Chin Med J 2007; 120 (1):46-49
Nonsense mutations in the PAX3 gene cause Waardenburg
syndrome type I in two Chinese patients
YANG Shu-zhi, CAO Ju-yang, ZHANG Rui-ning, LIU Li-xian, LIU Xin, ZHANG Xin,
KANG Dong-yang, LI Mei, HAN Dong-yi, YUAN Hui-jun and YANG Wei-yan
Keywords: Waardenburg syndrome type 1; PAX3 gene; mutation
Background Waardenburg syndrome type I (WS1) is an autosomal dominant disorder characterized by sensorineural
hearing loss, pigmental abnormalities of the eye, hair and skin, and dystopia canthorum. The gene mainly responsible for
WS1 is PAX3 which is involved in melanocytic development and survival. Mutations of PAX3 have been reported in
familiar or sporadic patients with WS1 in several populations of the world except Chinese. In order to explore the genetic
background of Chinese WS1 patients, a mutation screening of PAX3 gene was carried out in four WS1 pedigrees.
Methods A questionnaire survey and comprehensive clinical examination were conducted in four Chinese pedigrees of
WS1. Genomic DNA from each patient and their family members was extracted and exons of PAX3 were amplified by
PCR. PCR fragments were ethanol-purified and sequenced in both directions on an ABI_Prism 3100 DNA sequencer
with the BigDye Terminator Cycle Sequencing Ready Reaction Kit. The sequences were obtained and aligned to the wild
type sequence of PAX3 with the GeneTool program.
Results Two nonsense PAX3 mutations have been found in the study population. One is heterozygous for a novel
nonsense mutation S209X. The other is heterozygous for a previously reported mutation in European population R223X.
Both mutations create stop codons leading to truncation of the PAX3 protein.
Conclusions This is the first demonstration of PAX3 mutations in Chinese WS1 patients and one of the few examples
of an identical mutation of PAX3 occurred in different populations.
Chin Med J 2007;120(1):46-49
aardenburg syndrome (WS) is an autosomal
dominant disorder characterized by hearing loss
and pigmentation defects of the hair, skin and iris.1 It
accounts for 2% of congenital deafness.2 Four types of
WS have been classified depending on the presence or
absence of additional symptoms. Type 1 WS (WS1; MIM
193500) and type 2 WS (WS2; MIM 193510) are
distinguished by the presence or absence of dystopia
abnormalities separates type 3 WS (Klein-Waardenburg
syndrome, WS3; MIM 148820) from type 1. Type 4 WS
Hirschsprung disease, WS4;
characterized by the presence of an aganglionic
megacolon. WS shows high clinical variability and
genetic heterogeneity. The PAX3 (paired box gene 3) gene
accounts for most of WS1 and WS3, and the MITF
(microphthalmia associated transcription factor) gene is
responsible for about 15% of WS2.3 WS4 is
heterogeneous with reported mutations in EDN3
(endothelin-3) and its receptor EDNRB (endothelin
receptor type B) or sox10 (SRY (sex determining region
PAX3 is one of a family of nine human PAX genes coding
for DNA-binding transcription factors that are expressed
in the early embryo. PAX3 protein contains two highly
conserved DNA binding motifs, a paired domain and a
The presence of limb
paired homeodomain, as well as a highly conserved
octapeptide and a 3’ Ser-Thr-Pro rich region, which is
involved in transcriptional activation.4 PAX3 is expressed
in neural crest cells of the spinal ganglia, the craniofacial
mesectoderm, and the limb mesenchyme during
embryogenesis and plays an important role for the
migration and differentiation of melanocytes, which
originate from the embryonic neural crest. Over 50
different mutations in human the PAX3 gene have been
reported in familiar or sporadic patients with WS1/WS3
in several world populations except the Chinese.
