PAX6 gene variations associated with aniridia in south India.

Abstract

Mutations in the transcription factor gene PAX6 have been shown to be the cause of the aniridia phenotype. The purpose of this study was to analyze patients with aniridia to uncover PAX6 gene mutations in south Indian population.
Total genomic DNA was isolated from peripheral blood of twenty-eight members of six clinically diagnosed aniridia families and 60 normal healthy controls. The coding exons of the human PAX6 gene were amplified by PCR and allele specific variations were detected by single strand conformation polymorphism (SSCP) followed by automated sequencing.
The sequencing results revealed novel PAX6 mutations in three patients with sporadic aniridia: c.715ins5, [c.1201delA; c.1239A>G] and c.901delA. Two previously reported nonsense mutations were also found: c.482C>A, c.830G>A. A neutral polymorphism was detected (IVS9-12C>T) at the boundary of intron 9 and exon 10. The two nonsense mutations found in the coding region of human PAX6 gene are reported for the first time in the south Indian population.
The genetic analysis confirms that haploinsuffiency of the PAX6 gene causes the classic aniridia phenotype. Most of the point mutations detected in our study results in stop codons. Here we add three novel PAX6 gene mutations in south Indian population to the existing spectrum of mutations, which is not a well-studied ethnic group. Our study supports the hypothesis that a mutation in the PAX6 gene correlates with expression of aniridia.

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BioMed Central
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BMC Medical Genetics
Open Access
Research article
PAX6 gene variations associated with aniridia in south India
Guruswamy Neethirajan1, Subbaiah Ramasamy Krishnadas2,
Perumalsamy Vijayalakshmi3, Shetty Shashikant3 and
Periasamy Sundaresan*1
Address: 1Department of Genetics, Aravind Medical Research Foundation, Madurai, India, 2Department of Glaucoma, Aravind Eye Hospital,
Madurai, India and 3Department of Pediatric Ophthalmology and Strabismus, Aravind Eye Hospital, Madurai, India
Email: Guruswamy Neethirajan - neethirajan@aravind.org; Subbaiah Ramasamy Krishnadas - krishnadas@aravind.org;
Perumalsamy Vijayalakshmi - pvijayalakshmi@aravind.org; Shetty Shashikant - drshashikants@yahoo.com;
Periasamy Sundaresan* - sundar@aravind.org
* Corresponding author
Abstract
Background: Mutations in the transcription factor gene PAX6 have been shown to be the cause
of the aniridia phenotype. The purpose of this study was to analyze patients with aniridia to uncover
PAX6 gene mutations in south Indian population.
Methods: Total genomic DNA was isolated from peripheral blood of twenty-eight members of six
clinically diagnosed aniridia families and 60 normal healthy controls. The coding exons of the human
PAX6 gene were amplified by PCR and allele specific variations were detected by single strand
conformation polymorphism (SSCP) followed by automated sequencing.
Results: The sequencing results revealed novel PAX6 mutations in three patients with sporadic
aniridia: c.715ins5, [c.1201delA; c.1239A>G] and c.901delA. Two previously reported nonsense
mutations were also found: c.482C>A, c.830G>A. A neutral polymorphism was detected (IVS9-
12C>T) at the boundary of intron 9 and exon 10. The two nonsense mutations found in the coding
region of human PAX6 gene are reported for the first time in the south Indian population.
Conclusion: The genetic analysis confirms that haploinsuffiency of the PAX6 gene causes the
classic aniridia phenotype. Most of the point mutations detected in our study results in stop codons.
Here we add three novel PAX6 gene mutations in south Indian population to the existing spectrum
of mutations, which is not a well-studied ethnic group. Our study supports the hypothesis that a
mutation in the PAX6 gene correlates with expression of aniridia.
Background
Aniridia is a human congenital eye malformation with a
population frequency of 1 in 60,000–100,000 [1]. Muta-
tions in the transcription factor gene PAX6 cause blind-
ness [2] through a spectrum of ocular manifestations
among which aniridia and most probably foveal hypopla-
sia are the major signs [3-5].
