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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.

Chediak-Higashi综合征（CHS）的特征是眼皮肤白化病、许多细胞类型中的巨大包涵体和免疫缺陷。本研究采用马拉松cDNA扩增方法，成功地克隆了CHS全基因，并将其分为3、4、5 kb片段，克隆到TA克隆质粒中，获得了CHS全基因的cDNA序列，并对CHS基因的内含子-外显子结构和突变进行了分析。这些片段将被酶切并相互连接，形成一个全长cDNA，目前我们正在进行这一过程。我们能够找到一个含有该基因的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。我曾在1993年的《皮肤病学杂志》上报道过一例CHS患者的突变分析，该分析是通过PCR-SSCP和直接测序进行的。我们发现了一个4 bp的缺失，包括密码子3464-3465，这导致移码。由于患者为纯合突变，而其父母为杂合突变，因此我们克隆了CHS基因的整个编码区，并将其亚克隆到哺乳动物表达载体中，进行功能分析。我相信这项研究将是未来基因治疗这种致命疾病的关键一步。