Molecular analysis of the PAX6 gene for congenital aniridia in the Korean population: Identification of four novel mutations

Seoul St. Mary's Hospital, Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea.
Molecular vision (Impact Factor: 1.99). 02/2012; 18(53):488-94.
Source: PubMed
ABSTRACT
To analyze the paired box gene 6 (PAX6) in Korean patients with congenital aniridia.
Genomic DNA was isolated from peripheral blood leukocytes of 22 aniridia patients in 18 unrelated families. Polymerase chain reaction was performed for all 14 exons of PAX6 followed by bidirectional sequencing.
Fourteen different kinds of mutations were detected in 16 of 18 unrelated families (mutation detection rate: 88.9%), including four novel mutations; c.658G>T (p.Glu220*), c.464delG (p.Ser155Thrfs*52), c.87_90dupTGTA (p.Glu31Cysfs*26), and c.642A>C (p.Arg214Ser), among which the former three mutations induce premature termination of PAX6 protein translation. Approximately 92.9% of identified mutations lead to the premature termination of the protein resulting from 7 nonsense mutations (50.0%), 3 splicing errors (21.4%), 2 deletions (14.3%), and 1 insertion (7.1%).
Most of the mutations identified in Korean aniridia patients lead to the premature truncation of the PAX6 protein, supporting that PAX6 protein haploinsufficiency causes the classic aniridia phenotype. We also found four novel PAX6 mutations associated with aniridia.

Full-text

Available from: Myungshin Kim, Dec 06, 2015
Molecular analysis of the PAX6 gene for congenital aniridia in the
Korean population: Identification of four novel mutations
Shin Hae Park,
1
Man Soo Kim,
1
Hyojin Chae,
2
Yonggoo Kim,
2
Myungshin Kim
2
1
Seoul St. Mary’s Hospital, Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of
Korea, Seoul, Korea;
2
Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea
Purpose: To analyze the paired box gene 6 (PAX6) in Korean patients with congenital aniridia.
Methods: Genomic DNA was isolated
from peripheral blood leukocytes of 22 aniridia patients in 18 unrelated families.
Polymerase chain reaction was performed for all 14 exons of PAX6 followed by bidirectional sequencing.
Results: Fourteen different kinds of mutations were detected in 16 of 18 unrelated families (mutation detection rate:
88.9%), including four novel mutations; c.658G>T (p.Glu220*), c.464delG (p.Ser155Thrfs*52), c.87_90dupTGTA
(p.Glu31Cysfs*26), and c.642A>C (p.Arg214Ser), among which the former three mutations induce premature termination
of PAX6 protein translation. Approximately 92.9% of identified mutations lead to the premature termination of the protein
resulting from 7 nonsense mutations (50.0%), 3 splicing errors (21.4%), 2 deletions (14.3%), and 1 insertion (7.1%).
Conclusions: Most of the mutations identified in Korean aniridia patients lead to the premature truncation of the PAX6
protein, supporting that PAX6 protein haploinsufficiency causes the classic aniridia phenotype. We also found four novel
PAX6 mutations associated with aniridia.
Congenital aniridia (OMIM 106210)
is a rare
ocular
malformation that affects the development of multiple ocular
structures and is caused by a mutation in the paired box gene
6 (PAX6) located on chromosome 11p13 [1-3]. Iris hypoplasia
is the most obvious sign, but a broad spectrum of disorders
can manifest [2-4]. Many patients have corneal opacities,
cataracts, nystagmus, and foveal and optic nerve hypoplasia.
Aniridia typically causes severe visual impairment; the major
causative factor of this condition is foveal hypoplasia [5].
The incidence of congenital aniridia ranges from
1:64,000 to 1:96,000 [5]. In two-thirds of the cases, it is
inherited in an autosomal dominant fashion with almost
complete penetrance and variable expressivity; and the
remaining one-third of the cases are sporadic [1,6,7]. Some
sporadic cases have a risk of developing Wilms tumor as a
part of WAGR (Wilms tumor, aniridia, genitourinary
abnormalities, and mental retardation; OMIM 194072), which
is caused by deletion of both PAX6 and Wilms’ tumor gene
(WT1) in the 11p13 region.
PAX6 was isolated as a candidate gene for aniridia by
positional cloning in 1991 [8]. Heterozygous mutations are
found in about 40%–80% of all non-syndromic aniridia
patients [1,9-11]. Numerous PAX6 mutations have been
detected in aniridia patients (Online Human PAX6 Allelic
Database), and premature termination of the PAX6 protein is
the most frequent type of mutation [11].
