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Human Genetics

, Volume 131, Issue 3, pp 491–503 | Cite as

Functional analysis of Waardenburg syndrome-associated PAX3 and SOX10 mutations: report of a dominant-negative SOX10 mutation in Waardenburg syndrome type II

  • Hua Zhang
  • Hongsheng Chen
  • Hunjin Luo
  • Jing An
  • Lin Sun
  • Lingyun Mei
  • Chufeng He
  • Lu Jiang
  • Wen Jiang
  • Kun Xia
  • Jia-Da LiEmail author
  • Yong FengEmail author
Original Investigation

Abstract

Waardenburg syndrome (WS) is an auditory–pigmentary disorder resulting from melanocyte defects, with varying combinations of sensorineural hearing loss and abnormal pigmentation of the hair, skin, and inner ear. WS is classified into four subtypes (WS1–WS4) based on additional symptoms. PAX3 and SOX10 are two transcription factors that can activate the expression of microphthalmia-associated transcription factor (MITF), a critical transcription factor for melanocyte development. Mutations of PAX3 are associated with WS1 and WS3, while mutations of SOX10 cause WS2 and WS4. Recently, we identified some novel WS-associated mutations in PAX3 and SOX10 in a cohort of Chinese WS patients. Here, we further identified an E248fsX30 SOX10 mutation in a family of WS2. We analyzed the subcellular distribution, expression and in vitro activity of two PAX3 mutations (p.H80D, p.H186fsX5) and four SOX10 mutations (p.E248fsX30, p.G37fsX58, p.G38fsX69 and p.R43X). Except H80D PAX3, which retained partial activity, the other mutants were unable to activate MITF promoter. The H80D PAX3 and E248fsX30 SOX10 were localized in the nucleus as wild type (WT) proteins, whereas the other mutant proteins were distributed in both cytoplasm and nucleus. Furthermore, E248fsX30 SOX10 protein retained the DNA-binding activity and showed dominant-negative effect on WT SOX10. However, E248fsX30 SOX10 protein seems to decay faster than the WT one, which may underlie the mild WS2 phenotype caused by this mutation.

Keywords

High Mobility Group Nuclear Pore Complex Paired Domain SOX10 Protein Waardenburg Syndrome 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We would like to thank the patients for their participation in the study as well as their family members for their collaboration. We also thank Bondurand Nadege (INSERM, Unite U955,Creteil, France), Inoue Ken (National Institute of Neuroscience, NCNP, Tokyo, Japan), Goding Colin R. (Eukaryotic Transcription Laboratory, Marie Curie Research Institute, London, UK), Jiri Vachtenheim (Laboratory Molecular and Cell Biology, University Hospital, Charles University, Bulovka, Czech Republic.) for generously supplying the materials. This study was funded by a grant from Key Projects of Subordinate Hospital of Chinese Ministry of Health for Clinical Subjects and by grants from National Nature Science Foundation of China (30970958, 30971589, 81070481 and 81170923). JDL is a recipient of Lotus Scholar Professorship from Hunan Province, China.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

439_2011_1098_MOESM1_ESM.tif (103 kb)
FIG. 1 Effect of H186fsX5 or H80D mutants on WT PAX3 transactivity. Increasing amounts of mutant H186fsX5 or H80D expression plasmids were cotransfected with a fixed amount of WT PAX3 expression plasmid and the luciferase reporter plasmid MITF-Luc. The basal level of luciferase was set as 1. Data from all other transfections are presented as fold induction above this level. Luciferase activity was normalized by measuring β-galactosidase activity. Each value shown was the mean ± s.e.m. of three replicates from a single assay. The results shown were representative of at least three independent experiments (**P<0.01 by one way ANOVA with Dunnett's tests) (TIFF 102 kb)
439_2011_1098_MOESM2_ESM.doc (61 kb)
Supplementary material 2 (DOC 61 kb)

