Functional Analysis of Ret Mutations in Chinese Hirschsprung's Disease Patients

This dissertation, "Functional Analysis of RET Mutations in Chinese Hirschsprung's Disease Patients" by Yuk-yu, Leon, 梁毓裕, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled "Functional analysis of RET mutations in Chinese Hirschsprung's Disease patients" submitted by LEON, Yuk-yu for the degree of Doctor of Philosophy at The University of Hong Kong in August 2007 Hirschsprung's disease (HSCR) or congenital aganglionosis is attributed to a defective colonisation of the gut by the enteric neuron precursors. The proper development of the enteric nervous system mainly depends on the proper functioning of the RET receptor of the enteric neurons. Indeed, RET coding sequence (CDS) mutations account for 7%-35% of the sporadic and up to 50% of the familial HSCR cases. RET non-coding sequence (NCDS) mutations are also relevant to the phenotype. Both, RET CDS and NCDS mutations have reduced penetrance and led to variable phenotypes. To date, over 200 RET CDS and several RET NCDS mutations have been described, although not many of them have been functionally characterized. Therefore, assessment of the functional significance of HSCR-associated RET mutations/variants (either CDS or NCDS) is essential not only for the understanding of phenotypic differences, penetrance and recurrence risk estimations but also for shedding light on the disease mechanism. In this study, two RET NCDS single nucleotide polymorphisms (SNPs) and five RET CDS mutations have been functionally characterized. Two HSCR- associated RET promoter SNPs (located at -1 and -5 position with respect to the transcription start site) have been identified in Chinese HSCR patients. Sequence and transcription factor binding site analyses have shown that these RET promoter SNPs overlap with a thyroid transcription factor 1 (TTF1) binding site. We show that 1) the HSCR-associated RET promoter SNP alleles reduces the transcription activity of RET promoter; 2) TTF1 transactivates the RET transcription and the TTF1-activated RET transcription activity is reduced by the HSCR-associated RET promoter SNP alleles; 3) TTF1 physically interacts with the RET promoter and the interaction is disrupted by the HSCR-associated RET promoter SNP alleles. Given that TTF1 transactivates the RET transcription, hence, other transcription factors (TFs) (such as PHOX2B, SOX10 and PAX3), which are crucial for the RET expression, may co-operate with TTF1 in regulating the RET transcription. However, results show that TTF1 fails to co-operate with these TFs in regulating the transcription of RET. As for the RET CDS mutations, we have functionally characterized RET R114H V145G Y483X V636fsX F961L 9, RET 9, RET 9, RET 9 and RET 9 which had been previously identified in Chinese HSCR patients. We demonstrate that these mutations affect the RET function in diverse manners: 1) the glycosylation is V145G totally abolished by the RET 9 mutation; 2) the translocation is affected by V145G V636fsX RET 9 and RET 9 mutation; 3) the phosphorylation is affected by RET V145G R114H F961L 9, RET 9 and RET 9 mutations. To conclude, this study describes a novel model which TTF1 regulates the expression of RET gene. In addition, the functional consequences of different RET CDS mutations are also presented. Our data will help revealing the mechanisms underlying not only HSCR but all RET-related diseases. (448 words) DOI: 10.5353/th_b3963438 Subjects: Mutation (Biology) Hirschsprung's disease - Genetic aspects