Correspondence to: Myungshin Kim, M.D., Department of
Laboratory Medicine, College of
Medicine, The Catholic University
of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea;
Phone: 82-2-2258-1645; FAX: 82-2-2258-1719; email:
microkim@catholic.ac.kr
Although about 60 cases of congenital aniridia have been
reported in Korea since
the first report in 1977, little is known
about the molecular characterization of congenital aniridia in
Koreans [12-15]. Here, we analyzed PAX6 in 22 Korean
aniridic patients and identified the genetic aberrations and
genotype-phenotype correlations.
METHODS
This study was approved by the Ethics Committee of Seoul
St. Mary’s Hospital, The Catholic University of Korea
(KC11RISI0722). Informed consent was obtained from the
patients.
We evaluated 22 patients in 18 unrelated aniridia families
in Seoul St.Mary’s Hospital. The age, gender, visual acuity,
family history, and previous ocular history of the patients were
recorded. Thorough ocular examinations were performed,
including best-corrected visual acuity (BCVA), intraocular
pressure (IOP), and refractive measurement and slit lamp
biomicroscopy of the anterior segment and fundus. After
receiving informed consent, blood samples were collected
from all patients for DNA extraction and PAX6 analysis.
Genomic DNA was isolated from peripheral blood
leukocytes with the QIAmp DNA Mini Kit (Qiagen,
Hamburg, Germany). The DNA was quantified
spectrophotometrically using a ND-1000 (Nanodrop
Technologies Inc., Wilmington, DE). All 14 exons (including
an alternatively spliced exon 5a) of PAX6 were amplified
using the primers as previously described (Table 1) [10]. For
all amplicons, the genomic DNA was denatured at 95 °C for
5 min followed by 35 cycles of denaturation at 95 °C for 30
s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min,
Molecular Vision 2012; 18:488-494 <http://www.molvis.org/molvis/v18/a53>
Received 6 December 2011 | Accepted 16 February 2012 | Published 19 February 2012
© 2012 Molecular Vision
488
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and final extension at 72 for 5 min. The PCR products were
examined by agarose gel (1.5%) electrophoresis followed by
staining the gel in
ethidium bromide (0.5 μg/ml), which then
was visualized under ultraviolet (UV) light in a gel
documentation system (Gel Doc 1000; Biorad, Hercules, CA).
PCR amplicons were bidirectionally sequenced with the Big
Dye terminator v3.1 cycle sequencing kit (Applied
Biosystems, Foster City, CA) using the ABI PRISM 3100
Genetic Analyzer (Applied Biosystems). RefSeq ID:
NM_000280.3 was used for cDNA nucleotide numbering.
RESULTS
Ocular phenotypes: Table 2 shows the ocular phenotypes of
22 patients in 18 unrelated families tested. The male/female
ratio was 0.59. The percentage of sporadic cases was 36.4%.
The mean age of the patients was 19.4±14.7 years. Total
aniridia was demonstrated in 20 patients and partial aniridia
in 2 patients (patients 6-1 and 17). Glaucoma, cataracts,
keratopathy above grade II, and foveal hypoplasia were also
observed in addition to iris aplasia in the patients, as detailed
below. Nephroblastoma did not develop during the follow-up
period.
Glaucoma was observed in 7 of 22 patients (31.8%). Six
patients
could maintain IOP within the normal range with
topical anti-glaucoma medications, but one female (patient
12) required surgical treatment with Ahmed valve
implantation in her left eye at 12 years old.
Cataracts were seen in 18 of 22 patients (81.8%), and 6
had received cataract surgery. Congenital corneal opacity was
observed in two children (patients 2 and 14), who required
penetrating keratoplasty. A fundus examination was
performed in 20 patients who did not have severe corneal or
lens opacity. Foveal hypoplasia, defined as the absence of a
foveal reflex, was found in 17 patients (85%).
Genetic analysis of PAX6: The patients’ molecular findings
are summarized in Table 3. Fourteen different mutations were
detected in 16 of 18 unrelated families (88.9%). We found
four novel mutations, including c.87_90dupTGTA, c.
464delG, c.642A>C, and c.658G>T, in addition to 10 known
mutations: c.11–2A>G, c.19G>T, c.301delG, c.317T>A, c.
