Screening And Functional Study Of Pathogenic Mutations In Two Hereditary Pigment Abnormalities

Part â…  Mutational and Functional Analysis of ADAR1Gene in Three Dyschromatosis Symmetrica Hereditaria FamiliesDyschromatosis symmetrica hereditaria （DSH; OMIM#127400）, also called symmetric dyschromatosis of the extremities, is a rare autosomal dominant human pigmentary genodermatosis. It is characterized by hyperpigmented and hypopigmented macules on the dorsal aspects of the extremities and freckle-like macules on the face. Mutations in adenosine deaminase acting on RNA1（ADAR1） gene that encodes an A-to-I RNA editing enzyme have been revealed to be the causation of DSH. Human ADAR1protein includes two translation products due to alternative initiation of transcription. The type â… -or type â…¡-IFN-inducible ADAR1mRNA directs synthesis of a full-length1226amino acids p150protein, whereas the constitutively expressed transcript directs translation of a shorter931amino acids p110protein, corresponding to293-1226amino acids of the p150protein. However, the specific roles of these two isoforms in pathogenesis of DSH are poorly understood.In this study, three Chinese families with typical DSH were screened for mutation in ADAR1, and we aimed to investigate the functional significance of identified mutation. All exons and adjacent exon-intron sequences of ADAR1were amplified and sequenced. The possible influences of identified mutation on functionality of p150and p110were analyzed using minigene strategy and dual-luciferase reporter assay, respectively. Three mutations （c.271₂72delAG, c.3169delC and c.1491dupA） were identified in these three families, including a novel2bp of AG deletion mutation （c.271₂72delAG） in exon2of the ADAR1gene. The AG deletion caused a frameshift mutation in the p150 isoform, the mutant p150transcripts were subjected to nonsense-mediated mRNA decay （NMD）, while the deletion mutation did not alter the encoded amino acid sequence of p110protein due to its position in the5’-untranslated region （5’-UTR） of the p110isoform, and had no influence on the expression of p110. These results represented the first evidence that the ADAR1p150isoform was the determinant of DSH and might shed light on the yet unknown mechanisms involved in the development of DSH. Part â…¡ Roles of SASH1in Dyschromatosis Universalis Hereditaria PathogenesisDyschromatosis universalis hereditaria （DUH; OMIM%127500）, which occurs distinct from dyschromatosis symmetrica hereditaria （DSH; OMIM#127400）, is characterized by hyperpigmented and hypopigmented spots over much of the body. Mutations in SAM and SH3domain-containing protein1（SASH1） gene have been linked to DUH. SASH1, a member of the SH3/SAM family of signal adaptor proteins, contains a lymphocyte signaling adaptor （SLY） domain, a SRC homology3（SH3） domain, and two sterile alpha motif （SAM）.SASH1is a candidate tumor suppressor gene in multiple human cancers. Reduced expression of SASH1is correlated with tumor growth and metastasis formation. Although great achievements about SASH1have been made in oncology, the biological role of SASH1remains largely unknown, especially on organism level.In this study, we used a zebrafish model to investigate the roles of sashla in regulating the development of pigment cell and neural crest cell. We found the zebrafish Sashl a contains the same types of domains, and is highly similar to its human ortholog. We have also demonstrated that sash1a is widely expressed during the late gastrula period, and is highly expressed in the brain at the late stages of embryonic development, suggesting sash1a might play a role in the development of the gastrula and the brain. Morpholino oligonucleotides （MOs） were designed to effectively knockdown the sash1a expression. The sash1a knockdown zebrafish（sash1a morphant） displayed hypopigmentation, irregular pigment cells pattern, and decreased number of pigment cells, compared with controls. We also found sash1a morphants exhibited delayed neural crest cell migration and failed neural crest sublineages specification. Using gene expression microarray and quantitative PCR analysis, our results indicated that p53signaling pathway was specifically activated in sash1a morphants. The activation of the p53signaling pathway could lead to two consequences, triggering the neural tube apoptosis and disturbing the regulation of zebrafish background adaption, respectively. Thus, our results revealed the important roles of sash1a in regulating the development of pigment cells and neural crest cells in zebrafish, and might provide important theoretical basis for the role SASH1in DUH pathogenesis.