However, most mutations are unique.5 With the aim of
elucidating the clinical features and genetic basis of WS1
Department of Otolaryngology Head and Neck Surgery, First
Affiliated Hospital to Chinese General Hospital of PLA, Beijing
100037 (Yang SZ)
Institute of Otolaryngology, Chinese General Hospital of PLA,
Beijing 100853, China (Cao JY, Liu LX, Liu X, Zhang X, Kang DY,
Li M, Han DY, Yuan HJ and Yang WY)
Department of Otolaryngology Head and Neck Surgery, Central
Hospital, Yuncheng 044000, China (Zhang RN)
Correspondence to: Dr. YUAN Hui-jun and Dr. YANG Wei-yan,
Institute of Otolaryngology, Chinese General Hospital of PLA,
Beijing 100853, China (Tel: 86-10-66936753. Fax: 86-10-
68156974. Email: firstname.lastname@example.org; email@example.com)
This work was supported by a grant from National Natural Science
Foundation of China (No. 30371523) and Research Foundation
from Chinese PLA General Hospital (No. 03YZJJ003) to Dr.
Chinese Medical Journal 2007; 120 (1):46-49
Table. PCR primers for amplification of PAX3 exons
PCR fragment (bp)
Annealing temperature (℃)
in the Chinese population, we screened the entire coding
region of PAX3 for mutations in our patients who were
clinically diagnosed with WS1.
The subjects of this study were recruited from the
Otology Clinic at Chinese PLA General Hospital and
several schools for the deaf and mute in China. Informed
consent, blood samples and clinical evaluations were
obtained from all participating members, under protocols
approved by the Chinese PLA General Hospital ethics
Four WS1 subjects were diagnosed according to the
criteria for WS1 proposed by the Waardenburg
Consortium in 1992.6 A comprehensive clinical history
and neurotological, ophthalmologic and dermatologic
examinations were performed on all of the subjects. The
audiological and neurotological examination consisted of
otoscopy, pure-tone audiometry (Madsen522), immittance
(GSI33) and auditory brain-stem response (ABR)
(SmartEp IHS3099). The ophthalmologic examination
included visual acuity measurements, visual field
examination and fundus ophthalmoscope. Special
attention was given to the color of skin, hair, and iris, and
other developmental defects such as dystopia canthorum
and limb abnormalities. The degree of hearing loss was
defined according to the pure-tone averages (PTA), which
were based on the three frequencies (500, 1000, and 2000
Hz), as follows: normal<26 dB HL, mild 26-40 dB HL,
moderate 41-70 dB HL, severe 71-90 dB HL and
profound >90 dB HL.
The complete PAX3 coding region contains 10 exons
(NCBI accession No. NM_181459). Primers were
designed to amplify each exon including the intron-exon
boundaries with an on-line program PRIMER3
The primers used for amplification of these exons are
shown in Table.
Genomic DNA from each patient and their family
members was extracted from peripheral blood leukocytes
using the standard phenol/chloroform method. Coding
exons were amplified with the primers as described in
Table. PCR fragments were ethanol-purified and sequenced
in both directions on ABI_Prism 3100 DNA sequencer
with a BigDye Terminator Cycle Sequencing Ready
Reaction Kit (Applied Biosystems, Foster City, USA).
The sequences were obtained and aligned to the wild type
sequence of PAX3 with the GeneTool program.
Clinical features of WS1 patients
The four WS1 cases originated from different provinces
in China. While some phenotypic variations were
observed between them, the common features of WS1
they share were dystopia, heterochromia iridis, brown
freckles on the face and congenital, bilateral profound
sensorineural hearing loss.
hypopigmented skin were not found in any of them.
Premature graying hair and an abnormally shaped nose
with a high nasal root and bulbous tip were only found in
case 1, while synophrys was observed in case 2 only. W
indices of cases number 1 to 4 were calculated as 2.763,
2.929, 2.25 and 2.51, respectively.
Tracing the three generations of case 1 we found that 14
of his family members showed variability of depigmented
skin or hair but without hearing loss, heterochromia iridis
and dystopia (Fig. 1). In the other 3 cases, their own
parents were examined and they did not manifest any WS
White forelock and
Fig. 1. Pedigree of case 1. An arrow marks the proband with
WS1. Filled quadrants indicate phenotype associated with
WS1. Upper left: premature graying hair. Lower left: freckles
on the face. Upper right: hearing loss. Lower right:
Chin Med J 2007; 120 (1):46-49
Mutations in PAX3 gene
A novel nonsense mutation was identified in a family
with only one affected member. It was a 2-base pair
deletion at the 40-41 base position of exon 5 (40-
41delCT) (Fig. 2A), leading to a frame-shift mutation and
resulting in a premature termination codon at 209, ten
amino acids before the start of homeodomain (S209X).
