Detection Of The Mutations Of PINK1 Gene Using Real-time PCR And Direct Sequencing

Background: Parkinson's disease (PD) is a commonneurodegenerative disorder with the clinic features of tremor, rigidity anddyskinesia. The pathology feature of it is the formation of Lewy body indopaminergic neurons of the substantial nigral. 10-15% of PD patientshave a family history. Autosomal recessive early-onset Parkinsonism(AREP) indicates PD patients with a recessively hereditary backgroundand the age of onset is younger than 45-year old. Parkin, PINK1 andDJ-1 gene may be involved in the pathogenesis of AREP. DopamineReacted Dystonia (DRD) indicates a kind of dystonia with long lastingand prominent react to dopamine. The clinic manifestations of DRD maybe similar with AREP, and the related genes are GCH-1 and TH.Researchers all over the world have found many different mutations ofPINK1 including the gene dosage mutations. Direct sequencing is notcapable of detecting the gene dosage mutations, so researchers abroad areusing real-time PCR to detect the gene dosage mutations of PINK1. Therehas no report of domestic researcher detecting PINK1 gene dosagemutations by the method of real-time PCR.Objective: To establish the diagnostic platform of detecting the genedosage mutations of PINK1 using real-time PCR. To detect the mutationsof PINK1 in 21 AREP families and 6 DRD families using real-time PCRand direct sequencingMethods: Gene dosage mutations of PINK1 are detected usingreal-time PCR, point mutations are detected by direct sequencing. UseALB as the internal control, establish the standard curves of PINK1 andALB, and then get the relative copy number of PINK1Results: The relative copy number of each exon of PINK1 is 0.8-1.2.No gene dosage mutation is found in 21 AREP and 6 DRD families. Bydirect sequencing, we found 5 SNPs of PINK: c. 189C→T ,c. 1018G→A,c.1023G→A, c.1562A→C和3'-UTR37a→t, which are all reportedSNPs.Conclusions: At the first time, we have established the diagnosticplatform of detecting gene dosage mutations of PINK1 gene in Chinese patients with AREP or DRD using real-time PCR; we have found 5 SNPsof the PINK1 gene: c.189C→T, c.1018G→A, c.1023G→A, c.1562A→Cand 3 -UTR37a→t, which are all reported SNPs.