Chinese Medical Journal 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, YANG Wei-yan
YANG Shu-zhi (Department of Otolaryngology Head and Neck Surgery, First Affiliated Hospital to Chinese General Hospital of PLA, Beijing 100037)
CAO Ju-yang (Institute of Otolaryngology, Chinese General Hospital of PLA, Beijing 100853, China)
ZHANG Rui-ning (Department of Otolaryngology Head and Neck Surgery, Central Hospital, Yuncheng 044000, China)
LIU Li-xian (Institute of Otolaryngology, Chinese General Hospital of PLA, Beijing 100853, China)
LIU Xin (Institute of Otolaryngology, Chinese General Hospital of PLA, Beijing 100853, China)
ZHANG Xin (Institute of Otolaryngology, Chinese General Hospital of PLA, Beijing 100853, China)
KANG Dong-yang (Institute of Otolaryngology, Chinese General Hospital of PLA, Beijing 100853, China)
LI Mei (Institute of Otolaryngology, Chinese General Hospital of PLA, Beijing 100853, China)
HAN Dong-yi (Institute of Otolaryngology, Chinese General Hospital of PLA, Beijing 100853, China)
YUAN Hui-jun (Institute of Otolaryngology, Chinese General Hospital of PLA, Beijing 100853, China)
YANG Wei-yan (Institute of Otolaryngology, Chinese General Hospital of PLA, Beijing 100853, China)Correspondence to:YUAN Hui-jun,Institute of Otolaryngology, Chinese General Hospital of PLA, Beijing 100853, China (Tel: 86-10-66936753. Fax:86-10- 68156974. E-mail:email@example.com)
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.
Waardenburg 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 canthorum, respectively. The presence of limb abnormalities separates type 3 WS (Klein-Waardenburg syndrome, WS3; MIM 148820) from type 1. Type 4 WS (Shah-Waardenburg syndrome or Waardenburg- Hirschsprung disease, WS4; MIM 277580) is 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 Y)-box 10).
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 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 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 committee.
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 (www.genome.wi.mit.edu/cgi-bin/primer/ primer3_www.cgi). The primers used for amplification of these exons are shown in Table.
Table. PCR primers for amplification of PAX3 exons
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. White forelock and 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 phenotypic characteristics.
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: heterochromia iridum.
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 position 81.
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. dbSNP:12623857).
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 the variable clinical 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 variability.
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 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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