Cloning of the gene for Chediak-Higashi syndrome

眼皮肤白化病、多种细胞类型的巨大包涵体和免疫缺陷是Chediak-Higashi综合征(CHS)的特征。本研究的目的是克隆人CHS基因的全基因，并对其内含子-外显子组织和无序突变进行分析，通过马拉松式的cDNA扩增方法，成功地克隆了CHS基因的全长3kb、4kb和5kb三个片段，并克隆到TA克隆载体中。这些片段将被酶切割并相互连接，形成一个全长的DNA。目前，我们正在进行这一过程。我们找到了一个含有该基因的BAG克隆，并通过大规模制备BAG克隆对克隆进行了直接测序。结果发现，该基因由49个外显子组成。我们设计了扩增每个外显子的寡核苷酸。我曾在1993年的皮肤病杂志上报道过一例CHS患者的突变分析，通过PCR-SSCP和直接测序进行。我们发现了一个包含3464-3465密码子的4个碱基对的缺失，这导致了移码。由于是近交系，患者是纯合突变，父母是杂合突变，所以我们能够克隆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上，并对该基因进行功能分析。我相信，这项研究将是未来这种致命疾病的基因治疗的关键一步。

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.