Download Missense mutations in the PAX6 gene in aniridia.

Missense Mutations in the PAX6 Gene in Aniridia
Noriyuki Azuma,1 Yoshihiro Hotta,2 Hisako Tanaka,5 and Masao Yamada4
PURPOSE. Aniridia is caused by a mutation of the PAX6%ene. Haploinsufficiency of the gene product
is thought to result in the aniridia phenotype, because most mutations thus far detected have been
large deletions encompassing the entire gene and nonsense, frameshift, or splice errors that result
in premature translational termination on one of the alleles. Only two missense mutations have
been detected in aniridia pedigrees, each of which occurs in its paired domain or homeodomain.
In this study, four novel missense mutations were found in three aniridia pedigrees.
Polymerase chain reaction-single-strand conformation polymorphism analysis and sequencing of the PAX6 gene were performed using genomic DNA of three aniridia pedigrees and
more than 100 healthy control subjects.
METHODS.
Three mutations occurred in the N-terminal subdomain of the paired domain, namely
N17S, I29V, and R44Q, the first two of which were detected on the same allele of one patient. The
other mutation (Q178H) was in the linking portion of the paired domain and homeodomain.
RESULTS.
CONCLUSIONS. These
missense mutations give rise to haploinsufficiency by another route, because
the missense mutations presented here resulted in an aniridia phenotype indistinguishable from
that caused by a heterozygous deletion of the entire PAX6 gene. (Invest Ophthalmol Vis Sci. 1998;
39:2524-2528)
T
he Pax gene consists of a family of developmental
control genes; nine members have been isolated in
vertebrates since paired originally was identified as a
segmentation gene in Drosophila melanogaster.1-2 The encoded proteins are transcriptional regulators with DNA binding
through a conserved domain consisting of 128 amino acids
(paired box).3 Some Pax genes share another conserved domain, homeobox, which also provides DNA binding.4'5 The
PAX6 gene has been isolated as a candidate gene for aniridia by
positional cloning based on an overlapping region of chromosomal deletions at I l p l 3 that are observed in some patients
with aniridia, especially in those with Wilms' tumor, genitourinary abnormalities, and mental retardation (known as the
WAGR syndrome).6 In addition to large deletions encompassing the whole gene, 100 mutations have been detected in
patients with sporadic and autosomal dominant familial aniridia7"12 and are summarized in the database at http://www.
hgu.mrc.ac.uk/Softdata/PAX6/. Most of these mutations cause
translational termination by nonsense and frameshift mutations
and by splice errors. Because the mutation occurs on one of
the alleles, haploinsufficiency of the gene product has been
suggested to cause the aniridia phenotype.13 Phenotypes of
heterozygous and homozygous mutants of the small eye (Sey)
locus, where a mouse homologue of the PAX6 gene is located,
also support this hypothesis.14"16 In addition, two missense
mutations in the paired domain and homeodomain have been
found in aniridia pedigrees.1017 Other mutations of the PAX6
gene have been shown to develop various manifestations of
ocular morphogenesis. Missense mutations in the paired domain have been detected in Peters' anomaly18 and in isolated
foveal hypoplasia.19 A missense mutation in the proline-serinethreonine activation (PST) domain has been found in an anterior segment anomaly,20 and a nonsense mutation in the PST
domain causes corneal dystrophy.21 Thus, the PAX6 gene
seems to play a role in a variety of processes during ocular
morphogenesis, and the phenotype-genotype correlation must
be carefully examined to elucidate the functions of PAX6. We
report four novel missense mutations detected in patients with
aniridia that will provide useful information for studies of the
PAX6 gene.
MATERIALS AND METHODS
DNA Samples
From the 'Department of Ophthalmology, National Children's
Hospital, Tokyo, Japan; the 2Department of Ophthalmology, Juntendo
University School of Medicine, Tokyo, Japan; the 'Department of
Paediatric Ophthalmology, Osaka City Medical Center Hospital, Osaka,
Japan; and the ^National Children's Medical Research Center, Tokyo,
Japan.
Submitted for publication March 11, 1998; revised July 1, 1998;
accepted July 31, 1998.
