Cloning of the gene for Chediak-Higashi syndrome

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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.