Molecular Genetic Study Of Dopa-Responsive Dystonia In Han Chinese Patients

ObjectiveTo investigate the genotype-phenotype correlations in Han Chinese Dopa-Rsponsive Dystonia patients,we carried out mutation screening of GTP cyclohydrolase 1 and Tyrosine hydroxylase genes.Furthermore,we performed Multiple ligation-dependent probe amplification analysis(MLPA)and quantitative real-time PCR to investigate the exonic deletion of GCH1,TH and the epsilon-sarcoglycan encoding(SGCE)genes.MethodsWe collected 23 subjects,including 8 DRD patients and 5 unaffected family members in 4 DRD pedigrees,and 10 sporadic cases.Forty(40)unrelated normal controls were also collected for this study.DNA was extracted from ethylenediaminetetraaceticacid(EDTA)-treated whole blood samples using the standard high-salt method.All exons and splicing sites of GCH1 and TH genes were sequenced by PCR Sanger sequencing.Sequence alignments were performed by the Mutation Surveyor software(SOFTGENETICS)for mutation detection.All mutations were checked in 40 normal control subjects.To test if there was any exonic deletion in DRD subjects,MLPA and qPCR were employed to analyze all 22 exons from GCH1(6 exons),TH(5 exons),and SGCE(11 exons)genes in all 23 subjects and 3 unrelated normal controls.MLPA dosage quotients of GCH1,TH,SGCE genes exons were calculated and compared by the Coffalyser program.To further confirm the MLPA results,quantitative real-time PCR(qPCR)was performed on candidate gene exons using the SYBR Green method by the ABI 7500 Fast Real-Time PCR system.Exon deletions were determined by the comparative threshold cycle method(ddCt)as previously described.Results1 In family 9,the Ala98Val(454C>T)mutation in exon 1 of GCH1 was identified.Four of five siblings in this family carried the mutation,but the only male mutation carrier(also the youngest,46 years of age)had no symptoms of dystonia.2 In family 10,we found the Ile135Thr(37449T>C)mutation in GCH1 exon 2 that resulted in an isoleucine/threonine transition at codon 135.The proband was a 34-year-old female.The same mutations were found in the proband's father and elder brother,although neither was affected.3 In family 12,we have identified two siblings with heterozygous Tyr75Cys(385A.G)mutations in GCH1 exon 1,while the other sibling,who does not carry this mutation,is normal.4 Two heterozygous mutations were detected in TH exon 12(Gly397Arg)and exon 1(Ser1 9Cys)in sporadic DRD cases of our study.5 In a sporadic DRD patient,the signal ratio of exon 1 in GCH1 gene that ligated with the probes of 142nt and 256nt was around 0.5,however,ratios between 0.75 and 1.25 were considered normal.The decreased signal ratio(0.5)suggested a heterozygous deletion.No other deletion/duplications were observed in other samples.6 By qPCR,the ddCt value of the sporadic DRD sample with heterozygous deletion yielded a ddCt value of 0.9,while normal sample had ddCt values of 0.Conclusions1 The frequency of GCH1 gene mutations is relatively high in Han Chinese patients.Several novel missense mutations of the GCH1 gene were found in DRD pedigrees.On the other hand,incomplete penetration was not uncommon in DRD families.Symptoms and age of onsets varied in DRD patients who shared same mutations.We failed to identify GCH1 or TH mutations in some sporadic DRD subjects.Once DRD was suspected in patients,mutation screening may help establish the diagnosis.2 Exonic deletion of GCH1 may contribute to some DRD cases,but it was relatively rare;It is necessary to detect exonic deletions in DRD cases if no gene mutation was found.