Mutation Analysis Of Parkin And PINK1 Genes In Early-onset Parkinson'sdisease In China

Background:Parkinson's disease(PD) is one of the most common neurodegenerative disorders, with a prevalence of more than 1%among persons older than 65 years of age.Clinical features of PD include resting tremor,rigidity,bradykinesia and a good response to levodopa;the main features in pathology are loss of dopaminergic neurons in substantia nigra pars compacta and emergence of Lewy bodies in surviving dopaminergic neurons. To date,the definite reason of PD is unknown,but more and more genes are regarded as PD-caused factors,mutations in genes of a-synuclein,UCH-L1,and LRRK2 are reported to be the causative genes for familial autosomal dominant PD,and mutations in genes of parkin,PINK1,D J-1,and ATP13A2 are reported to be the causative genes for autosomal recessive early-onset PD.To our knowledge,PD is usually divided into early-onset PD(EOPD) and late-onset PD(LOPD),or familial PD and sporadic PD,or autosomal dominant PD and autosomal recessive PD.Onset age of PD less than 50 years was definited as EOPD in most literatures.In 1997,parkin gene was mapped to chromosome 6q25.2-q27 by Matsumine et al. by linkage analysis.The mutation frequency of parkin gene is very high among EOPD. Excepting Japan in Asia,China,Korea,and Taiwan of China also reported the research results of parkin gene in EOPD patients.Chung EJ et al.detected compound heterozygous mutations in 3 patients among 94 EOPD patients with gene dosage analysis;Guo et al.analyzed 45 EOPD patients in 29 families with direct sequencing and real-time quantitative PCR,and detected 25 parkin patients in 14 families with different mutation types,including point mutation,heterozygous mutation,homozygous mutation,and compound heterozygous mutation;Wu RM et al.detected 4 parkin mutation carriers in 41 EOPD patients.PINK1 gene was found during the study of exploring the mechanism of tumor suppressor PTEN.To date,the mutation of PINK1 gene mainly is point mutation,the exon rearrangement is very rare.So when to detect mutation of PINK1,mostly researchers performed DHPLC(denaturing high performance liquid chromatography) and direct-sequencing.The mutation frequency of PINK1 was different in different groups,mostly between 1%and 9%,and became the second mutation gene of EOPD after parkin gene.To date,study of detecting parkin and PINK1 mutation in EOPD patients is still very few in Zhejiang province in China,furthermore the correlation analysis between genotype and phenotype is lack.So the study of detecting parkin and PINK1 mutation in EOPD patients in Zhejiang province in China is necessary,and the correlation analysis between genotype and phenotype is very necessary.Objective:Analyzing point mutations of parkin and PINK1 genes in EOPD patients in Zhejiang province of China with Denaturing High Performance Liquid Chromatography (DHPLC) and direct-sequencing,and analyzing exon rearrangements of parkin gene with real time quantitative PCR,also we analyzed correlation between phenotype and genotype of PD patients. Methods:We selected 69 Chinese patients according to the following criteria:(1) age at onset＜50 years;(2) the presence of at least two signs of the four cardinal features (resting tremor,bradykinesia,rigidity and postural instability);(3) a good response to the treatment of levodopa;(4) excluding the patients with evidence of secondary parkinsonism caused by other neurologic diseases or known drugs or toxins.Firstly,we detected the rearrangement mutations of 12 exons in parkin gene with real time quantitative PCR in 69 patients;then detected point mutations with DHPLC and direct-sequencing in those patients who did not carry rearrangement mutations or only carry heterozygous rearrangements;lastly detected point mutations in 8 exons of PINIK1 gene in those patients who did not carry mutations or only carry heterozygous mutations.Results:First,by using real time quantitative PCR,we found that 19 cases carried different exon rearrangement mutations in parkin gene.M630 and M631 are sisters,both carried heterozygous deletions in exon 3,4,and 7 ofparkin;M5 carried homozygous deletions in exon 3 and 4 ofparkin;M6 carried heterozygous deletions in exon 2 and 6 ofparkin; M10 carried homozygous deletion in exon 4 of parkin;M30 carried heterozygous deletion in exon 6 and heterozygous duplication in exon 11 of parkin.In addition to these,6 cases carried heterozygous deletion in different single exon ofparkin;and 7 cases carried heterozygous duplication in different single exon ofparkin.Second,we found a new heterozygous point mutation c.2T＞C(p.M1T) in parkin gene in one patient by using DHPLC and direct-sequencing.In addition,several SNPs were found in parkin and PINK1 genes.Third,patients with homozygous or compound heterozygous mutations in the parkin gene mostly had a history of symmetrical onset,such as resting tremor,rigidity and bradykinesia;and the onset age of patients with mutation in parkin is earlier significantly than that of patients without mutation in parkin.Conclusion:First,the mutational frequency ofparkin is 28%in this study,which is lower than 50%in many other studies.The reason for this probably is:most patients are sporadic PD,not familial PD in our study.Second,excepting SNP,no mutation was found in PINK1 gene,this result indicates that the mutational frequency of PINK1 is very low,especially in Chinese PD patients.Third,the onset age of PD patients with parkin mutation is significantly earlier than those without parkin mutation,in addition to this,there is no other special clinical feature,so we can not judge whether a patient has mutation in parkin or PINK1 gene according to the clinical symptom.