The human PAX6 gene located on chromosome 11p13,
was isolated by positional cloning as a candidate gene for
aniridia [6]. Most of the point mutations in this gene will
lead to premature truncation of protein [7]. Aniridia
occurs due to decreased dosage of the PAX6 gene, which
controls early events in the morphogenesis of the eye and
brain [8,9]. It exists in both sporadic and familial forms.
Published: 16 April 2004
BMC Medical Genetics 2004, 5:9
Received: 05 December 2003
Accepted: 16 April 2004
This article is available from: http://www.biomedcentral.com/1471-2350/5/9
© 2004 Neethirajan et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in
all media for any purpose, provided this notice is preserved along with the article's original URL.

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One third of the aniridia cases are sporadic and two thirds
are familial [3]. The sporadic cases can be part of WAGR
syndrome (Wilms' tumor, aniridia, genitourinary anoma-
lies, mental retardation), which is caused by hemizygous
deletions of the PAX6 and WT1 genes. The PAX6 gene
spans 22 kb in size and consists of 14 exons and 13
introns. The translation initiation codon is in exon 4 and
the termination codon is in exon 13 [8]. The human PAX6
gene has paired box and homeobox motifs and encodes a
highly conserved protein of 422 amino acids among
metazoans. The function of the PAX6 protein is crucial for
the development of human eye. The paired domain and
the homeodomain are the two DNA binding domains,
which are separated by a linker segment (LNK) and fol-
lowed by a C-terminal region rich in proline, serine and
threonine (PST). The presence of two DNA binding
domains in PAX6 strongly suggests that the protein func-
tions as a transcription factor to regulate the expression of
other genes during embryogenesis [10,11].
Mutations in the PAX6 gene have been reported in a grow-
ing number of aniridia patients and patients with other
ocular malformations [12,5]. The mutations are scattered
throughout the gene. The vast majority of mutations
reported so far are nonsense mutations, frameshift muta-
tions or splicing errors that are predicted to cause prema-
ture truncation of the
haploinsufficiency [12].
PAX6 protein, causing
Even though the PAX6 gene has been studied in various
ethnic populations, Indian aniridia mutations are not
well studied. For this reason we decided to study PAX6
variations in an Indian population. Here we report five
coding mutations of which three are novel mutations in
sporadic cases of aniridia in south Indian population. Our
study supports the hypothesis that the mutations in the
human PAX6 gene appear to correlate with classic aniridia
phenotype.
Methods
Clinical evaluation
This was done with informed consent and in accordance
to the tenets of the Declaration of Helsinki. Twenty-eight
members of six unrelated aniridia families and 60 healthy
normal controls were recruited for this study. Examina-
tion included slit lamp, gonioscopy, IOP measurement
and biomicroscopy.
Mutation screening and sequence analysis
Total genomic DNA was isolated by the salt precipitation
method from peripheral blood [13]. The amplification of
exons 4–13 to detect PAX6 gene mutations was carried out
in a 20 µl reaction mixture containing 100 ng of genomic
DNA, 100 pM of each primer, 1X PCR buffer (Promega
USA) and 0.5 U of Taq DNA polymerase (Promega USA).
The coding region of the human PAX6 gene was amplified
with previously published primers [8]. PCRs were carried
out in a MJ Research thermocycler for 35 cycles, consisting
of 1 min at 95°C for denaturation, 1 min at 58°C for
primer annealing and 10 min at 72°C for primer exten-
sion. Single strand conformation polymorphism analysis
was carried out in 10% polyacrylamide with or without
2.5% glycerol as previously described [14]. The PCR prod-
ucts were sequenced to confirm the exact nucleotide
change by ABI prism (Microsynth, Switzerland).
Allele specific clones
The superimposed mutant PCR products were amplified
and cloned into the pGEM-T vector (Promega USA). Each
transformed colony contains the mutant allele or the nor-
mal allele [15]. Exon-specific PCR-SSCP was performed
from the transformed colonies. Two PCR reactions were
performed in parallel. In the first reaction the 5' primer is
complementary to the wild type sequence and in the sec-
ond reaction the 5' primer is complementary to the
mutant sequence. The elongation occurs only when prim-
ers and target sequence match completely, so only one
allele of either mutant or wild type DNA is amplified
[16,17]. The plasmids were used as templates in amplifi-
cation to generate PCR products for SSCP. The SSCP band-
ing pattern was used to select allele-specific clone for
sequencing (data not shown). The restriction digestion
was performed for exon 6 with RsaI enzyme to differenti-
ate between mutant and normal allele and visualized on a
2.5% agarose gel.
