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

基本信息

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

项目摘要

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。鼠NCX（ENX，HOX11L1）基因在神经元的衍生组织的神经元子集中特异性表达。为了阐明组织特异性表达的调节DNA元素，我们测序了NCX基因的5'-频乘区域。在ATG起始密码子（+1）的297个核动脉（-297）上确定转录启动位点。视黄酸反应元件位于-1163和-1150之间的区域。与荧光素酶基因融合的5'-倾斜序列的瞬时翻译测定表明，-1387和-1368之间的区域对于组织特异性增强子活性至关重要。此外，从神经元顶衍生的细胞（如鼠和人类神经母细胞瘤细胞）中提取的核蛋白与-1387和-1368之间的DNA区域结合。该DNA元件在人NCX基因的5'-频偏区域也保守。我们的观察结果强烈表明，DNA元素（-1387和-1368）有助于组织SP…在鼠类和人类中，NCX基因的更为表达。2。我们通过基于PCR的随机寡核苷酸选择确定了特异性NCX蛋白结合共识DNA序列。最佳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。 NCX和HOX11都可以通过电泳迁移率转移测定法在体外特异性地与TAAT和TAAG核心寡核苷酸结合。但是，它们可以有效地反式激活与TAAT核心序列相关但与TAAG核心序列相关的荧光素酶报告基质粒。这是NCX和HOX11通过其目标序列（5'-cggtaattgg-3'）充当转录激活器。现在，我们正在尝试通过表示差异分析（RDA）识别NCX基因的下游.3。我们克隆了人类NCX基因并确定其结构。人NCX基因由3个外显子组成，NCX cDNA序列与米琳NCX高度同源。我们通过描述几组引物可以扩增每个外显子和外显子内边界来建立PCR-SSCP方法。从33例HIRSCHSPRUNG相关疾病的患者的外周血中分离出基因组DNA。我们可以在编码区域中确定一些无声突变或介绍中一些核动肽变化。但是，我们找不到任何引起氨基酸取代或停止密码子的显着突变。较少的