524–2A>G, c.607C>T (n=3), c.718C>T, c.901C>T, c.
949C>T (n=2), and c.1183+2T>C [9,16-26].
The types of mutations were as follows: 11 single
nucleotide substitutions, including 1 missense mutation
TABLE 1. LIST OF PRIMERS USED TO PERFORM AMPLIFICATION AND SEQUENCING OF THE 14 PAX6 EXONS.
PAX6 gene
Primer sequences for PAX6 (5′→3′) Tm (°C)
Exon 1 F-CTCATTTCCCGCTCTGGTTC 56
R-AAGAGTGTGGGTGAGGAAGT
Exon 2 F-TTATCTCTCACTCTCCAGCC 56
R-AAGCGAGAAGAAAGAAGCGG
Exon 3 F-TCAGAGAGCCCATCGACGTAT 56
R-CTGTTTGTGGGTTTTGAGCC
Exon 4 F-TTGGGAGTTCAGGCCTACCT 56
R-GAAGTCCCAGAAAGACCAGA
Exon 5 F-CCTCTTCACTCTGCTCTCTT 56
R-ATGAAGAGAGGGCGTTGAGA
Exon 5a F-TGAAAGTATCATCATATTTGTAG 50
R-GGGAAGTGGACAGAAAACCA
Exon 6 F-GTGGTTTTCTGTCCACTTCC 56
R-AGGAGAGAGCATTGGGCTTA
Exon 7 F-CAGGAGACACTACCATTTGG 56
R-ATGCACATATGGAGAGCTGC
Exon 8 F-GGGAATGTTTTGGTGAGGCT 56
R-CAAAGGGCCCTGGCTAAATT
Exon 9 F-GTAGTTCTGGCACAATATGG 54
R-GTACTCTGTACAAGCACCTC
Exon 10 F-GTAGACACAGTGCTAACCTG 56
R-CCCGGAGCAAACAGGTTTAA
Exon 11 F-TTAAACCTGTTTGCTCCGGG 56
R-TTATGCAGGCCACCACCAGC
Exon 12 F-GCTGTGTGATGTGTTCCTCA 56
R-TGCAGCCTGCAGAAACAGTG
Exon 13 F-CATGTCTGTTTCTCAAAGGGA 54
R-GAACAATTAACTTTTGCTGGCC
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T
ABLE
2. O
CULAR
FINDINGS
IN K
OREAN
PATIENTS
WITH CONGENITAL ANIRIDIA
.
Case No
Age/gender
Inheritance
BCVA (OD/OS) Nystagmus Keratopathy
Cataract Glaucoma
Macular
hypoplasia
Comments
1-1 27/F Familial 0.1/0.1 + Grade II + - +
1-2 3Mon/F Familial F&F (+) + Grade IV - - uncheckable Corneal opacity
2 34/M Sporadic 0.2/0.16 + Grade I + - -
3 15/M Sporadic 0.25/0.16 + - + - -
4 1/F Sporadic F&F (+) + Grade II + - +
5 24/M Familial 0.04/0.04 + Grade II + +, eyedrops +
6-1 31/F Sporadic HM/0.02 + Grade IV + +, eyedrops + Partial aniridia
6-2 1/M Familial F&F (+) + - + - +
7-1 8/F Familial 0.1/FC 30 cm + Grade III + - +
7-2 40/M Familial FC 30 cm /0.1 + Grade IV + +, eyedrops +
8-1 48/F Familial 0.02/0.02 + Grade IV + - +
8-2 15/F Familial 0.16/0.06 + Grade I + +, surgery + Valve implant
9 21/M Sporadic 0.32/0.2 + - - - +
10 3/M Sporadic FC10 cm/LP- + Grade IV + +, eyedrops uncheckable Corneal opacity
11 30/M Familial 0.1/FC 30 cm + Grade IV + - +
12 15/F Familial FC50 cm/0.1 + Grade I + - +
13 8/M Sporadic 0.16/0.2 + Grade II + - +
14 4/M Familial 0.16/0.16 + - - +, eyedrops +
15 30/M Familial 0.02/0.1 + Grade III + +, eyedrops +
16 48/F Familial 0.04/0.04 + Grade IV + - +
17 16/M Familial 0.06/0.06 + Grade I + - + Partial aniridia
18 8Mon/M Sporadic F&F (+) + - - - -
M: Male; F: Female; F&F: Fix and follow; PD: paired domain; LNK: linker domain; HD: Homeodomain; PST: proline-, serine-, and threonine-rich transregulatory
domain. Keratopathy was graded as follows: grade 0, clear; grade 1, peripheral mudding with ingrowth of neovascular tissue not exceeding 1 mm from the limbal
arch; grade II, peripheral neovascularization in at least the peripheral half of the cornea, corneal clouding, and subepithelial fibrosis; grade III, involvement of the
central cornea.
