Study On Gene Diagnosis Of Human Keliods

Background:Fas was confirmed to be one of the most important factors in the process of apoptosis.Our previous studies have testified there were some mutations in the exon6,8,9 in Fas gene which was thought to be closely relative to the defect in fibroblasts apoptosis.Objective:To establish a convenient and fast detection of mutations mPCR-SSCP was performed to investigate the mutation situations of exon6, 8, 9 of Fas gene and then set the experimental fundation for the future clinical gene diagnosis and the prediction of operation in keloids patients.Methods:!. mPCR-SSCP technique was carried out in this research. After deep analysis on the sequence of Fas gene exon6, 8,9 we designed three pairs of primers according to the principle of mPCR;2. After every fragment of Fas gene from normal skins,hypertrophic scars and keloids of human was amplified successfully the appropriate condition of multiplex PCR was explored;3.the mPCR products were analyzed by mSSCP with silver stained with the control of single exon SSCP to detect gene mutations;4. All amplified exons were sequenced and compared with the standard ones in GenBank.Results:!, the bands of three products were seen in agarose gel electrophoresis clear and unique,without nonspecific amplified fragment, the DNA sequences of these three bands were in coincidence with lengthes and sequences which had been predicted;2. By mPCR-SSCP analysis shifts in electrophoreticmobility of the Fas gene were detected in 18 of 23 (78%) keloids, but none in all hypertrophic scars and normal skin tissues;3. Direct nucleotide sequencing and the comparison to GenBank showed the highest positive ratio was 52%(12 of 23 keloids with Fas gene mutations in single exon by SSCP) in exon 6,8 and 9,74% (17 of 23 in two exons by mSSCP together), 78% (18 of 23 in three exons by mSSCP together) .Obviously the mSSCP could get the higher positive ratio. Furthermore, some new mutations were found.Conclusions:1. An economical and fast multiplex PCR-SSCP method for detection of Fas gene mutations in keloids had been established, which could screen mutations of three exons at one time and the positive ratio was higher than that detecting respectively.So it was useful for detecting gene point mutation and further study on mutations of Fas gene and even gene diagnosis of keloids;2. The results verified mutations in Fas gene from keloids lead to defect of apoptosis of fibroblasts; the mutations of eoxnS, 9 in Fas gene could play a important role in pathogenesis of keloids. 3. None mutations were found in five keloids, this suggests the progression of keloids is correlated to the alterations of multiple genes.