Cloning of the gene for Chediak-Higashi syndrome

Chediak-Higashi 综合征基因的克隆

基本信息

批准号：
09470190
负责人：
FUKAI Kazuyoshi
金额：
$ 6.72万
依托单位：
Osaka City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470190/
关键词：
Chediak-Higashi syndrome Immunodeficiency gene therapy albinism 白皮症

项目摘要

Oculocutaneous albinism, giant inclusion bodies in many cell types, and immunodeficiency characterize Chediak-Higashi syndrome (CHS). Previously, others and we have mapped and cloned the gene for human CHS.In this study, we aimed to clone the whole gene and characterize the intron-exon organization of the gene and mutation analysis of the disorder.By marathon cDNA amplification method, we succeeded in cloning the whole gene for CHS.The gene is now fragmented in three parts, about 3kb, 4kb, and 5kb in length and cloned in TA cloning plasmids. These fragments will be cut with enzyme and ligated each other to form one-full-length cDNA.Currently, we are working in that process. We were able to find a BAG clone containing the gene, and we direct-sequence the clone by large-scale preparation of the BAG clone. The gene turned out to be composed of 49 exons. We designed oligonucleotides for amplifying each exon. Mutation analysis of a patient with CHS, which I previously reported in the Journal of Dermatology in 1993, was performed by PCR-SSCP and direct sequencing. We found a 4 bp-deletion encompassing codon 3464-3465, which results in frameshift. Being inbred, the patient was homozygous for that mutation and the parents were heterozygous for that mutation.Thus we were able to clone the whole coding region of the gene for CHS.We are going to sub-clone the gene to mammalian expression vector and functional analysis of the gene. I believe that this study will be a critical step for the future gene therapy of this fatal disorder.

眼皮肤白化病、多种细胞类型中存在巨大包涵体以及免疫缺陷是 Chediak-Higashi 综合征 (CHS) 的特征。此前，我们和其他人已经绘制并克隆了人类中枢性低通气综合症的基因。在本研究中，我们旨在克隆整个基因并表征该基因的内含子-外显子组织以及该疾病的突变分析。通过马拉松式cDNA扩增方法，我们成功克隆了 CHS 的整个基因。该基因现已片段化为三部分，长度约为 3kb、4kb 和 5kb，并克隆到 TA 克隆质粒中。这些片段将被酶切并相互连接，形成一个全长的cDNA。目前，我们正在进行这一过程。我们能够找到含有该基因的 BAG 克隆，并通过大规模制备 BAG 克隆来直接测序该克隆。该基因由 49 个外显子组成。我们设计了用于扩增每个外显子的寡核苷酸。我曾于 1993 年在《皮肤病学杂志》上报道过一名中枢性低通气综合症患者的突变分析，该分析是通过 PCR-SSCP 和直接测序进行的。我们发现包含密码子 3464-3465 的 4 bp 缺失，导致移码。由于是近交系，患者的该突变是纯合的，而父母的该突变是杂合的。因此，我们能够克隆 CHS 基因的整个编码区。我们将把该基因亚克隆到哺乳动物表达载体中，并发挥功能基因分析。我相信这项研究将是这种致命疾病未来基因治疗的关键一步。