Results
We used the SSCP technique to search for mutations in
exons 4–13 of the PAX6 gene (OMIM 106210) in
probands with aniridia and normal controls. The clinical
information for each sporadic aniridia patient is given in
Table 1. Altered mobility shifts detected by SSCP were
confirmed by repeating two or three times. All the
mutants we describe here (except two that were analysed
by direct sequencing) were cloned in to the pGEM-T vec-
tor followed by allele-specific PCR to pick up the correct
clone using the plasmid DNA as template because the
genomic PCR gave superimposed signals. Sequencing
reactions were then carried out. Although this plasmid
based SSCP analysis is time consuming, it reliably allows
identification of each allele. The mutant sequences are
deposited in Genbank [18]. (AY307164, AY289493,
AY337853, AY337852, AY342394) and also in the PAX6
Allelic Variant database [19]. The summary of our find-
ings is given in Table 2.
Novel mutations in the PAX6 gene
In proband 28–1 the plasmid-based clones were
sequenced, revealing an insertion with duplication of five
bases at position 715 in codon 118 (c.715ins5), which

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resulted in a novel frameshift mutation in PAX6 gene. The
phenotypic expression of aniridia is shown in Fig. 1A. The
pedigree and the SSCP pattern and the unusual band shift
detected in exon 6 is given in Fig. 2A. The insertion of 5
bases duplicates the cDNA sequence (Genbank M93650)
from bases 711–715 of PAX6 gene (Fig. 3A). De novo
change was observed at position 715 in the proband,
which is a site for most common reported mutations [20-
22]. The PCR product of 300 bp was digested with RsaI
enzyme, which resulted in a 220 bp and 80 bp products in
normal whereas the mutant gives 220 bp and 85 bp (data
not shown).
The phenotypic expression of proband 27–1 is shown in
Fig. 1B. The patient showed an abnormal band shift when
compared with the unaffected family members in SSCP
analysis (Fig. 2B). The plasmid-based sequencing results
showed a deletion of single base at position 1201; codon
280 (c.1201delA) in PST region is shown in Fig. 3B. The
deletion of adenosine residue at exon 10 predicts the pre-
mature truncation at exon 12 alters the conservation of
amino acids from serine at codon 280. The proband had
another unique change, A>G at position 1239
(c.1239A>G), 38 bases downstream of the deletion on the
same allele in exon 10 (data not shown). The two changes
were identified by allele specific PCR with repeated
sequencing of different clones, since the direct sequencing
of genomic PCR gave superimposed signals. Two changes
on one allele have been previously reported [23].
The morphology of aniridia due to deletion in the LNK
region is shown in Fig. 1C. In proband 10–1, an obvious
bandshift on the SSCP gel was seen (Fig. 2C), which on
sequencing (plasmid-based) revealed the mutation
c.901delA in the coding region of exon 8 at codon 180
(Fig. 3C). The single adenosine residue deletion results in
a frameshift mutation, generating a stop codon within the
LNK region. The consequences of this mutation would be
to produce a truncated protein, lacking the homeobox
domain.