Proprietary interest category: N.
Reprint requests: Noriyuki Azuma, Department of Ophthalmology, National Children's Hospital, 3-35-31, Taishido, Setagaya-ku, Tokyo 154-8509, Japan.
These studies were conducted in accordance with the World
Medical Association Declaration of Helsinki. Our use of human
subjects was conducted with patients' informed consent, approved by the National Children's Hospital Experimental Review Board, and deemed exempt from human subject regulations. We analyzed PAX6 mutations in eight families
(autosomal dominant trait) and in 26 sporadic patients with
aniridia. After obtaining informed consent from all patients,
blood samples from 1 patient with familial aniridia and 3
patients with sporadic aniridia were collected from peripheral
veins into lithium-heparin tubes. Unaffected individuals who
were immediate family members also were examined.
Genomic DNA was prepared from isolated leukocytes using a
standard phenol-chloroform procedure.22 The DNA samples
from healthy control subjects have been described pre-
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Investigative Ophthalmology & Visual Science, December 1998, Vol. 39, No. 13
Copyright © Association for Research in Vision and Ophthalmology
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PAX6 Missense Mutations in Aniridia
IOVS, December 1998, Vol. 39, No. 13
viously.22 Chromosomal analysis was also performed. Highresolution G-banded chromosomes (750 bands) were obtained
from phytohemoagglutinin synchronized blood lymphocyte
culture for 72 hours. Twenty metaphases were analyzed.
Polymerase Chain Reaction-Single-Strand
Conformation Polymorphism Assay and
Sequencing
Polymerase chain reaction (PCR) primers used for amplification of 14 exons of PAX6 were synthesized using a DNA/RNA
synthesizer (model 392; Applied Biosystems, Urayasu, Japan) as
described in a previously published report.7 The PCR conditions we used were essentially the same as in our previous
report22 and were modified as follows. The annealing temperature was adjusted to 55°C for exons 5a and 13 and to 60°C for
all others, and Mg2*** concentration was 1.5raM.Single-strand
conformation polymorphism (SSCP) analyses were carried out
using automated mini-gel electrophoresis coupled with silver
staining (Phastsystem; Pharmacia, Little Chalfont, UK).22 Because DNA fragments generated by PCR for exon 13 were
large, the products were digested into two fragments with
Nindlll before being subjected to electrophoresis. SSCP analyses also were performed after radiolabeling with a-32P-dATP
in the PCR with conventional-sized gels of 5% polyacrylamide
under at least three different conditions with various concentrations of glycerol and running temperatures. Nucleotide sequences were determined after cloning on pUC18 using a
Sequenase version 2 kit (Amersham, Cleveland, OH) with PCR
primers or universal primers in pUC18. After 10 independent
subclones had been analyzed in each case, positive was scored
when at least four subclones showed the same mutation. The
mutations were also confirmed by direct sequencing from the
genomic DNA.
CASE REPORTS
In the following three pedigrees with aniridia, missense mutations of the PAX6 gene were identified.
Patient 1 was a 9-year-old girl with visual impairment and
nystagmus. She had bilateral total aniridia, cataract, and foveal
hypoplasia with visual acuities of 0.1 and 0.1 in the right and
left eyes. She had no systemic abnormalities, was of normal size
and intelligence for her age, and had a normal karyotype
(46,XX). Her parents and siblings had no ocular abnormalities.
Patient 2 was a 21-year-old man with visual impairment
and nystagmus. He had bilateral total aniridia, cataract, and
foveal hypoplasia with visual acuities of 0.1 and 0.1 in the right
and left eyes. He had no systemic abnormalities, was of normal
size and intelligence for his age, and had a normal karyotype
(46,XY). His parents and siblings had no ocular abnormalities.
Patient 3 was a 6-year-old boy with visual impairment and
nystagmus. He had bilateral total aniridia, cataract, and optic
nerve hypoplasia with visual acuities of 0.03 and 0.1 in the
right and left eyes, respectively. He had no systemic abnormalities, was of normal size and intelligence for his age, and had a
normal karyotype (46,XY). His 35-year-old mother also had
bilateral total aniridia and a cataract with a visual acuity of 0.02
in her left eye. Her right eye had no light perception vision
because of a vitreous hemorrhage of unknown origin. The
fundus of her right eye had optic nerve hypoplasia; the fundus
of her left eye was not visualized because of hazy media. She
2525
was otherwise normal. No other members of the immediate
family had ocular abnormalities.