The truncated protein product lacks the homeodomain,
the Ser-Thr-Pro rich region and the normal carboxyl
terminus. This mutation was in a heterozygous state and
was not found in the patient’s parents.
One single base substitution in exon 5, 81T>C, was
detected in the heterozygous state in another patient (Fig.
2B). This mutation resulted in a premature termination
codon at 223, five amino acids after the start of the
homeodomain (R223X). The truncated protein product
lacks the bulk of the homeodomain, the Ser-Thr-Pro rich
region and the normal carboxyl terminus. Although 14
family members of this patient showed variability of
depigmented skin or hair with normal hearing, none of
them carried the R223X mutation. We did not find
mutation in the other two WS1 patients.
Fig. 2. Identification of the S209X mutation of PAX3 in one
family. Partial sequence chromatograms of exon5 from the
proband and a married-in-control family member (A). An
arrow indicates the location of the base changed at position
40-41. Identification of the R223X mutation of PAX3 in
another family (B). Partial sequence chromatograms of exon
5 from the proband and a married-in-control family member.
An arrow indicates the location of the base changed at
A synonymous mutation in exon 2, 43C>T, was detected
in homozygous state in all of 4 patients and previously
described as a polymorphism (NCBI accession No.
PAX3 mutations had been found to be responsible for
about 33% to 80% of WS1 cases.7,8 To date, over 50
different mutations had been identified throughout the
whole PAX3 gene. The majority of these mutations
associated with WS are apparently unique to single
families except for E210X, R223X, W274X and
R271C.8-10 S209X is a novel mutation and believed to
have a pathological effect. R223X has been described
previously by Baldwin and Panda et al in the European
population.9,11,12 This is its first confirmation in the
Chinese population. Both S209X and R223X occurred in
exon 5 and were truncating mutations with deletion of the
homeodomain, the Ser-Thr-Pro rich region and the
normal carboxyl terminus.
The phenotypic features of WS1 are highly variable in
families even with the same mutation. R223X had been
reported in two families. Comparing the phenotypic
features of the syndrome we found that one family only
had a high proportion of affected individuals with white
forelock, while the other family contained a high
proportion of affected individuals with premature graying
and heterochromia irides, but no white forelock. In our
family, white forelock was not found either.
The PAX3 protein belongs to the family of paired domain
proteins that bind DNA and regulate gene expression.
Four structural motifs have been identified in PAX3: a
paired domain, a highly conserved octapeptide sequence,
a paired homeodomain, and a Ser-Thr-Pro rich region.13,14
The homeodomain has three helices. Helices 2 and 3 fold
into a structure related to the helix-turn-helix motif found
in prokaryotes.15 Structural studies of the homeodomain
bound to DNA have shown that helix 3, also known as the
recognition helix, lies in the major groove, and makes
sequence-specific contacts with bases, as well as several
contacts with the sugar-phosphate backbone. The S209X
and R223X mutations result in deletion of the
homeodomain, the Ser-Thr-Pro rich region and the
normal carboxyl terminus, and are predicted to decrease
DNA binding affinity and/or specificity; the mutant PAX3
protein cause clinical symptoms.
A threshold hypothesis for the pathogenesis of WS1 and
the Splotch mouse (the mouse homologue of human WS1)
phenotypes may explain
manifestations. It is likely that the critical threshold
values may be different in different tissues and even
among individuals, and when the amount of the mutant
PAX3 product surpasses the threshold, it may lose the
action as a transcription factor.13,16 Environmental and
genetic factors may also account for the phenotypic
variation. Genetic factors include allelic heterogeneity
within PAX3, modifier genes, or polygenic background.
Recent studies in both mice and humans have produced
strong evidence for genes modifying the severity of WS4
patients.17-19 Eventual identification of the modifier genes
may contribute to better understanding of the phenotypic
As far as we know from the study, WS1 is not a rare
disease in the Chinese population and PAX3 is a good
candidate gene for screening mutations. This is the first
demonstration of PAX3 mutations in Chinese WS1
the variable clinical
Chinese Medical Journal 2007; 120 (1):46-49 Download full-text
patients and one of the few examples of an identical
mutation of PAX3 occurring in different populations.
Acknowledgements: We thank all these family members for their
kind participation in the study.
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(Received August 23, 2006)
Edited by GUO Li-shao