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/primer3www.cgi). 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. 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.
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.
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.
We thank all these family members for their kind participation in the study.
1. Pardono E, van Bever Y, van den Ende J, Havrenne PC, Iughetti P, Maestrelli SP, et al. Waardenburg syndrome: clinical differentiation between types I and II. Am J Med Genet 2003; 117: 223-235.
2. Newton VE. Clinical features of the Waardenburg syndromes. Adv Otorhinolaryngol 2002; 61: 201-208.
3. Read AP, Newton VE. Waardenburg syndrome. J Med Genet 1997; 34: 656-665.
4. Edgar D, Haruhiko K, Rudi B. PAX genes and organogenesis. Bioessays 1997; 19: 755-765.
6. Farrer LA, Grundfast KM, Amos J, Arnos KS, Asher JHJ, Beighton P, et al. Waardenburg syndrome (WS) type 1 is caused by defects at multiple loci, one of which is near ALPP on chromosome 2: first report of the WS Consortium. Am Hum Genet 1992; 50: 902-913.
7. Farrer LA, Arnos KS, Asher JH, Baldwin CT, Diehl SR, Friedman TB, et al. Locus heterogeneity for Waardenburg syndrome is predictive of clinical subtypes. Am Hum Genet 1994; 55: 728-737.
8. Tassabehji M, Newton VE, Liu XZ, Brady A, Donnai D, Walasek KM, et al. The mutational spectrum in Waardenburg syndrome. Hum Mol Genet 1995; 4: 2131-2137.
9. Pandya A, Xia XJ, Landa BL, Arnos KS, Israel J, Lloyd J, et al. Phenotypic variation in Waardenburg syndrome: mutational heterogeneity, modifier genes or polygenic background? Hum Mol Genet 1996; 5: 497-502.
10. DeStefano AL, Cupples LA, Arnos KS, Asher JHJ, Baldwin CT, Blanton S, et al. Correlation between Waardenburg syndrome phenotype and genotype in a population of individuals with identified PAX3
mutations. Hum Genet 1998; 102: 499-506.
11. Baldwin CT, Lipsky NR, Hoth CF, Cohen T, Mamuya W, Milunsky A, et al. Mutation
association with Waardenburg syndrome type 1
. Hum Mutat 1994; 3: 205-211.
12. Baldwin CT, Hoth CF, Macina RA, Milunsky A. Mutations in PAX3
that cause Waardenburg syndrome type 1
: ten new mutations and review of the literature. Am J Med Genet 1995; 58: 115-122.
13. Tassabehji M, Newton VE, Leverton K, Turnbull K, Seemanova E, Kunze J, et al. PAX3
gene structure and mutations: close analogies between Waardenburg syndrome and the Splotch mouse. Hum Mol Genet 1994; 3: 1069-1074.
14. Tassabehji M, Read AP, Newton VE, Harris R, Balling R, Grass P, et al. Waardenburg syndrome patients have mutaions in the human homologue of the Pax-3 paired box gene. Nature 1992; 355: 635-636.
15. Kissinger CR, Liu BS, Martin-Blanco E, Kornberg TB, Pabo CO. Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: A framework for understanding in the homeodomain-DNA interactions. Cell 1990; 63: 579-590.
16. Strachan T, Read AP. PAX genes. Curr Opin Genet Dev 1994; 4: 427-438.
17. Puffenberger EG, Kauffman ER, Bolk S, Matise TC, Washington SS, Angrist M, et al. Identity-by-descent and association mapping of a recessive gene for Hirschsprung disease on human chromosome 13q22. Hum Mol Genet 1994; 3: 1217-1225.
18. Puffenberger EG, Hosoda K, Washington SS, Nakao K, de Wit D, Yanagisawa WC, et al. A missense mutation
of the endothelin-B receptor gene in multigenetic Hirschsprung's disease. Cell 1994; 79: 1257-1266.
19. Pavan WJ, Mac S, Cheng M, Tilghman SM. Quantitative trait loci that modify the severity of spotting in piebald mice. Genome Res 1995; 5: 29-41.