Detect Parkin Gene Dosage Mutation By Real Time Quantitive PCR And DNA Direct Sequencing

Objective:To establish the clinical diagnostic platform of detecting Parkinson'S disease parkin gene rearrangement mutations at hot spot(exon 2,3,4)by quantitive real—time PCR.To detect the mutations of parkin in 21 AREP families using real—time PCR and DNA direct sequencing。Methods:Gene rearrangement of parkin gene are detected by real—time PCR,point mutations are detected by direct sequencing.Use beta globin gene as the internal control,establish the standard curves of parkin gene and beta globin gene,and then get the relative copy number ratio of parkin gene. The relative ratio of parkin gene to theβ-globin gene concentration was calculated for all DNA samples.Results:Based on the observed variability in the values of the ratios in normal individuals and in positive controls with heterozygous parkin gene rearrangements,w.e considered ratios between 0.8 and 1.2 as normal.Values lower than 0.6 or higher than 1.4 were interpreted as heterozygous deletions or duplications of the assessed exon,respectively.In the case of homozygous deletions,the ratio was 0.1 or lower.And finally,we confirmed 1 0 dosage mutations of parkin gene at exon 2,3,4,which have found by semi—quantitative method.and we found other three gene rearrangement in the same Chinese patients with AREP.Conclusions:At the first time,we have established the diagnostic platform of detecting gene dosage mutaions of parkin gene in Chinese patients with AREP using real—time PCR;and we have found 2 SNPs of the parkin gene exon 4:(heterozygosis)c.500G→A; (homozygosis) c.500G→A.which are all reported SNPs.