RESULTS
Chromosomal Anomalies and PAX6 Mutations
In 34 pedigrees with aniridia, chromosomal analysis demonstrated deletions at I l p l 3 in one respective allele of 3 sporadic
patients. Heterozygous mutations of 6 nonsenses, 4 frameshifts
(3 insertions and 1 deletion of a nucleotide), and 3 splice
junction errors were found in 3 families and 8 sporadic patients. One nonsense mutation and 1 splice error were present
on the same allele of 1 sporadic patient. These taincation
mutations will be reported elsewhere with correlation to their
phenotypes. Four missense mutations were also found in 1
family and in 2 sporadic patients described above.
Missense Mutations
We analyzed genomic DNA isolated from leukocytes of the
family members. Genomic DNA covering each of 14 exons was
amplified by PCR and then subjected to SSCP analysis. Abnormal patterns in exon 5 were found in patients 1 and 2 and in
exon 8 of patient 3 and his affected mother but not in unaffected members of the immediate families or in more than 100
healthy control subjects. The SSCP pattern indicated a heterozygous mutation. Sequencing analyses revealed the following mutations on one allele in each patient; no other changes
were detected after careful analysis of PCR products from
patients' genomic DNA. Patient 1 had two nucleotide substitutions and insertion of a short oligonucleotide on the same
allele; nucleotide substitutions from A to G occurred at the
467th and 502nd positions of its cDNA form (in this study, the
numbers of the nucleotide and amino acid were based on the
sequence of GenBank Accession No. M93650) that caused
amino acid substitutions N17S and I29V, respectively. The
mutated allele had an additional insertion of 12 nucleotides in
intron 5, and 10 nucleotides after the splice donor site of exon
5 (Fig. 1). Patient 2 had a nucleotide substitution from G to A
at the 548th position, which resulted in R44Q (Fig. 2). The
affected individuals of family 3 had a nucleotide substitution
from G to T at the 951st position, which resulted in Q178H
(Fig. 3).
DISCUSSION
We detected four novel missense mutations in three pedigrees
with aniridia, three of which occurred in the paired domain of
the PAX6 gene. The paired domain, through which the Pax
protein binds DNA and functions as a transcriptional regulator,
consists of the N-terminal subdomain and the C-terminal subdomain. ''2-23'24 The N-terminal subdomain, which contains
two j3 turns and three a helices, is well conserved among the
Pax family members, whereas amino acids of the C-terminal
subdomain, which contains three a helixes, are considerably
diversified. The N-terminal subdomain is in extensive contact
with minor and major DNA grooves. Most missense mutations
thus far detected in the Pax family genes occur in this region
(R26G in PAX6 in Peters' anomaly,l8 five mutations in PAX3 in
Waardenburg's syndrome,25"27 and splotch-delayed (5/?cl)
mice,28 and one mutation of Paxl in a murine skeleton anom-
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2526
Azuma et al.
IOVS, December 1998, Vol. 39, No. 13
Normal
CTAG
Mutant
CTAG
Normal Mutant
GATCGATC
31
T
lie
T
A
.
lie
A
Val
Arg
Asn
Ser
A
A
C
C '
C
T
Gin
Ser
FIGURE 2. Mutation analysis of the PAX6 gene from patient 2.
Sequencing of the normal and mutant alleles from the patient
identified a G to A nucleotide substitution at the 548th position
in exon 5, which resulted in R44Q.
exon
exon
intron
intron
3-
m
—m
m
3
FIGURE 1. Mutation analysis of the PAX6 gene for patient 1.
