Evaluation of the thyroid transcription factor-1 gene (TITF1) as a Hirschsprung's disease locus

Hirschsprung's disease (HSCR, colonic aganglionosis) is an oligogenic entity that usually requires mutations in RET and other interacting loci. Decreased levels of RET expression may lead to the manifestation of HSCR. We previously showed that RET transcription was decreased due to alteration of the TITF1 binding site by two HSCR-associated RET promoter single nucleotide polymorphisms (SNPs). This prompted us to investigate whether DNA alterations in TITF1 could play a role in HSCR by affecting the RET - regulatory properties of the TITF1 protein. Our initial study on 86 Chinese HSCR patients revealed a Gly322Ser amino acid substitution in the TITF1protein. In this study we have examined an additional 102 Chinese and 70 Caucasian patients, and 194 Chinese and 60 Caucasian unselected, unrelated, subjects as controls. The relevance of the DNA changes detected in TITF1 by direct sequencing were evaluated using bioinformatics, reporter and binding-assays, mouse neurosphere culture, immunohistochemistry and immunofluorescence techniques. Met3Leu and Pro48Pro were identified in 2 Caucasian patients and 1 Chinese patient, respectively. In vitro analysis showed that Met3Leu reduced the activity of the RET promoter by 100% in the presence of the wild-type or HSCR-associated RET promoter SNP alleles. The apparent binding affinity of the TITF1 mutated protein was not decreased. The Met3Leu mutation may affect the interaction of TITF1 with its protein partners. The absence of Titf1 expression in mouse gut but not in human gut suggests that the role of TITF1 in gut development differs between the two species. TITF1 mutations could contribute to HSCR by affecting RET expression through defective interactions with other transcription factors. © 2007 The Authors Journal compilation © 2007 University College London.link_to_OA_fulltex

Evaluation of the NK2 homeobox 1 gene (NKX2-1) as a hirschsprung's disease locus

Hirschsprung's disease (HSCR, colonic aganglionosis) is an oligogenic entity that usually requires mutations in RET and other interacting loci. Decreased levels of RET expression may lead to the manifestation of HSCR. We previously showed that RET transcription was decreased due to alteration of the NKX2-1 binding site by two HSCR-associated RET promoter single nucleotide polymorphisms (SNPs). This prompted us to investigate whether DNA alterations in NKX2-1 could play a role in HSCR by affecting the RET-regulatory properties of the NKX2-1 protein. Our initial study on 86 Chinese HSCR patients revealed a Gly322Ser amino acid substitution in the NKX2-1 protein. In this study, we have examined 102 additional Chinese and 70 Caucasian patients and 194 Chinese and 60 Caucasian unselected, unrelated, subjects as controls. The relevance of the DNA changes detected in NKX2-1 by direct sequencing were evaluated using bioinformatics, reporter and binding-assays, mouse neurosphere culture, immunohistochemistry and immunofluorescence techniques. Met3Leu and Pro48Pro were identified in 2 Caucasian and 1 Chinese patients respectively. In vitro analysis showed that Met3Leu reduced the activity of the RET promoter by 100% in the presence of the wild-type or HSCR-associated RET promoter SNP alleles. The apparent binding affinity of the NKX2-1 mutated protein was not decreased. The Met3Leu mutation may affect the interaction of NKX2-1 with its protein partners. The absence of NKX2-1 expression in mouse but not in human gut suggests that the role of NKX2-1 in gut development differs between the two species. NKX2-1 mutations could contribute to HSCR by affecting RET expression through defective interactions with other transcription factors. © 2007 The Authors Journal compilation © 2007 University College London.link_to_subscribed_fulltex