Establishment And Application Of A Multiple Ligation-dependent Probe Amplification In Parkin

BackgroundParkinson’s disease (PD) is the second most common progressive neurodegenerative disorder beyond Alzheimer’s disease, affecting approximately1%of people over the age of65. The major pathological hallmarks of PD are significant loss of nigrostriatal dopaminergic (DA) neurons and the presence of intraneuronal protein inclusions termed Lewy bodies. Sporadic cases represent more than90%of total patients with PD, some are caused by gene mutations. Mutations in parkin are the most common cause in autosomal-recessive parkinson’s disease, and explain up to half of the cases with a clinical diagnosis of familial PD compatible with recessive inheritance and disease onset before the age of45years, and also～15%of the sporadic cases with onset before45Nowadays, Diverse frequency and a wide spectrum of mutations including point mutations, insertions and deletions within the parkin gene have been reported in different ethnic populations.50%of parkin mutations are gene rearrangement, they cann’t be identified in common DNA-sequencing. So it is neccessary for doseage analysis in order to achieve a high sensitivity in the mutational screening of parkin. MLPA(multiple ligation-dependent probe amplification), is a simple, rapid, and sensitive tool to detect exon dosage alterations and specific point mutations in selected genes. The establishment of MLPA in parkin is benefit for a rapid,sensitive clinical diagnosis of parkinsonism.ObjectiveTo invent agentia box for detecting rearrangement mutations of parkin gene in sporadic parkinson’s disease by using multiple ligation-dependent probe amplification (MLPA).Methods1.Designing parkin probes according to the probe-designing principles and establishing agentia box for detecting rearrangement mutations of parkin using multiple ligation-dependent probe amplification (MLPA);2.Comparing with the front real-time PCR results to test the reliability of the MLPA agentia box;3.Detecting rearrangement mutations of parkin in Chinese sporadic parkinson’s disease patients, and then using MLPA to test the positive results again.Results1. Inventing parkin agentia box of MLPA after searching the best concentration of probes and testing the stability and reliability of probes;2. After comparing with the results of real-time PCR of78PD patients, we found that76patients have the same results while2with different mutations, the false results are two heterozogous duplication which can’t be identified by MLPA;3.755index patients were screened for gene rearrangement of parkin in MLPA, the positive results were tested in real-time PCR again. There were25patients with rearrangement of parkin gene, the frequency of mutations was3.3%, including18males and7females, the onset aging is14-72years. There were176early-onset PD patients with onset aging <45years,20patients took multiplication mutations of parkin, the frequency of mutations was11.4%.579patients is later-onset aging patients,5with rearrangement sequences of parkin, the frequency of mutation was0.86%. Moreover, there were18patients with sole heterozygous deletions of parkin exons,1patient with sole exon heterozygous duplication,2patients with sole homozygous deletion, there were7patients with multiple exons rearrangment. In them, one patient with multiple heterozygous deletions of exon2and3; one patient with multiple heterozygous deletions of exon2and4; one with multiple heterozygous deletions of exon2,3,4,5and6; one with multiple heterozygous deletions of exon4,5,6and7; one with homozygous deletions of exon3and4, one with multiple heterozygous deletions of exon11and12; one with multiple heterozygous deletions of exon1,2,3,4and5. and the multiple deletion is the main mutation style.Conclusion1. Constructing agentia box to detect rearrangement mutations of parkin gene by using MLPA successfully;2. Applying the designed MLPA kit to detect SPD patients and founding exon rearrangement mutation is3.3%;3. The earlier onset age the higher possibility of gene mutaion;4. We found exon deletion may be the main style of parkin rearrangment mutation.5. Exon2～4may be the hot spot for exon rearrangement of parkin.