ANALYSIS OF DOMINANT MEGACOLON--ANOTHER MODEL FORHIRSCHSPRUNG DISEASE

显性巨结肠分析--先天性巨结肠症的另一种模型

• 批准号: 7968850
• 负责人: William J Pavan
• 金额: $ 100.42万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968850
• 关键词: Animals; Back; Cleft Lip; Colon; Complement; Congenital Disorders; Congenital Megacolon; DNA; Defect; Development; Diagnosis; Disease; Embryo; Exhibits; Functional RNA; Ganglia; Gene Targeting; Gene Transfer; Genes; Genetic; Genome; Goals; Human; Hypopigmentation; In Vitro; Investigation; Lead; Maps; Megacolon; Modeling; Mus; Mutagenesis; Mutant Strains Mice; Mutation; Myxoid cyst; Nature; Neural Crest; Pathway interactions; Patients; Peripheral Nervous System; Sampling; Severity of illness; Skin; Stem Cell Development; Stem cells; System; Testing; Transgenic Mice; Variant; Waardenburg syndrome; deafness; gene cloning; in utero; interest; melanocyte; melanoma; mouse model; mutant; overexpression; programs; therapy development; transcription factor; vector

Animals heterozygous for mutants in the SOX10 transcription factor exhibit multiple defects in neural crest development including reduced numbers of melanocytes in the skin, an absence of myenteric ganglion in the colon and can be associated with deafness. Homozgous animals die in utero and there is extensive defects in the entire peripheral nervous system. A human congenital disorder, Hirschsprung disease also exhibits rectocolic aganglionosis and can be associated with hypopigmentation and casued by SOX10 mutations. Thus SOX10 mice, as well as the other neural crest mutant mice, serve as mouse models for this disease. We have found that the SOX10 defects disrupt expression of early neural crest genes, MITF, DCT and EDNRB placing the SOX10 gene early in the neural crest development pathway. We are using additional markers and lineage directed gene transfer to determine the mode of action of SOX10 and its effects on downstream targets. Investigation of the involvement of SOX10 in Hirschsprung disease and other neural crest related disorders will be explored.We have demonstrated that SOX10 directly controls the expression of MITF and DCT. We have shown that the effect on target genes is semindomnant in nature. We have made transgenic mice that overexpress Sxo10 to analyze its effects on neural crest stem cell development. We have also established a system for adding genes back to neural crest stem cells in order to complement genetic defects. We used this system to test hierarchial relationships between SOX10 and its target genes. We have demonstrated that we can use this system to correct SOX10 defects in vitro. We have generated vectors to make this very efficient. We have shown that MITF is not sufficient to complete repalce SOX10 in development. We have also established a whole genome mutagenesis program to identifying SOX10 genetic interaction factors. We have identified 7 heritable loci, mapped five and cloned the mutation in four of the genes. These may become human modifier loci and interesting contributors to neural crest developmental pathways. We are testing humand diseases for mutations in these genes. We have extended the screen to look for earlier embryonic neural crest defects and have mapped four mutants and cloned the gene for three. In addition we have sequenced non-coding DNA from multiple samples of HSCR disease and are looking for correlations of sequence variants disease severity.

Sox10转录因子突变杂合子的动物在神经脊发育中表现出多种缺陷，包括皮肤中黑素细胞数量减少，结肠中缺乏肌间神经节，并可能与耳聋有关。同卵动物在子宫中死亡，整个周围神经系统都有广泛的缺陷。先天性巨结肠症是一种人类先天性疾病，也表现为直肠无神经节细胞增多症，可与色素减退有关，并由Sox10突变引起。因此，Sox10小鼠和其他神经脊突变小鼠一样，是这种疾病的小鼠模型。我们发现，Sox10缺陷干扰了早期神经脊基因的表达，MITF、DCT和EDNRB将Sox10基因置于神经脊发育的早期途径。我们正在使用额外的标记和谱系定向的基因转移来确定Sox10的作用模式及其对下游靶点的影响。Sox10在先天性巨结肠症和其他神经脊相关疾病中的作用将被探索。我们已经证明Sox10直接控制MITF和DCT的表达。我们已经证明，对靶基因的影响在自然界中是半显性的。我们制造了过表达Sxo10的转基因小鼠，以分析其对神经脊干细胞发育的影响。 我们还建立了一个系统，将基因重新添加到神经脊干细胞中，以弥补遗传缺陷。我们使用这个系统来测试Sox10和它的目标基因之间的层次关系。我们已经证明，我们可以使用这个系统来纠正Sox10的体外缺陷。我们已经生成了矢量，以使这一过程非常有效。我们已经证明，MITF不足以在开发中完成替代Sox10。 我们还建立了一个全基因组突变程序来鉴定Sox10的遗传互作因子。我们已经确定了7个可遗传的基因座，定位了5个，并在其中4个基因上克隆了突变。这些基因可能成为人类修饰性基因座，也可能成为神经脊发育途径的有趣贡献者。我们正在测试人类疾病中这些基因的突变。我们扩大了筛查范围，寻找早期胚胎神经脊缺陷，并绘制了四个突变体的图谱，并克隆了三个基因。此外，我们已经对HSCR疾病的多个样本中的非编码DNA进行了测序，并正在寻找序列变异与疾病严重程度的相关性。