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(7.1%), 7 nonsense mutations (50.0%), and 3 intronic
mutations that lead to splicing errors (21.4%), in addition to
3 indel mutations that resulted in frameshifts (21.4%).
The DNA-binding domains (DBDs) of the PAX6 protein
were composed of the 128 amino acid paired domain (PD) and
the 61 amino acid homeodomain (HD) separated by a linker
region (LNK). The proline-, serine-, and threonine-rich
transregulatory (PST) domain in the COOH-terminal region
was composed of 152 amino acids. In this study, 5 kinds of
mutations (35.7%) occurred in the PD, and 3 kinds of
mutations (21.4%) occurred in the LNK, HD, and PST
domains. Relatively fewer mutations have been detected in
the PST domain considering its long size.
Four novel mutations were detected in this study (Table
3, Figure 1). Patient 3 showed the c.87_90dupTGTA
(p.Glu31Cysfs*26) mutation in exon 5 within the PD, which
resulted in a premature termination due to the frame-shift.
Patients 6–1 (mother) and 6–2 (son) possessed a 1-bp deletion,
c.464delG (p.Ser155Thrfs*52), in exon 7, which causes a
frameshift and premature termination of translation in the
LNK domain of the PAX6 protein. Patient 10 was 3 year-old
male and had a novel missense mutation, c.642A>C
(p.Arg214Ser), in exon 8 within the HD. This mutation was
predicted to be not tolerable by SIFT analysis and possibly
damaging by PolyPhen analysis. This child had a severe
clinical manifestation of aniridia with marked corneal opacity
and increased IOP in both eyes. This mutation was not
detected in his unaffected mother. Patient 11 showed a novel
nonsense mutation, c.658G>T (p.Glu220*), in exon 8, which
results in premature termination within the LNK domain.
Single nucleotide variation (SNV) c.766–12C>T
(rs667773) was detected in 3 patients in 2 probands (7–1, 7–
2, 10) who represented c.524–2A>G and c.642A>C,
respectively.
Genotype-phenotype correlation: Ophthalmic and genetic
findings exhibited inter- and intrafamilial phenotypic
variabilities of the disease. Patient 1–2 had bilateral congenital
corneal opacities presumed to be accompanying Peters
anomaly, which were not observed in her mother (Patient 1–
1) with the same splicing error mutation (c.11–2A>G). In the
family with the c.464delG mutation (Patients 6–1 and 6–2),
one showed complete aniridia, and the other showed partial
aniridia. Patient 8–2 had juvenile onset glaucoma in both eyes,
which was not detected in Patients 8–1 and 9 with the same
nonsense mutation (c.607C>T).
We did not find any phenotypic differences according to
the location of the identified genotype. Nineteen patients had
total aniridia irrespective of the domain of identified
mutations, except for Patient 6–1 with the LNK domain
frameshift mutation. The ocular phenotypes in patients with
three truncating mutations in the PST domains, which retained
the intact DNA-binding domains, were comparable to that
with mutations within the PD and HD domains.
TABLE 3. MOLECULAR FINDINGS IN KOREAN PATIENTS WITH CONGENITAL ANIRIDIA.
Case No Mutation Exon/Intron Domain mRNA/protein effect
1-1 c.11-2A>G Intron 4 PD Splicing error
1-2 c.11-2A>G Intron 4 PD Splicing error
2 c.19G>T Exon 5 PD p.Gly7*
3 c.87_90dup TGTA† Exon 5 PD p.Glu31Cysfs*26
4 c.301delG Exon 6 PD p.Glu101Lysfs*23
5 c.317T>A Exon 6 PD p.L106*
6-1 c.464delG† Exon 7 LNK p.Ser155Thrfs*52
6-2 c.464delG† Exon 7 LNK p.Ser155Thrfs*52
7-1 c.524-2A>G Intron 7 LNK Splicing error
7-2 c.524-2A>G Intron 7 LNK Splicing error
8-1 c.607C>T Exon 8 LNK p.Arg203*
8-2 c.607C>T Exon 8 LNK p.Arg203*
9 c.607C>T Exon 8 LNK p.Arg203*
10 c.642A>C† Exon 8 HD p.Arg214Ser
11 c.658G>T† Exon 8 HD p.Glu220*
12 c.718C>T Exon 9 HD p.Arg240*
13 c.901C>T exon10 PST p.Gln310*
14 c.949C>T Exon 11 PST p.Arg317*
15 c.949C>T Exon 11 PST p.Arg317*
16 c.1183+2T>C Intron 12 PST Splicing error
17 Not detected
18 Not detected
A mark with † indicates the four novel mutations identified in this study.