Table 1: Clinical and common phenotypes associated with the aniridic probands
Proband Age/ SexBest VisionRefractive ErrorNystagmusKeratopathy Iris CataractGlaucomaOptic nerve
Hypoplasia
Macular
Hypoplasia
Glaucoma
Medical
treatment
Surgical
treatment
RELE RELERELE RELERE LERELE RE LERELE RE LE
28–1
27–1
17 yrs/F
8 yrs/M
6/24
6/60
6/24
6/60
+7.0
-1.0 -1.0
× 90
NI
-
NI
NI
+7.0
-1. 50
NA
+
+
-
+
-
A
A
+
-
+
-
+
+
+
+
-
-
-
-
NA
+
NA
+
+
+
+
+
4,1
3
1,2
3
10–1
21–1
16–1
18–1
13 yrs/M
2 mon/M
15 yrs/M
30 yrs/M
PL
-
6/60
PL
HM
-
HM
PL
NI
-
NI
NI
+
+
+
+
-
-
+
+
-
-
+
+
A
R
R
R
+
-
+
+
+
-
+
+
NA
-
+
+
+
-
+
+
-
-
-
-
-
-
-
-
NA
+
+
NA
NA
+
+
NA
-
-
+
+
+
-
+
+
5
-
-
-
-
-
-
-
RE-Right Eye; LE-Left Eye; HM-Hand Movement; PL-Perception of Light; A-Absent; R-Remnant; NA-Not Available; NI-No Improvement; 1-
Trabeculectomy; 2-Cyclocryocoagulation; 3-Awaiting cataract surgery; 4-Cataract surgery; 5-Surgery for Retinal detachment.
Table 2: Molecular analysis of PAX6 mutations with their predicted products.
Proband Exon/ IntronSystematic NameCodon Domain Type of MutationPredicted amino acid
change
References
23–1
14–1
28–1
9
10
6
c.1080C>T
c.1180insA
c.715ins5
240
273
118
HD
PST
PD
Nonsense
Frame shift Due to insertion
Frame shift due to insertion
with duplication
Deletion
Deletion
Nonsense
Nonsense
Transition
Arginine to stop Codon
Premature termination
Addition of two amino acids
[14]
[14]
Present Report
27–1
10–1
21–1
16–1
18–1
10
8
5
7
c.1201delA
c.901delA
c.482C>A
c.830G>A
IVS 9-12C>T
280
180
40
156
--
PST
LNK
PD
LNK
HD
Termination codon at Exon 12
Premature termination at exon 8
Cysteine to stop codon
Tryptophan to stop codon
Neutral polymorphism
Present Report
Present Report
Present Report
Present Report
Present Reportintron 9
PA-Paired domain; HD-Homeo domain; LNK-linker region; PST-Proline serine threonine region.

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The PCR-SSCP followed by direct sequencing revealed a
nucleotide substitution in proband 21–1 (Fig. 2D). The
heterozygous transversion (C>A) was observed at position
482 (c.482C>A), which disrupts the cysteine (TGC) to
stop (TGA) at codon 40 (C40X) in exon 5 in the paired
domain (Fig. 3D). As a consequent, the nonsense muta-
tion C40X produced a truncated protein, lacking the LNK,
homeobox and PST regions.
Another nonsense mutation (W156X), which was
detected in proband 16–1, resulted in expression of ani-
ridia phenotype (Fig. 1D). The SSCP gel analysis (Fig. 2E),
and the chromatogram revealed G>A transition at posi-
tion 830 (c.830G>A) which changes the tryptophan
(TGG) to stop (TGA) at codon 156 in LNK region (Fig.
3E). We report these nonsense mutations for the first time
in a south Indian population, although they have been
reported before in French population [24,25].
PAX6 gene polymorphism in aniridia
SSCP analysis of exon 10 in the family of proband 18–1 is
shown in Fig. 2F. The sequencing analysis showed an
intronic change at position -12 of intron 9 (IVS9-12C>T)
is shown in Fig. 3F. This polymorphism has been previ-
ously reported in Indian [26] and Caucasian [27] popula-
tions. Even though the mutation falls in the splice
acceptor site of exon 9 it seems to be a neutral polymor-
phism. The unaffected parents have normal sequences in
all the exons of PAX6 gene.
Discussion
Mutations of the PAX6 gene are known to cause many
cases of inherited and sporadic aniridia. The PAX6 gene is
clearly vital for the normal development of the eye [28].
The phenotype presumably results from heterozygous
insufficiency such that if only one copy of the gene is
active insufficient protein is produced to support normal
development of the eye. The protein products of the
mutant genes could theoretically have DNA binding activ-
ity and compete with the wild type protein. However, the
fact that aniridia caused by intragenic mutation is pheno-
typically indistinguishable from aniridia caused by dele-
tion of entire PAX6 gene makes it likely that the mutant
alleles are in fact null alleles [4,25].
