Disease Gene Positional Cloning Of Disseminated Superficial Porokeratosis

Objective Using the located locus for DSP , we perform a cosmical mutation screening and the mutation analysis for candidate genes in order to find the disease gene causing DSP ,and then clarify the pathogenesis of PK more clearly. Method We collected the pedigree members' clinical data , blood samples and the skin tissue of the healthy person. Afterwards the genomic DNA of peripheral blood and the RNA of the skin tissue were extracted. Firstly, we screened the human genomic database (http://www.genomic.ucsc.edu/ )and fixed 78 candidate genes at this region . Then,we designed PCR primers by Primer Express? 2.0 software ourselves.A cosmical mutation screening was beginning.First of all,we processed RT-PCR with RNA extracted from normal skin tissues by classical method ,and selected the genes expressed in skin to performe PCR amplification with genomic DNA of individuals of pedigree 4.Afterwards, the PCR products were purified with Shrimp Alkaline and Exonuclease .Then, sequenced with 2 μ l purified product(with ABI Prism^R BigDye^TM Terminator Cycle Sequencing Ready Reaction Kit), the mutation analysis was performed for the sequence in order to find the disease gene causing DSP.Result We found 24 genes are expressed in skin in the candidate region by RT-PCR. We screened the 60 candidate genes including 736 exons. In these genes, we found 203 snps including 3 locus which are tightly linkage to the disease. (1) KIAA0701 exon20 161bp;a G/T heterozygote; (2) FLJ10620 the upstream of exon8 a two-G-deletion; (3)FLJ34221 exon25 22bp a G/A heterozygote. But no mutation was found.Conclusion 60 candidate genes were excluded. 3 SNPs were detected linking to the disease in this family.Significance 3 SNPs were found highly linkage to pedgree 4, suggesting the locus we identified was linked to the disease gene causing DSP. And 60 candidate genes were excluded that will give an insight into the pathogenesis of DSP.