References

  1. Baldwin CT, Hoth CF, Amos JA, da-Silva EO, Milunsky A (1992) An exonic mutation in the HuP2 paired domain gene causes Waardenburg’s syndrome. Nature 355(6361):637–638. doi: 10.1038/355637a0 PubMedCrossRefGoogle Scholar
  2. Bondurand N, Pingault V, Goerich DE, Lemort N, Sock E, Le Caignec C, Wegner M, Goossens M (2000) Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome. Hum Mol Genet 9(13):1907–1917PubMedCrossRefGoogle Scholar
  3. Bondurand N, Dastot-Le Moal F, Stanchina L, Collot N, Baral V, Marlin S, Attie-Bitach T, Giurgea I, Skopinski L, Reardon W, Toutain A, Sarda P, Echaieb A, Lackmy-Port-Lis M, Touraine R, Amiel J, Goossens M, Pingault V (2007) Deletions at the SOX10 gene locus cause Waardenburg syndrome types 2 and 4. Am J Hum Genet 81(6):1169–1185. doi: 10.1086/522090 PubMedCrossRefGoogle Scholar
  4. Cao Y, Wang C (2000) The COOH-terminal transactivation domain plays a key role in regulating the in vitro and in vivo function of Pax3 homeodomain. J Biol Chem 275(13):9854–9862PubMedCrossRefGoogle Scholar
  5. Chen H, Jiang L, Xie Z, Mei L, He C, Hu Z, Xia K, Feng Y (2010) Novel mutations of PAX3, MITF, and SOX10 genes in Chinese patients with type I or type II Waardenburg syndrome. Biochem Biophys Res Commun 397(1):70–74. doi: 10.1016/j.bbrc.2010.05.066 PubMedCrossRefGoogle Scholar
  6. Corry GN, Underhill DA (2005) Pax3 target gene recognition occurs through distinct modes that are differentially affected by disease-associated mutations. Pigment cell research/sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society 18(6):427–438. doi: 10.1111/j.1600-0749.2005.00275.x PubMedGoogle Scholar
  7. Epstein DJ, Vogan KJ, Trasler DG, Gros P (1993) A mutation within intron 3 of the Pax-3 gene produces aberrantly spliced mRNA transcripts in the splotch (Sp) mouse mutant. Proc Natl Acad Sci USA 90(2):532–536PubMedCrossRefGoogle Scholar
  8. Fortin AS, Underhill DA, Gros P (1997) Reciprocal effect of Waardenburg syndrome mutations on DNA binding by the Pax-3 paired domain and homeodomain. Hum Mol Genet 6(11):1781–1790 dda234[pii]PubMedCrossRefGoogle Scholar
  9. Galibert MD, Yavuzer U, Dexter TJ, Goding CR (1999) Pax3 and regulation of the melanocyte-specific tyrosinase-related protein-1 promoter. J Biol Chem 274(38):26894–26900PubMedCrossRefGoogle Scholar
  10. Gorlich D, Mattaj IW (1996) Nucleocytoplasmic transport. Science 271(5255):1513–1518PubMedCrossRefGoogle Scholar
  11. Goulding MD, Chalepakis G, Deutsch U, Erselius JR, Gruss P (1991) Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J 10(5):1135–1147PubMedGoogle Scholar
  12. Herbarth B, Pingault V, Bondurand N, Kuhlbrodt K, Hermans-Borgmeyer I, Puliti A, Lemort N, Goossens M, Wegner M (1998) Mutation of the Sry-related Sox10 gene in dominant megacolon, a mouse model for human Hirschsprung disease. Proc Natl Acad Sci USA 95(9):5161–5165PubMedCrossRefGoogle Scholar
  13. Hoth CF, Milunsky A, Lipsky N, Sheffer R, Clarren SK, Baldwin CT (1993) Mutations in the paired domain of the human PAX3 gene cause Klein-Waardenburg syndrome (WS-III) as well as Waardenburg syndrome type I (WS-I). Am J Hum Genet 52(3):455–462PubMedGoogle Scholar
  14. Inoue K, Khajavi M, Ohyama T, Hirabayashi S, Wilson J, Reggin JD, Mancias P, Butler IJ, Wilkinson MF, Wegner M, Lupski JR (2004) Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations. Nat Genet 36(4):361–369. doi: 10.1038/ng1322 PubMedCrossRefGoogle Scholar
  15. Iso M, Fukami M, Horikawa R, Azuma N, Kawashiro N, Ogata T (2008) SOX10 mutation in Waardenburg syndrome type II. Am J Med Genet A 146A(16):2162–2163. doi: 10.1002/ajmg.a.32403 PubMedCrossRefGoogle Scholar
  16. Jun S, Desplan C (1996) Cooperative interactions between paired domain and homeodomain. Development 122(9):2639–2650PubMedGoogle Scholar
  17. Kamachi Y, Cheah KS, Kondoh H (1999) Mechanism of regulatory target selection by the SOX high-mobility-group domain proteins as revealed by comparison of SOX1/2/3 and SOX9. Mol Cell Biol 19(1):107–120PubMedGoogle Scholar
  18. Kubic JD, Young KP, Plummer RS, Ludvik AE, Lang D (2008) Pigmentation PAX-ways: the role of Pax3 in melanogenesis, melanocyte stem cell maintenance, and disease. Pigment Cell Melanoma Res 21(6):627–645. doi: 10.1111/j.1755-148X.2008.00514.x PubMedCrossRefGoogle Scholar
  19. Kuhlbrodt K, Herbarth B, Sock E, Hermans-Borgmeyer I, Wegner M (1998a) Sox10, a novel transcriptional modulator in glial cells. J Neurosci 18(1):237–250PubMedGoogle Scholar
  20. Kuhlbrodt K, Schmidt C, Sock E, Pingault V, Bondurand N, Goossens M, Wegner M (1998b) Functional analysis of Sox10 mutations found in human Waardenburg-Hirschsprung patients. J Biol Chem 273(36):23033–23038PubMedCrossRefGoogle Scholar
