and one unaffected relative from this family. The DNA

PCR product of the affected heterozyshows an 11-bp insertion (TGCGGACCTCC), and the last four (CTCC) bases from this insertion are a duplication of 4 bp of the contiguous wild-type sequence. The insertion of these 11 bp at the threonine codon (ACC) results in a frameshift mutation ultimately changing codon 6 (valine-GTA) into a stop codon TAG. This is predicted to result in the truncation of the protein product within the paired domain; the mutation segregates with the aniridia phenotype in four generations. The second mutation was found in family AN1W, which had seven affected family members (fig. 3A). The

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VMR1 Figure 6 Structure of PAX6 cDNA, and relative sizes of the paired box, homeobox, and pro-ser-thr-rich domains. Comparison of the positions of the expected termination of the polypeptide chain and translation of truncated mRNA in ANIW, ANS3, AN015, AN017, and VMR-1, respectively, by relative sizes, are shown. The box with horizontal lines in AN1W, AN015, AN017, and VMR-1 represents altered reading frames, from the site of mutation indicated by arrows.

phenotype in this family segregates with a mutation found in exon 7, detected by altered SSCP mobility of PCR products (fig. 3B). Sequencing of the PCR product (fig. 3C) detected a single base deletion (AA) at codon 124 (AAC), and the consequent frameshift produces a stop codon (TAA) at the leucine (CTA) site, adjacent to the paired domain, i.e., in the glycine rich-domain at 22 codons downstream from the site of deletion. This mutation also was predicted to result in truncation of the protein. The mutation analyzed in this family segregates with the aniridia phenotype in four generations. The small family, ANS3 (fig. 4A), which has three members, two of whom are affected, revealed altered mobility of PCR products amplified from exon 6 of the paired domain (fig. 4B). Sequence analysis of the PCR products from individuals 1-2 and 11-1 detected a base substitution, G-*.A, transition in exon 6 (fig. 4C). Whereas the normal allele encodes tryptophan (TGG) at codon 100, the mutant allele encodes a stop codon (TAG) at this position. This nonsense mutation should result in polypeptide chain termination in the paired

domain and is concordant with the aniridia phenotype in the affected individuals. Two additional mutations were detected in two sporadic aniridia cases (fig. SA). SSCP analysis (fig. SB) followed by sequence analysis (fig. 5C) revealed a single base (C) insertion at codon 94 in exon 6 in patient ANO1S, and this insertion causes a translational frameshift in the paired domain. The resulting stop codon (TAA) at 22 amino acids downstream from the inserted position should terminate the polypeptide chain in exon 6 of the paired domain. Finally, SSCP analysis of patient AN017 revealed altered mobility in PCR products amplified from exon 6 of the paired domain (fig. SB). Sequence analysis of the PCR products indicated a single base deletion (AT) at codon 56 (ATT) (fig. SD). The result of this deletion is a frameshift mutation that results in a stop codon (TAG) at 22 codons from the point of deletion. The result of this mutation is predicted to be a truncated protein product, which will ultimately exclude part of the paired domain and the remainder of the PAX6 protein.

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