published_or_final_version / Psychiatry / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Research in Medicine

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Hirschsprung's disease, or aganglionic megacolon, is a common cause of intestinal obstruction in neonates and is associated with the congenital absence of intrinsic ganglion cells in the myenteric and submucosal plexuses of the gastrointestinal tract. The affected area is usually restricted to the distal part of the colon (short segment disease), but total colonic or intestinal involvement occurs in some patients (long segment disease). DNA analysis was performed on samples from 53 unrelated sporadic HSCR patients to search for mutations in RET proto-oncogene, endothelin-B receptor (EDNRB) and endothelin-3 (EDN3) genes. The patients were from different ethnic groups in South Africa, including 29 coloured, 14 white (Caucasian) and 9 black individuals. The origin of 1 patient was unknown. PCR HEX-SSCP analysis of the RET protooncogene revealed one previously described (P973L) and five novel mutations (V202M, E480K, IVS10-2A1G, D771N, IVS19-9Crr), likely to cause or contribute to the HSCR phenotype. Nine polymorphisms were also identified in the RET protooncogene, of which four were novel (IVS6+56deIG, IVS13-29Crr, IVS16-38deIG, X1159) and five previously described (A45, A432, L769, S904, R982). All the mobility shifts detected in the EDNRB gene represented polymorph isms (A60T, S184, 1187, V234, L277, IVS3-6Crr, IVS4+3A1G). No sequence variants were identified in the EDN3 gene. The majority of mutations in the RET proto-oncogene (28.6%) were identified in coloured patients while no mutations were identified in black patients. A mutation in RET was identified in two of 14 patients (14%) presenting with HSCR and Down's syndrome compared to 6 mutations identified in 9 of 39 patients (23%) with only HSCR. The fact that Down's syndrome patients have a high chance of developing HSCR, implies the involvement of modifier gene(s) in a HSCR/Oown's syndrome phenotype. This study demonstrated that, within the South African HSCR patient population, the RET proto-oncogene is the major susceptibility gene, whereas EDNRB and EDN3 may contribute only to a minority of cases. In 81% of patients no disease-causing mutation could be identified, which is in keeping with the heterogeneous nature of HSCR. The identification of mutations in HSCR patients would in future lead to improved and accurate counselling of South African HSCR patients and their families. / AFRIKAANSE OPSOMMING: Hirschsprung se siekte (HSCR), ook bekend as aganglionosis megakolon, is 'n algemene oorsaak van intestinale obstruksie in pasgeborenes en word geassosieer met die kongenitale afwesigheid van intrinsieke ganglion selle, in die miênteries en submukosa pleksus van die gastrointestinale kanaal. Alhoewel die aangetaste deel hoofsaaklik by die distale area van die kolon geleê is (kort segment siekte), kom totale koloniese of intestinale betrokkenheid ook in sommige pasiënte voor (lang segment tipe). Molekulêre ONS analise van 53 nie-verwante Suid Afrikaanse sporadiese HSCR pasiênte (29 kleurlinge, 14 blankes, 9 swartes en 1 individu van onbekende oorsprong) is uitgevoer in die RET proto-onkogeen, endoteel-B reseptor (EDNRB) en endoteel-3 (EDN3) gene. Heterodupleks-enkel string konformasie polimorfisme (HEX-SSCP) analise van polimerase ketting reaksie (PKR) geamplifiseerde produkte van die RET proto-onkogeen het gelei tot die identifikasie van vyf nuwe mutasies (V202M, E480K, IVS10-2A1G, D771N, IVS19-9CIT) en een bekende mutasie (P973L). Vier nuwe polimorfismes (IVS6+56deIG, IVS13-29Crr, IVS16-38deIG, X1159) en vyf bekende polimorfismes (A45, A432, L769, S904, R982) is ook aangetoon. Sewe polimorfismes (A60T, S184, 1187, V234, L277, IVS3-6CIT, IVS4+3A1G) is in die EDNRB geen geïdentifiseer. Geen veranderinge is in die EDN3 geen waargeneem nie. Die meerderheid mutasies waargeneem in die RET protoonkogeen is in die kleurling populasie (28.6%) waargeneem, terwyl geen mutasies in die swart populasie geïdentifiseer is nie. 