The pathological mutations will be found throughout the
gene including the promoter and the other regulatory
regions. In these patients all the mutations if translated,
would result in truncated protein products. The translated
proteins would be unable to carry out normal PAX6
function because functional domains are deleted. The two
DNA binding domains are important for the normal activ-
ity of the protein [7,10]. Proband 27–1 with c.1201delA
also has another change, c.1239A>G downstream of the
deletion, however its impact on transcriptional product is
unknown. The downstream mutation may be less impor-
tant because it is likely that the other mutation
(c.1201delA) is the one responsible for aniridia. Though
the two nonsense mutations C40X and W156X were pre-
viously reported in other population, we report for the
first time in aniridic patients in south Indian population
and thus it could be a recurrent mutation.
The change in the splice acceptor region of intron 9 (IVS9-
12C>T) is a neutral polymorphism that has been observed
before and the C>T substitution in the splice acceptor con-
sensus sequence is not predicted to have any effect on
function [29]. In addition, other studies with this change
in patients with aniridia have a clear pathological
mutation elsewhere in the gene [19]. It is likely that the
patient 18–1 might have a pathological mutation
elsewhere in the PAX6 gene, perhaps in one of the cis reg-
ulatory sequences.
Analysis of PAX6 mutants showed that the pathogenicity
effects appear to be loss-of-function mutations, which
leads to aniridia (Fig. 1) and other variable phenotypes
[25]. The mutations showed varying phenotypic severity
of the disease. The proband with c.715ins5 mutation
showed the presence of sclerocornea with nystagmus in
both eyes. A similar phenotype (Foveal hypoplasia) was
observed in probands with c.1201delA and c.482C>A.
Proband 10–1 with c.901delA revealed ptosis, micro-
cornea with dislocated cataractous lens. Marfan syndrome
and ectopia lentis was observed in both eyes of the
Phenotypic expression of aniridia with PAX6 gene mutation
Figure 1
Phenotypic expression of aniridia with PAX6 gene mutation.
The aniridic probands showed typical features of sclero-
cornea with nystagmus in proband 28–1 (A); Foveal hypopla-
sia in proband 27–1 (B); Ptosis, microcornea with dislocated
cataractous lens in proband 10–1 (C); Ectopia lentis in
proband 16–1 (D).

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proband 16–1 with c.830G>A. Even though Marfan syn-
drome is rarely associated with aniridia [3] we observed
the syndrome in the proband. The peripheral corneal pan-
nus with bullous keratopathy and corneal ectasia was
observed in IVS9-12C>T. It is worthwhile to note that all
the mutations observed in this study are associated with
common phenotypes.
Our data provide evidence that some of the PAX6 gene
mutations identified in aniridia patients lead to disrup-
tion of PAX6 gene expression due to the premature termi-
nation and most of which are assumed to cause loss of
activity of one allele [30]. The Nonsense mutations would
be predicted to result in truncated proteins due to a mech-
anism called 'nonsense-mediated decay' which degrades
mRNAs containing premature stop codons (nonsense
mutations). Therefore, PAX6 mRNAs that contain non-
sense mutations would be degraded and would not be
translated [31]. Identifying new mutations contributes
valuable information for carrier detection for specific
effect mutation and genetic counseling.
Conclusions
In summary we add three novel frameshift mutations, two
nonsense mutations and a polymorphism to the existing
spectrum of PAX6 mutations in south India in patients
with the characteristic phenotype of aniridia. The two
nonsense mutations are identified for the first time in
south Indian population, which is not a well-studied
ethnic group. Our genetic analysis provides further evi-
dence that haploinsuffiency of the PAX6 gene causes the
classic aniridia.
The pedigree and SSCP analysis of aniridia probands
Figure 2
The pedigree and SSCP analysis of aniridia probands. Pedigree shows the affected (arrow) and unaffected individuals to the cor-
responding SSCP gel; unaffected family members (1,2,3), filled square (male) and filled circle (female) are affected members. N-
unrelated healthy controls. Arrow indicates the mobility shift in SSCP gel.