POSTER NO: 404

Functional characterization of SOX10 mutations for understanding the molecular pathogenesis of Waardenburg-Hirschsprung syndrome

¹Kwok Keung Chan, ¹Corinne K.Y. Wong, ²Vincent C.H. Lui, ²Paul K.H. Tam, ¹Mai Har Sham
¹Department of Biochemistry, The University of Hong Kong, Sassoon Road, Pokfulam, Hong Kong SAR, China., ²Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Pokfulam, Hong Kong SAR, China.

SOX10 is a member of the SOX gene family which encodes HMG domain containing transcription factors that share sequence homology to the testis-determining gene SRY. Mutations of the SOX10 gene lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans and to neural crest defects in the spontaneous mouse mutant Dominant megacolon (Dom). A number of SOX10 mutations have been identified in WS4 patients, who suffer from different extent of intestinal aganglionosis, pigmentation and hearing abnormalities. Though less frequently, some patients also exhibit signs of deficiency of myelination in the central nervous system and peripheral nervous system. Several classes of SOX10 mutations which result in truncation of the protein before or after the HMG domain, or affect the C-terminal trans-activation domain have been found. In order to analyse the impact of these mutations on the function of the SOX10 protein and to further understand the genotype-phenotype relationship between the types of mutations and the manifestation of symptoms, we studied several SOX10 mutations by yeast one-hybrid system and transfection assays in different neural and glial cell lines. To test for trans-activation effects of the mutant proteins, three different SOX binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; Sox10 binding sites identified in the c-ret promoter; and binding sites identified in the P0 promoter. Interestingly, among the seven mutant SOX10 proteins tested, those with an additional C-terminal peptide (X467C, X467K) have much higher trans-acting activities, about 5-fold higher than wild type proteins when tested on the Sox10 binding sites from c-ret promoter. Of the two mutations X467C and X467K, only the X467C mutant protein exhibited a 5.5-fold increase of trans-activation through binding sites from the P0 promoter. Our results support the idea that mutations affecting the C-terminal transactivation domain of SOX10 might produce mutant proteins that have a dominant negative effect, which account for a more severe intestinal aganglionosis phenotype in the patients. Furthermore, in agreement with the dysmyelination phenotype observed in the patient, we have demonstrated that the X467C mutant protein might act on the P0 promoter and produce a dominant negative phenotype.