  21. Lang D, Epstein JA (2003) Sox10 and Pax3 physically interact to mediate activation of a conserved c-RET enhancer. Hum Mol Genet 12(8):937–945PubMedCrossRefGoogle Scholar
  22. Lange A, Mills RE, Lange CJ, Stewart M, Devine SE, Corbett AH (2007) Classical nuclear localization signals: definition, function, and interaction with importin alpha. J Biol Chem 282(8):5101–5105. doi: 10.1074/jbc.R600026200 PubMedCrossRefGoogle Scholar
  23. Pingault V, Bondurand N, Kuhlbrodt K, Goerich DE, Prehu MO, Puliti A, Herbarth B, Hermans-Borgmeyer I, Legius E, Matthijs G, Amiel J, Lyonnet S, Ceccherini I, Romeo G, Smith JC, Read AP, Wegner M, Goossens M (1998) SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat Genet 18(2):171–173. doi: 10.1038/ng0298-171 PubMedCrossRefGoogle Scholar
  24. Pingault V, Ente D, Dastot-Le Moal F, Goossens M, Marlin S, Bondurand N (2010) Review and update of mutations causing Waardenburg syndrome. Hum Mutat 31(4):391–406. doi: 10.1002/humu.21211 PubMedCrossRefGoogle Scholar
  25. Potterf SB, Furumura M, Dunn KJ, Arnheiter H, Pavan WJ (2000) Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3. Hum Genet 107(1):1–6PubMedCrossRefGoogle Scholar
  26. Read AP, Newton VE (1997) Waardenburg syndrome. J Med Genet 34(8):656–665PubMedCrossRefGoogle Scholar
  27. Rehberg S, Lischka P, Glaser G, Stamminger T, Wegner M, Rosorius O (2002) Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10-mediated transactivation. Mol Cell Biol 22(16):5826–5834PubMedCrossRefGoogle Scholar
  28. Sanchez-Mejias A, Watanabe Y, MF R, Lopez-Alonso M, Antinolo G, Bondurand N, Borrego S (2010) Involvement of SOX10 in the pathogenesis of Hirschsprung disease: report of a truncating mutation in an isolated patient. J Mol Med 88(5):507–514. doi: 10.1007/s00109-010-0592-7 PubMedCrossRefGoogle Scholar
  29. Southard-Smith EM, Kos L, Pavan WJ (1998) Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model. Nat Genet 18(1):60–64. doi: 10.1038/ng0198-60 PubMedCrossRefGoogle Scholar
  30. Sudbeck P, Scherer G (1997) Two independent nuclear localization signals are present in the DNA-binding high-mobility group domains of SRY and SOX9. J Biol Chem 272(44):27848–27852PubMedCrossRefGoogle Scholar
  31. Tassabehji M, Read AP, Newton VE, Harris R, Balling R, Gruss P, Strachan T (1992) Waardenburg’s syndrome patients have mutations in the human homologue of the Pax-3 paired box gene. Nature 355(6361):635–636. doi: 10.1038/355635a0 PubMedCrossRefGoogle Scholar
  32. Watanabe A, Takeda K, Ploplis B, Tachibana M (1998) Epistatic relationship between Waardenburg syndrome genes MITF and PAX3. Nat Genet 18(3):283–286. doi: 10.1038/ng0398-283 PubMedCrossRefGoogle Scholar
  33. Wegner M (1999) From head to toes: the multiple facets of Sox proteins. Nucleic Acids Res 27(6):1409–1420. doi: gkc266[pii] PubMedCrossRefGoogle Scholar
  34. Wilson DS, Guenther B, Desplan C, Kuriyan J (1995) High resolution crystal structure of a paired (Pax) class cooperative homeodomain dimer on DNA. Cell 82(5):709–719 0092-8674(95)90468-9[pii]PubMedCrossRefGoogle Scholar
  35. Wollnik B, Tukel T, Uyguner O, Ghanbari A, Kayserili H, Emiroglu M, Yuksel-Apak M (2003) Homozygous and heterozygous inheritance of PAX3 mutations causes different types of Waardenburg syndrome. Am J Med Genet Part A 122A(1):42–45. doi: 10.1002/ajmg.a.20260 PubMedCrossRefGoogle Scholar
  36. Zlotogora J, Lerer I, Bar-David S, Ergaz Z, Abeliovich D (1995) Homozygosity for Waardenburg syndrome. Am J Hum Genet 56(5):1173–1178PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Hua Zhang
    • 1
    • 4
  • Hongsheng Chen
    • 1
    • 2
  • Hunjin Luo
    • 3
  • Jing An
    • 3
  • Lin Sun
    • 3
  • Lingyun Mei
    • 1
    • 2
  • Chufeng He
    • 1
    • 2
  • Lu Jiang
    • 1
    • 2
  • Wen Jiang
    • 5
  • Kun Xia
    • 3
  • Jia-Da Li
    • 3
    Email author
  • Yong Feng
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Otolaryngology, Xiangya HospitalCentral South UniversityChangshaPeople’s Republic of China
  2. 2.Province Key Laboratory of Otolaryngology Critical DiseasesChangshaPeople’s Republic of China
  3. 3.State Key Laboratory of Medical GeneticsCentral South UniversityChangshaPeople’s Republic of China
  4. 4.Department of Otolaryngology, 1st Affiliated HospitalXinjiang Medical UniversityUrumqiPeople’s Republic of China
  5. 5.Department of OtolaryngologyNo. 163 Hospital of PLAChangshaPeople’s Republic of China

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