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

1.鼠Ncx（Enx，Hox 11 L1）基因在神经嵴衍生组织的神经元亚群中特异性表达。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。转录起始位点确定在ATG起始密码子（+1）上游的297个核苷酸（-297）处。维甲酸反应元件位于-1163和-1150之间的区域。用与荧光素酶基因融合的5 '侧翼序列进行的瞬时转染试验表明，-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，从神经嵴衍生的细胞如鼠和人神经母细胞瘤细胞提取的核蛋白结合到-1387和-1368之间的DNA区域。该DNA元件在人NCX基因的5 '侧翼区也是保守的。我们的观察结果强烈地表明，DNA元件（-1387和-1368）有助于组织特异性表达。 关于我们 Ncx基因在小鼠和人类物种中的特异性表达。我们通过基于PCR的随机寡核苷酸选择确定了特异性Ncx蛋白结合共有DNA序列。Ncx的最佳结合序列为5 '-CGGTAATTGG-3'（TAAT核心）和5 '-CGGTAAGTGG-3'（TAAG核心），与Hox 11的结合序列一致。电泳迁移率变动分析表明，Ncx和Hox 11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与TAAT核心序列连接的荧光素酶报告质粒，但不能反式激活与TAAG核心序列连接的荧光素酶报告质粒。因此，Ncx和Hox 11通过其靶序列5 '-CGGTAATTGG-3'作为转录激活因子。我们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基因并测定了其结构。人NCX基因由3个外显子组成，其cDNA序列与小鼠NCX高度同源。通过设计多对引物，分别扩增每个外显子和外显子-内含子边界，建立了PCR-SSCP方法。从33例先天性巨结肠相关疾病患者的外周血中分离基因组DNA。我们可以发现一些编码区的沉默突变或内含子中的核苷酸改变。但是我们没有发现任何引起氨基酸替换或终止密码子的显著突变。少

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