Sequencing of the normal and mutant alleles of the patient
identified two A to G nucleotide substitutions at the 467th and
502nd positions in exon 5, which resulted in N17S and I29V,
respectively. The same allele also had an insertion of 12 nucleotides at the +10 nucleotide position after the 3' splice site of
exon 5.
aly29) by which the DNA binding ability may be altered. Three
PAX6 missense mutations have been found in the C-terminal
subdomain (I87R in aniridia,17 R128C in isolated foveal hypoplasia, ' 9 and I103N in Caenorhabditis elegans30). It has been
suggested that the C-terminal subdomain also is in contact with
DNA because of a sequence similarity between a recognition
helix of Hin (VSTLYR of residues 173 to 178) and the 6th a
helix of Pax6 (VSSINR of residues 120 to 125).2'f This also has
been experimentally demonstrated by methylation interference analysis, DNase footprint analysis, and x-ray diffraction
study on the co-crystal complex of the expressed Drosophila
prd protein and target oligonucleotides.24 3 '" 3 3
Three of the four missense mutations identified in our
patients with aniridia occurred in the N-terminal subdomain of
the paired domain. N17S is in the second j3 turn, I29V is in the
first a helix, and R44Q is in the second a helix. The isoleucine
residue at 29 and arginine at 44 are conserved throughout all
Pax family members identified to date, and the same can be
said of asparagine at 17, 24 which only changes to glycine in
Drosophila ey (GenBank Accession No. X79492). The crystal
structure study 24 demonstrated that asparagine at 17 is in
contact with the sugar phosphate backbone of DNA and with
the base of the minor groove and that arginine at 44 also is in
contact with the sugar phosphate backbone of DNA, Thus,
mutations at these positions may abolish or alter normal DNA
binding.
Mutations detected in patient 1 provide an interesting
phenomenon. In addition to the two missense mutations described above, an insertion of 12 nucleotides occurred on the
same allele. Multiple mutations within a small region are very
interesting considering the mechanisms of the generating mutations; however, the insertion itself may not affect PAX6
function because it occurs in intron.
The haploinsufficiency theory of the aniridia phenotype
seems to be consistent, but interpretation of PAX6 missense
mutations with respect to their function (in the direction of
31
Normal Mutant
GATCGATC
31
Gin
Gin
Gin
His
Cys
Cys
FIGURE 3- Mutation analysis of the PAX6 gene for patient 3
and the family with aniridia. Sequencing of the normal and
mutant alleles of the patient identified a G to T nucleotide
substitution at the 951th position in exon 8, which resulted in
Q178H.
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IOVS, December 1998, Vol. 39, No. 13
phenotypic ocular manifestations) is still controversial. Recently, interesting data have been garnered from an in vivo
transcriptional assay and a quantitative electrophoretic mobility shift assay using the mutations previously detected in Peters' anomaly (R26G in the N-terminal subdomain) and in
aniridia Q87R in the C-terminal subdomain).17 The R26G-mutated protein failed to bind to a subset of the consensus sequences for the PAX6 binding but still kept binding to another
set, and even transactivated some promoters. The I87R mutant
lost DNA binding to all the consensus sequences tested. The
findings seem to be consistent with the haploinsufficiency
theory for aniridia in the case of I87R and with a hypomorphic
change in Peters' anomaly by R26G. However, it is still unclear
how a mutation in the C-terminal subdomain affects DNA
binding through the N-terminal subdomain, because the two
subdomains are believed to have distinct DNA-binding abilities
and to act independently. Another experimental analysis postulated autoregulation in DNA binding through the paired
domain.34 The isolated N-terminal and C-terminal subdomains
do possess distinct DNA binding. However, when both subdomains are linked as in the PAX6 protein, they negatively regulate the other's function, that is, some mutations in the Nterminal subdomain not only loose a function controlled by the
N-terminal subdomain but also gain a function controlled by
the C-terminal subdomain and vice versa. The role of the other
mutation, Q178H, that was positioned in the linking portion of
the paired domain and homeodomain is also controversial.
Functional analyses are under way using PAX6 constructs with
mutations detected in this study.
