Garcia-Barceló Maria-Mercè, Lau Danny Ko-chun, Ngan Elly Sau-wai, Leon Thomas Yuk-yu, Liu Ting-ting, So Man-ting, Miao Xiao-ping, Lui Vincent Chi-hang, Wong Kenneth Kak-yuen, Ganster Raymond William, Cass Daniel Thomas, Croaker Geoffrey David Hain, Tam Paul Kwong-hang
Division of Paediatric Surgery, Department of Surgery, Li Ka Shing Faculty of Medicine of the University of Hong Kong, Hong Kong SAR, China.
Ann Hum Genet. 2007 Nov;71(Pt 6):746-54. doi: 10.1111/j.1469-1809.2007.00384.x. Epub 2007 Jul 19.
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.
先天性巨结肠症（HSCR，结肠无神经节症）是一种寡基因疾病，通常需要RET基因及其他相互作用位点发生突变。RET表达水平降低可能导致HSCR的表现。我们之前表明，由于两个与HSCR相关的RET启动子单核苷酸多态性（SNP）改变了TITF1结合位点，导致RET转录减少。这促使我们研究TITF1中的DNA改变是否会通过影响TITF1蛋白的RET调节特性而在HSCR中起作用。我们对86名中国HSCR患者的初步研究发现TITF1蛋白中存在Gly322Ser氨基酸替代。在本研究中，我们又检测了另外102名中国患者和70名高加索患者，以及194名中国和60名高加索未选择、无亲缘关系的对照受试者。使用生物信息学、报告基因和结合测定、小鼠神经球培养、免疫组织化学和免疫荧光技术评估了通过直接测序在TITF1中检测到的DNA变化的相关性。分别在2名高加索患者和1名中国患者中鉴定出Met3Leu和Pro48Pro。体外分析表明，在存在野生型或与HSCR相关的RET启动子SNP等位基因的情况下，Met3Leu使RET启动子的活性降低了100%。TITF1突变蛋白的表观结合亲和力并未降低。Met3Leu突变可能影响TITF1与其蛋白伴侣的相互作用。小鼠肠道中不存在Titf1表达，但人类肠道中存在，这表明TITF1在肠道发育中的作用在这两个物种之间有所不同。TITF1突变可能通过与其他转录因子的缺陷相互作用影响RET表达，从而导致HSCR。
