Molecular Analyses Of GCH1, TH And Parkin Genes In Chinese DRD Families And Research On Function Of Mutation GTP Cyclohydrolase Ⅰ

PARTⅠMolecular analyses of GCH1, TH and parkin genes in Chinese DRD families Background and ObjectivesDopa-responsive dystonia (DRD) is an inherited dystonia characterized by marked diurnal fluctuation, exquisite responsiveness to levodopa, and mild parkinsonian features.It is a rare disorder with a prevalence of 1 per 2 million and can be treated successfully with low dose levodopa. This potential for therapy emphasizes the importance of diagnosis of DRD families. However, clinical manifestations of DRD vary widely, Due to its highly variable expressivity, the early diagnosis of DRD is difficult and many cases are misdiagnosed. Therefore, molecular analyses of DRD families are of crucial importance for clarifying the molecular genetics and enlarging our knowledge of its highly variable expressivity.The locus of DRD has been assigned to the GTP-cyclohydrolase 1 (GCH1) and tyrosine hydroxylase (TH),Sometimes, it is difficult to distinguish DRD from autosomal recessive juvenile parkinsonism (ARJP)caused by parkin mutations, thus it is necessary to screen for parkin mutations in the DRD patients without GCH1 and TH mutations. However, these analyses have not been extensively studied in Chinese DRD patients.Here we reported the molecular analyses of the GCH1, TH and parkin genes in 10 DRD of Chinese Han ethnic groupMethods1. Subjects is fourteen DRD patients and 28 clinically unaffected relatives from 10 unrelated families of Han ethnic group were included in the present study..2. GCH1 mutations were screening in 10 index cases by direct sequencing. The entire GCH1 coding region including exons 1-6 and the intron-exon boundaries was amplified as previously described and the 5'-untranslated region was amplified. semiquantitative PCR experiments were used to detect heterozygous exon deletions in GCHI .To confirm the presence of the mutation in relatives or to verify its absence in the control population, 10 microlitres of PCR product were digested with the appropriate restriction enzyme3. Ten index cases with or without GCH1 mutation were all screened for TH mutations by direct sequencing and semiquantitative PCR. The novel variations were verified in 200 unrelated healthy controls (ages≥50 years old) with no known family history of DRD from a random Chinese population.4. If no GCH1 and TH mutations were identified, the index patients were further screened for parkin mutations by direct sequencing and semiquantitative PCR.5. Clinical analyses of the DRD families.Results:We have identified 6 novel mutations and 3 known mutations. The novel mutations are Leu91Val, Pro95Leu, Val204Gly and 628delC in GCH1 gene, Gly216Ser in TH gene and Cys253Phe in parkin gene. After molecular analyses of 7 families with identified GCH1 mutations, 9 asymptomatic cases were found among 23 relatives, which confirmed the low penetrance of DRD. Unlike previous publications, male patients with GCH1 mutations have early onset ages while some female patients have very late onset ages in this medium-size seriesConclusions1,Altogether, we have identified 6 novel mutations in the present study, further broadening the mutational spectrum of the GCH1, TH and parkin genes.2,It is necessary to know the genetic defects of DRD patients in clinics, which will help to elucidate the mode of inheritance, facilitate causal therapy with levodopa。.3,The process screen in GCH1, TH, parkin gene for diagnosis of DRD. PARTⅡResearch on function of mutation GTP cyclohydrolase I Background and ObjectivesGuanosine 5'-triphosphate cyclohydrolase I (GCH 1) catalyzes a rate-limiting step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of tyrosine, phenylalanine, and tryptophan hydroxylases. GCH1 protein defects caused by GCH1 gene mutations will lead to dopa-responsive dystonia disease.however ,the mechanism of protein disfunction cause by gene mutation is different.In our previous study, GCH1 gene mutations were screened by direct sequencing in DRD familes, and Leu91Val, Val204Gly, 628delC mutations were identified. Leu91Val and Val204Gly caused dystonia phenotypes and 628delC caused parkinson. However, the molecular mechanism and pathogenesis of the phenotype difference is unclear. In this study, we transfected Hela cells with Leu91Val, Val204Gly, 628delC and wild type GCH1/Pcmv-myc eukaryon expressional plasmid, and construct transient cell lines, further study on the function of GCH1 protein, in order to elucidate the molecular mechanism and pathogenesis of the phenotype difference.Methods1,Leu91Val, Val204Gly, 628delC and wild type GCH1cDNA/pCMV/myc eukaryon expressional plasmid were constructed by restriction enzyme digestion and site-directed mutagenesis, and full length sequences of the target gene were detected.2,Hela cells were transfected with Leu91Val, Val204Gly, 628delC and wild type GCH1cDNA/pCMV/myc eukaryon expressional plasmid by Lipofectamine2000 kit..3,Western blotting methods for analysis wild-type and mutant GCH1 protein quantitative4,High-performance liquid chromatography detect wild-type and mutant protease activity.Results:1,Leu91Val, Val204Gly, 628delC and wild type GCH1cDNA/pCMV/myc eukaryon expressional plasmid were constructed successfully.2,Western Blot confirmed the eukaryotic cell transient transfection cell model can successfully expressed wild-type and mutant GCH1 protein.3,Western Blot semi-quantitative analysis showed that three mutant and wild-type proteins have no difference expression. in eukaryotic cell transient and co-transfection cell model4,High-performance liquid chromatography is performed to detect protease activity in in eukaryotic cell transient and co-transfection cell model and showed three kinds of mutant proteins have decreased activity and Leu91Val decrease .similar with the level of protein activity Val204Gly. 628delC protein decreased activity the most serious. .Conclusions1,Several cell model for DRD have been constructed and provide a good tool for research on DRD protein function and pathogenesy.2,We proved that Leu91Val, Val204Gly, 628delC mutation in GCH1 gene may not cause a decline in protein expression.3,We proved that Leu91Val, Val204Gly, 628delC mutations lead to disactivity of GCH1 protein. GCH1 protein and varying degrees of disactivity may lead to differences in the clinical phenotype.which elucidate the mechanism of the results of genotype-phenotype analysis.