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DISCUSSION
In this report, we described PAX6
mutations in 22 Korean
aniridia patients from 18 unrelated families. The mutation
detection rate was 88.9% (16/18). The mutation spectrum of
PAX6 in aniridia was highly biased, as 92.9% of identified
mutations included 7 nonsense mutations (50.0%), 3 splicing
errors (21.4%), 2 deletions (14.3%), and 1 insertion (7.1%),
leading to the premature truncation of the protein and one
missense mutation inducing an amino acid change in the HD
domain. Interestingly, 4 novel mutations were identified in
this study, including 3 mutations (c.87_90dupTGTA, c.
464delG, and c.658G>T) leading to the premature termination
of the PAX6 protein and one missense mutation (c.642A>C).
The phenotype of aniridia could be explained by the
haploinsufficiency of the PAX6 protein, in which the mutated
PAX6 protein does not have any transcriptional activity and
the remaining single normal copy of PAX6 is not enough to
produce a sufficient threshold level of biologically active
PAX6 protein to initiate the transcription of its target genes
[11,27,28]. A critical dose of PAX6 protein is required to
initiate the transcription of its downstream target genes for
normal eye development [28]. Nonsense-mediated decay,
which is the process in which mRNAs containing premature
termination codons are degraded before they produce large
amounts of truncated proteins, is relevant to the
pathomechanism of aniridia because the major mutations
detected in aniridia are truncations. Haploinsufficiency in the
mutants in the COOH-terminal half of the PAX6 protein could
be explained by dominant-negative effects, which could be
caused by competition for DNA-binding between truncated
PAX6 proteins and wild-type PAX6 proteins. Some truncated
mutants have 3–5 fold higher affinities to various DNA
binding sites when compared with the wild-type PAX6 [27].
The clinical manifestations associated with aniridia
express variable phenotypes. We did not find any phenotypic
differences according to the location of the identified
genotypes. Our results also exhibited interfamilial (Patients
8–2 and 9) and intrafamilial (Patients 1–1 and 1–2, Patients
6–1 and 6–2, and Patients 8–1 and 8–2) phenotypic
variabilities of the disease. Atchaneeyasakul et al. reported
that the total aniridia phenotype was associated with
mutations at the COOH-terminus, whereas partial aniridia
patients carried mutations that resulted in a loss of the
homeodomain with or without a loss of the paired domain
[29]. However, our results are not consistent with that finding.
Total aniridia was observed in 19 patients irrespective of the
domain of identified mutations, except for patient 6–1 with
the frameshift mutation within the LNK. The reason for
variable phenotypes among individuals with the same
mutation is unclear. The variable expressivity could be
explained by subtle differences in PAX6 protein levels and
the ratio of mutant to wild type PAX6 protein or the
Figure 1. Four novel PAX6 mutations were identified in
this study. A-D: Sequencing chromatograms of c.658G>T, c.642A>C, c.
87_90dupTGTA, and c.464delG, respectively.
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interactions with other factors [27]. Mutations behind the HD
could potentially lead to more severe phenotypes than
truncating mutations within the DNA-binding domains, such
as the PD and HD [11,28]. Generally, the PAX6 missense
mutation occurs less frequently in aniridia and has a tendency
to be associated with milder phenotypes [18,30]. A PAX6
protein with an amino acid substitution could still retain some
residual activity and result in partial haploinsufficiency. Some
missense mutations might have the potential to impair the
proper folding of the PAX6 protein and compromise the
normal three dimensional structure. In our 3-year old male
(Patient 10) carrying the novel missense mutation c.642A>C
(p.Arg214Ser), other factors than the mutated PAX6 protein
might contribute to his severe phenotype.
