interactions with other factors . 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 .
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
, 49% (34/70) in Caucasian , 30% (9/30) in Mexican
, and 67% (4/6) in Thai patients . 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.
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
c.766-12C>T (rs667773) was found in 2 probands. This
SNV has been considered as probably a neutral polymorphism
and defined as variation in 1000 Genomes. Various mutations
were detected with this SNV including c.949C>T, c.277G>A,
c.607C>T, c.1267dupT in the PAX6 mutation database
(Online Human PAX6 Allelic Database), and we cannot find
any cosegregation pattern.