An expression pattern of the PAX6 gene indicates the
multiple function of the gene. The gene expresses first in the
optic sulcus, subsequently in the eye vesicle, in the lens placode invaginating from the surface ectoderm, in the differentiating retina, and, finally, in the cornea.35 Peters' anomaly
occurs at an early gestational age of 4 to 5 weeks when the lens
separates from the surface ectoderm and mesenchymal cells
invade the anterior space; however, aniridia occurs at 8 to 10
weeks when mesenchymal cells and the anterior rim of the
optic cup differentiate to the iris. Foveal hypoplasia that often
is associated with aniridia occurs at a late stage, because the
fovea starts to develop at 30 weeks' gestation and is complete
at 4 months after birth.36'37 The same master control gene and
isoforms probably are used repeatedly in the morphogenesis of
various ocular tissues. It is established that aniridia affects the
entire eye, whereas Peters' anomaly is restricted to the anterior
segment and foveal hypoplasia to the posterior fundus. Because numerous ocular tissues differentiate with mutual correlation during development, various phenotypes occur as a
result of mutations of the same gene.
References
1. Walther C, Guenet JL, Simon D, et al. Pax: a murine multigene
family of paired box-containing genes. Genomics. 1991 ;11:424434.
2. Stapleton P, Weith A, Urbanek P, Kozmik Z, Busslinger M. Chromosomal localization of seven PAX genes and cloning of a novel
family member, PAX9. Nat Genet. 1993;3:292-298.
3- Treisman J, Haris E, Desplan C. The paired box encodes a second
DNA-binding domain in the paired homeo domain protein. Genes
Dev. 1991;5:594-604.
4. Gehring WJ. Homeo boxes in the study of development. Science.
1987;236:1245-1252.
PAX6 Missense Mutations in Aniridia
2527
5. Desplan C, Theis J, O'Farrell PH. The sequence specificity of
homeodomain-DNA interaction. Cell. 1988;54:1081-1090.
6. Ton CTT, Hirvonen H, Miwa H, et al. Positional cloning and
characterization of a paired box-containing gene from the aniridia
region. Cell. 1991 ;67:1059 -1074.
7. Glaser T, Walton DS, Maas RL. Genomic structure, evolutionary
conservation and aniridia mutation in the human PAX6 gene. Nat
Genet. 1992; 1:232-238.
8. Jordan T, Hanson I, Zaletayev D, et al. The human PAX6 gene is
mutated in two patients with aniridia. Nat Genet. 1992; 1:328 -332.
9. Hanson IM, Seawright A, Hardman K, et al. PAX6 mutations in
aniridia. Hum Mol Genet. 1993;2:915-920.
10. Davis A, Cowell JK. Mutations on the PAX6 gene in patients with
hereditary aniridia. Hum Mol Genet. 1993;2:2093-2097.
11. Martha A, Ferrell RE, Mintz-Hittner H, Lyons LA, Saunders GF.
Paired box mutations in familial and sporadic aniridia predicts
truncated aniridia proteins. Am J Hum Genet. 1994;54:801-8ll.
12. Glaser T, Jepeal L, Edwards JG, et al. Pax6 gene dosage effect in a
family with congenital cataracts, aniridia, anophthalmia and central nervous system defects. Nat Genet. 1994;6:463-471.
13. Fisher E, Scambler P. Human haploinsufficiency—one for sorrow,
two for joy. Nat Genet. 1994;7:5-6.
14. Hill RE, Favor J, Hogan BLM, et al. Mouse Small eye results from
mutation in a paired-like homeobox-containing gene. Nature.
1991354:522-525.
15- Schmahl W, Knoedlseder M, Favor J, Davidson D. Defects of
neuronal migration and the pathogenesis of the cortical malformations are associated with small eye (Sey) in the mouse, a point
mutation at the PAX-6 locus. Acta Neuropathol. 1993;86:126 -135.
16. Quiring R, Walldorf U, Kloter U, Gehring WJ. Homology of the
eyeless gene of Drosophila to the small eye gene in mice and
aniridia in humans. Science. 1994;265:785-78917. Tang HK, Chao L, Saunders GF. Functional analysis of paired box
missense mutations in the PAX6 gene. Hum Mol Genet. 1997;6:
381-386.
