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

1. 小鼠Ncx （Enx,Hox11L1）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'侧区进行了测序。转录起始位点位于ATG起始密码子（+1）上游297个核苷酸（-297）处。维甲酸响应元件位于-1163和-1150之间的区域。荧光素酶基因5'侧序列的瞬时转染实验表明，-1387和-1368之间的区域对组织特异性增强子的活性至关重要。此外，从神经嵴衍生细胞（如小鼠和人类神经母细胞瘤细胞）中提取的核蛋白结合到-1387和-1368之间的DNA区域。该DNA元件也保守在人类NCX基因的5'侧区。我们的观察结果有力地表明，DNA元素（-1387和-1368）对组织特异性的表达有贡献。我们通过PCR随机寡核苷酸选择来确定特异性Ncx蛋白结合的一致DNA序列。最佳Ncx结合序列为5′- cggtaattg -3′（TAAT核心）和5′-CGGTAAGTGG-3′（TAAG核心），与Hox11结合序列一致。Ncx和Hox11均能通过电泳迁移位移法特异性结合TAAT和TAAG核心寡核苷酸。然而，它们可以有效地反激活与TAAT核心序列相连的荧光素酶报告质粒，而不是与TAAG核心序列相连的荧光素酶报告质粒。因此，Ncx和Hox11通过其靶序列5‘-CGGTAATTGG-3’发挥转录激活因子的作用。我们现在正试图通过代表性差异分析（RDA）来确定Ncx基因的下游。我们克隆了人类NCX基因并确定了其结构。人NCX基因由3个外显子组成，cDNA序列与鼠NCX高度同源。我们通过设计多组引物来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例巨结肠相关疾病患者的外周血中分离基因组DNA。我们可以在编码区发现一些沉默突变或在内含子中发现一些核苷酸的变化。然而，我们没有发现任何引起氨基酸取代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