Analysis of NCX gene mutation in patients with Hirschsprung-related disease

先天性巨结肠相关疾病患者NCX基因突变分析

• 批准号: 10670132
• 负责人: HATANO Masahiko
• 金额: $ 2.37万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670132/
• 关键词: NCX; Hirschsprung-related disease; transcription factor; homebox; DNA binding; SSCP; RDA; 遺伝子発現調節; 神経堤細胞; 疾患モデル動物

1. The murine Ncx (Enx,Hox11L1) gene is specifically expressed in a neuronal subset of neural crest derived tissues. In attempts to elucidate the regulatory DNA element of the tissue-specific expression, we sequenced the 5'-flanking region of the Ncx gene. The transcriptional initiation site was determined at the 297 nucleotides (-297) upstram from the ATG start codon (+1). A retinoic acid response element was located on the region between -1163 and -1150. Transient transfection assays with the 5'-flanking sequences fused to the luciferase gene showed that the region between -1387 and -1368 was crucial for the tissue-specific enhancer activity. Furthermore, nuclear proteins extracted from neural crest derived cells such as murine and human neuroblastoma cells bind to the DNA region between -1387 and -1368. This DNA element was also conserved in the 5'-flanking region of the human NCX gene. Our observations strongly suggest that the DNA element (-1387 and -1368) contributes to tissue-sp … More ecific expression of the Ncx gene in murine and human species.2. We determined specific Ncx protein binding consensus DNA sequences by PCR based random oligonucleotide selection. Optimal Ncx binding sequences were 5'-CGGTAATTGG-3' (a TAAT core) and 5'-CGGTAAGTGG-3' (a TAAG core), which coincided with the Hox11 binding sequence. Both Ncx and Hox11 could specifically bind to the TAAT and the TAAG core oligonucleotide in vitro by electrophoretic mobility shift assay. However, they could efficiently transactivate the luciferase reporter plasmid linked to the TAAT core sequence but not to the TAAG core sequence. Thus, Ncx and Hox11 act as a transcriptional activator via their target sequence, 5'-CGGTAATTGG-3'. We are now trying to identify downstream of Ncx gene by representational difference analysis (RDA).3. We cloned human NCX gene and determined its structure. Human NCX gene consists of 3 exons and NCX cDNA sequence was highly homologous to mirine Ncx. We established PCR-SSCP method by desinging several sets of primers which can amplify each exons and exon-intron boundaries. Genomic DNA was isolated from peripheral blood of 33 patients with Hirschsprung-related disease. We could identify some silent mutations in coding region or some nucleotides changes in introns. However we could not find any significant mutations which cause amino acid substitution or stop codon. Less

1.小鼠NCX(ENX，Hox11L1)基因在神经脊源性组织的神经元亚群中特异表达。为了阐明组织特异性表达的调控DNA元件，我们对NCX基因的5‘侧翼区进行了测序。转录起始点位于ATG起始密码子(+1)上游297个核苷酸(-297)。维甲酸反应元件位于-1163和-1150之间。与荧光素酶基因融合的5‘侧翼序列的瞬时转染分析表明，-1387和-1368之间的区域对于组织特异性增强子活性是至关重要的。此外，从神经脊来源的细胞，如小鼠和人神经母细胞瘤细胞中提取的核蛋白结合到-1387到-1368之间的DNA区域。该DNA元件在人类NCX基因的5‘侧翼区也是保守的。我们的观察有力地表明dna元件(-1387和-1368)对组织SP…有贡献。Ncx基因在小鼠和人类物种中的更特异表达。我们通过基于PCR的随机寡核苷酸选择确定了特定的NCX蛋白结合共识DNA序列。最佳的NCX结合序列为5‘-CGGTAATTGG-3’(Taat核心)和5‘-CGGTAAGTGG-3’(TAAG核心)，与Hox11结合序列一致。经凝胶迁移率改变分析，NCX和Hox11在体外均能与TaAT和TAAG核心寡核苷酸特异性结合。然而，它们可以有效地反式激活连接到Taat核心序列但不连接到TAAG核心序列的荧光素酶报告质粒。因此，NCX和Hox11通过其靶序列5‘-CGGTAATTGG-3’作为转录激活因子。我们目前正在尝试通过代表性差异分析(RDA)来鉴定NCX基因的下游。我们克隆了人NCX基因，并对其结构进行了测定。人NCX基因由3个外显子组成，与Mirine NCX基因有很高的同源性。通过设计多对能扩增各外显子及外显子-内含子边界的引物，建立了聚合酶链式反应-单链构象多态性方法。从33例先天性巨结肠患者外周血中提取基因组DNA。我们可以发现编码区的一些沉默突变或内含子的一些核苷酸变化。然而，我们没有发现任何导致氨基酸替换或终止密码子的显着突变。较少

项目成果

Honda, A. et al.: "HCV-core protein accelerates recovery from the insensitivity of liver cells Fas-mediated apoptosis in mice."J. Hepatology.. (in press). (2000)

Honda, A. 等人：“HCV 核心蛋白可加速小鼠肝细胞 Fas 介导的细胞凋亡不敏感的恢复。”

Okada S.,et al.: "A physiological role of Bcl-xL induced in activated macrophages." J.Immunol.160. 2590-2596 (1998)

Okada S.,et al.：“Bcl-xL 在激活的巨噬细胞中诱导的生理作用。”

Iitsuka, Y et al.: "An enhancer element for expression of the Ncx (Enx, Hox11L1) gene in neural crest-derived cells."J. Biol. Chem.. 274. 24401-24407 (1999)

Iitsuka, Y 等人：“神经嵴衍生细胞中 Ncx（Enx，Hox11L1）基因表达的增强子元件。”J.

Iizuka, J. et al.: "Intriduction of an osteopontin gene confers the increase in B1 cell population and the production of anti-DNA autoantibodies."Lab. Invest.. 78. 1523-1533 (1998)

Iizuka, J. 等人：“骨桥蛋白基因的引入导致 B1 细胞群的增加和抗 DNA 自身抗体的产生。”实验室。

Okabe S.,et al.: "BAZF,a novel Bc16 homolog,functions as a transcriptional repressor." Mol.Cell.Biol.18. 4235-4244 (1998)

Okabe S. 等人：“BAZF 是一种新型 Bc16 同源物，具有转录抑制因子的功能。”