18. Hanson I, Fletcher JM, Jordan T, et al. Mutations at the PAX6 locus
are found in heterogeneous anterior segment malformations including Peters' anomaly. Nat Genet. 1994;6:168-173.
19- Azuma N, Nishina S, Okuyama T, Yanagisawa H, Yamada M. PAX6
missense mutation in isolated foveal hypoplasia. Nat Genet. 1996;
13:141-142.
20. Azuma N, Yamada M. Missense mutation at the C terminus of the
PAX6 gene in ocular anterior segment anomalies. Invest Ophthalmol VisSci. 1998;39:828-830.
21. Myrzayans F, Pearce WG, MacDonald IM, Walter MA. Mutation of
the PAX6 gene in patients with autosomal dominant keratitis. Am]
Hum Genet. 1995;57:539-548.
22. Yanagisawa H, Fujii K, Nagafuchi S, et al. A unique origin and
multistep process for the generation of expanded DRPLA triplet
repeats. Hum Mol Genet. 1996;5:373-379.
23. Bopp D, Burri M, Baumgartner S, Frigerito G, Noll M. Conservation
of a large protein domain in the segmentation gene paired and
functionally related genes of Drosophila. Cell. 1986;47:10331040.
24. Xu W, Rould MA, Jun S, Despan C, Pabo CO. Crystal structure of
a paired domain-DNA complex at 25 A resolution reveals structural
basis for Pax developmental mutations. Cell. 1995;80:639-650.
25. Baldwin CT, Hoth CF, Amos JA, da-Silva EO, Milunsky A. An
exonic mutation in the HuP2 paired domain gene causes Waardenburg's Syndrome. Nature. 1992;355:637-638.
26. Tassabehji M, Read AP, Newton VE, et al. Waardenburg's Syndrome patients have mutations in the human homologue of the
Pax-3 paired box gene. Nature. 1992;355:635-636.
27. Tassabehji M, Read AP, Newton VE, et al. Mutations in the Pax-3
gene causing Waardenburg's Syndrome type 1 and type 2. Nat
Genet. 1993;3:26-30.
28. Vogan KJ, Epstein DJ, Trasler DG, Gros P. The splotcb-delayed
(spd~) mouse mutant carries a point mutation within the paired box
of the pax3 gene. Genomics. 1993;17:364-369.
29- Balling R, Deutsch U, Gruss P. Undulated, a mutation affecting the
development of the mouse skeleton, has a point mutation in the
paired box of Pax-1. Cell. 1988;55:531-535.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933204/ on 08/03/2017
2528
Azuma et al.
30. Chisholm AD, Horvitz HR. Patterning of the Caenorhabditis elegans head region by the Pax-6 family member vab-3- Nature.
1995:377:52-55.
31. Czerny T, Schaffner G, Busslinger M. DNA sequence recognition
by Pax proteins: bipartite structure of the paired domain and its
binding site. Genes Dev. 1993;7:2048-206l.
32. Epstein JA, CaiJ, GlaserT, Jepeal L, Maas RL. Identification of a Pax
paired domain recognition sequence and evidence for DNA-depenclent conformational changes. / Biol Chem. 1994;269:8355-836l.
33- Epstein JA. Glaser T, Cai J, et al. Two independent and interactive
DNA-binding subdomains of the Pax6 paired domain are regulated
by alternative splicing. Genes Dev. 1994;8:2022-2034.
IOVS, December 1998, Vol. 39, No. 13
34. Yamaguchi Y, Sawada J, Yamada M, Handa H, Azuma N. Autoregulation of Pax6 transcriptional activation by two distinct DNAbinding subdomains of the paired domain. Genes Cells. 1997;2:
255-261.
35. Walther C, Gruss P. Pax6, a murine paired box gene, is expressed
in the developing CNS. Development. 1991;113:l435-l449.
36. Mann I. The Development of the Human Eye. New York: Grime &
Stratton; 1964.
37. Ozanics V, Jakobiec FA. Prenatal development of the eye and
its adnexa. In: Duane TD, Jaeger EA, eds. Biomedical Foundation of Ophthalmology, Vol 1. Philadelphia: Harper &: Row;
1985.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933204/ on 08/03/2017