Accodring to the PAX6 mutation database, the most
frequent PAX6 mutations in aniridia are c.607C>T, c.718C>T,
c.949C>T, and c.1267dupT (Online Human PAX6 Allele
Database) [9,11]. The former three muations were also
identified in this study. The distribution of the identified
mutations was as follows: 35.7% in the PD, 21.4% in the LNK,
21.4% in the HD, and 21.4% in the PST domains. Definite
mutational hot spots were not observed in our study. In two
patients, a PAX6 mutation was not identified with direct DNA
sequencing throughout the whole gene. Exon deletions and
deletions of control regions can be the cause of isolated
aniridia, so that tests used to identify gene copy number, such
as quantitative PCR, mutiplex ligation-dependent probe
amplification (MLPA), and array comparative genomic
hybridization, may be helpful to clarify such cases.
Generally, it is recommended to perform several analyses
in anridia to obtain the maximum detection yield, as aniridia
could be caused by different type of genetic aberrations.
Chromosomal rearrangement and deletion can be detected by
karyotype analysis espcially in the cases of WAGR or the
aniridia patients presenting other malformation [24].
Fluorescence in situ hybridization and MLPA can detect
cryptic deletion of PAX6 effectively [24,31]. Detection of
PAX6 mutations was performed using direct sequencing
method combined with or without mutation detection
screening tools such as DHPLC (denaturing high performance
liquid chromatography) or SSCP (single-strand conformation
polymorphism). Mutation detection rate by direct sequencing
of PAX6 was variable as follows; 47% (18/38) in Chinese
[32], 49% (34/70) in Caucasian [31], 30% (9/30) in Mexican
[9], and 67% (4/6) in Thai patients [29]. To our best
knowledge, our PAX6 mutation detection rate of 88.9% is the
one of highest rates by single test alone. One of the estimated
reason of our high mutation detection rate is the characteristics
of patients included in this study. Most of patients had
clinically definite non-syndromic aniridia with total absence
of iris (20/22). The other possible reason to improve detection
rate is that we performed bidirectional sequencing in all
samples because the mutations in aniridia patients were
distributed throughout the whole exon and intron of PAX6.
Based on our result, the bidirectional DNA sequencing
including whole exon and intron-exon boundary of PAX6
could be recommended as the first screening test for the
molecular confirmation of aniridia, especially when it is not
combined with other systemic abnomalities such as renal
tumor, genitourinary abnormalities, and mental retardation.
In conclusion, most of the mutations identified in Korean
aniridia patients lead to the premature truncation of the PAX6
protein, supporting that haploinsufficiency of the PAX6
protein causes the classic aniridia phenotype. Also, we found
four novel PAX6 mutations associated with aniridia.
ACKNOWLEDGMENTS
This work was supported by “Laboratory reagent
development and evaluation for clinical application
(10024719)” under the Industrial Source Technology
Development Programs of the Ministry of Knowledge
Economy (MKE) of Korea.
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Molecular Vision 2012; 18:488-494 <http://www.molvis.org/molvis/v18/a53> © 2012 Molecular Vision
Articles are provided courtesy of Emory University and the Zhongshan Ophthalmic Center, Sun Yat-sen University, P.R. China.
The print version
of this article was created on 16 February 2012. This reflects all typographical corrections and errata to the
article through that date. Details of any changes may be found in the online version of the article.
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    • "This result also clarified the recurrence risk advice for affected family members for this autosomaldominant condition. The proband in Family 18 had a de novo missense mutation in the paired domain of PAX6 (Figs. 4F and 5C), adding to the increasing evidence in the literature that missense mutations in the paired domain of PAX6 can lead to a milder nonaniridia phenotype [Park et al., 2012] , possibly by affecting DNA binding to transcriptional targets. Similar to the PAX6 frameshift family, this result has important implications for this patient. "
    [Show abstract] [Hide abstract] ABSTRACT: Congenital cataracts are a significant cause of lifelong visual loss. They may be isolated or associated with microcornea, microphthalmia, anterior segment dysgenesis (ASD) and glaucoma, and there can be syndromic associations. Genetic diagnosis is challenging due to marked genetic heterogeneity. In this study, next-generation sequencing (NGS) of 32 cataract-associated genes was undertaken in 46 apparently non-syndromic congenital cataract probands, around half sporadic and half familial cases. We identified pathogenic variants in 70% of cases, and over 68% of these were novel. In almost two-thirds (20/33) of these cases, this resulted in new information about the diagnosis and/or inheritance pattern. This included identification of: new syndromic diagnoses due to NHS or BCOR mutations; complex ocular phenotypes due to PAX6 mutations; de novo autosomal dominant or X-linked mutations in sporadic cases; and mutations in two separate cataract genes in one family. Variants were found in the crystallin and gap junction genes, including the first report of severe microphthalmia and sclerocornea associated with a novel GJA8 mutation. Mutations were also found in rarely reported genes including MAF, VIM, MIP, and BFSP1. Targeted NGS in presumed non-syndromic congenital cataract patients provided significant diagnostic information in both familial and sporadic cases. This article is protected by copyright. All rights reserved.