'n RET mutasie is in twee van 14 (14%) pasiênte met 'n HSCR en Down's sindroom fenotipe waargeneem, in vergelyking met mutasies geïdentifiseer in 9 van 39 pasiënte (23%) met slegs HSCR. Die algemene voorkoms van Down's sindroom met HSCR, impliseer die rol van ander gene in die HSCRI Down's sindroom fenotipe. Die meerderheid mutasies wat aanleiding gee tot die HSCR fenotipe kom voor in die RET proto-onkogeen (19%), terwyl slegs polimorfismes in die EDNRB geen waargeneem is. Geen HEX-SSCP bandpatroon veranderinge is in die EDN3 geen waargeneem nie. Ongeveer 81% van die Suid Afrikaanse HSCR pasiënte was mutasie-negatief wat dui op die heterogene aard van die siekte. In die toekoms sal analise van siekte-verwante mutasies in die RET geen lei tot akkurate diagnose en verbeterde genetiese voorligting van HSCR in die Suid-Afrikaanse populasie.

Hirschsprung’s disease (HSCR) is the major enteric nervous system anomaly affecting newborns with high incidence in Asians. HSCR is a congenital complex genetic disorder characterized by a lack of enteric ganglia along a variable length of the intestine. The receptor tyrosine kinase gene (RET) is the major HSCR gene and cis-elements in the promoter and intron of RET gene are crucial for RET expression. Abnormal RET expression leading to insufficient RET activity causes defective development of the enteric nervous system and is implicated in the pathogenesis of the Hirschsprung’s disease. The human homeobox B5, HOXB5, has an important role in the development of enteric neural crest cells, and perturbation of HOXB5 signaling causes reduced RET expression and HSCR phenotypes in mice. To investigate the roles of HOXB5 in the regulation of RET expression and in the aetiology of HSCR, I sought to(i) elucidate the underlying mechanisms that HOXB5 mediates RET expression, and (ii) to examine the interactions between HOXB5 and other transcription factors including SOX10 and NKX2-1 that have been implicated in RET expression and HSCR. In this study, I demonstrated that HOXB5 binds to the RET promoter and regulates RET expression. HOXB5 and NKX2-1 forma protein complex and mediate RET expression in a synergistic manner. In contrast, HOXB5 cooperates in an additive manner with SOX10in trans-activation from RET promoter. ChIP assay further revealed that HOXB5 and NKX2-1 interact with the same chromatin region proximate to the transcription start site of RET, suggesting that these two factors may interact with each other and regulate the transcription of RET. In silico analysis, EMSA and ChIP analysis showed that HOXB5 also binds to an enhancer element (MCS+9.7)in the intron 1 of RET gene, and HSCR-associated SNPs have been identified in this enhancer element. To further access the HOXB5 trans-activity onMCS+9.7, RET mini-gene was constructed by ligating the RET promoter to the 5’and MCS+9.7 to the 3’of a luciferase gene. Luciferase assay indicated that MCS+9.7 enhances the HOXB5 trans-activation from the RET promoter. In addition, previously identified HSCR-associated SNPs inintron 1 markedly reduce the HOXB5 trans-activation from the RET mini-gene. Moreover, the result of IP-LC-MS/MS indicated that HOXB5 could form protein-protein complexes with nuclear proteins involved in the transcription initiation of genes with TATA-less promoter. This evidence suggested that HOXB5 may cooperate with other activators or co-factors in the remodeling of chromatin conformation, local histone modification and recruitment of essential transcription factors for RNA Polymerase II based transcription from TATA-less promoter, such as RET. My data indicated that HOXB5 in coordination with other transcription factors mediates RET expression. Therefore, defects in cis-or trans-regulation of RET by HOXB5 could lead to a reduction of RET expression and contribute to the manifestation of the HSCR phenotype. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Surgery / Master / Master of Philosophy