    Full-text · Article · Dec 2015 · Human Mutation
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    • "These results conform to genotype–phenotype correlation analysis and we verified that the deletion caused the aniridia in this family and haplo-insufficiency of the PAX6 gene may underlie the aniridia phenotype. Deletion of PAX6 in aniridia patients had been previously described in several studies (Cai et al., 2010; Zhang et al., 2011; Chen et al., 2012 Chen et al., , 2013 Lin et al., 2012; Park et al., 2012; Wawrocka et al., 2013; Lee et al., 2014 ). According to the PAX6 Allelic Variant Database, over three-quarters of aniridia cases are caused by introduction of PTC in the open reading frame of PAX6, just as our study has demonstrated. "
    [Show abstract] [Hide abstract] ABSTRACT: Aniridia is a rare, congenital ocular disorder with the characteristics of incomplete formation of the iris caused by the mutations of the paired box gene-6 (PAX6). To investigate the clinical characterization and the underlying genetic defect in a Chinese family with autosomal dominant aniridia, we recruited the family members who underwent comprehensive ophthalmic examination. A novel heterozygous PAX6 deletion mutation c.796 del G (p.A266 fs) (GenBank ID: KP255960) in exon 10 was exclusively observed in all affected individuals but not in any of the unaffected family members or unrelated controls. The PAX6 mRNA level was about 50% lower in patients with aniridia than in unaffected family members, indicating that this mutation caused nonsense-mediated mRNA decay. In conclusion, we identified a novel deletion mutation in the PAX6 gene resulting in an abnormal PAX6 COOH-terminal extension in the Chinese family with aniridia. Our study further expands the mutation spectrum of PAX6. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Mar 2015 · Gene
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    • "In the present study, the mutation detection rate was 43.3% (13/30), which is comparable to previous reports [36,383940. However, several other studies have also described higher mutation detection rates [35,414243. In 17 patients with aniridia, a PAX6 mutation was not identified with the direct DNA sequencing approach. "
    [Show abstract] [Hide abstract] ABSTRACT: Aniridia is a rare panocular disorder characterized by iris hypoplasia and other associated eye anomalies. Heterozygous null mutations in paired box gene 6 (PAX6) are the major cause of the classic aniridia phenotype. This study aims to detect the mutational spectrum of PAX6 and associated phenotypes in southern Indian patients with sporadic and familial aniridia. Genomic DNA was isolated from peripheral blood from all participants. The coding regions and flanking intronic sequences of PAX6 were screened with Sanger sequencing in 30 probands with aniridia. The identified variations were further evaluated in available family members and 150 healthy controls. The pathogenic potential of the mutations were assessed using bioinformatics tools. Thirteen different mutations were detected in eight sporadic and five familial cases. Eleven novel mutations, including five insertions (c.7_10dupAACA, c.567dupC, c.704dupC, c.868dupA and c.753_754insTA), two deletions (c.242delC and c.249delT), and four splicing variants (c.10+1G>A, c.141G>A, c.141+4A>G and c.764A>G) were identified in this study. Clinical findings of the patients revealed phenotypic heterogeneity with the same or different mutations. This study reported 11 novel mutations and thus expanded the spectrum of PAX6 mutations. Interestingly, all mutations reported in this study were truncations, which confirms the hypothesis that haploinsufficiency of PAX6 causes the aniridia phenotype. Our observations revealed inter- and intrafamilial phenotypic variability with PAX6 mutations. The common ocular findings associated with PAX6 mutations were iris hypoplasia, nystagmus, and foveal hypoplasia reported in almost all cases, with cataract, glaucoma, and keratopathy reported in approximately 50% of the patients.
    Full-text · Article · Jan 2015 · Molecular vision
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