Screening For DJ-1 Gene Mutation By Real-time PCR And Direct Sequencing

BackgroundParkinson's disease (PD) is a common neurodegenerative disorder.Clinically, most patients suffer from bradykinesia, resting tremor, rigidity,and postural instability. Pathologically, PD is characterized byprogressive loss of dopaminergic neurons and the presence of Lewybodies in the substantia nigra. Autosomal-recessive earlyparkinsonism(AREP) refers to parkinsonism with a mode of inheritancecompatible with autosomal recessive transmission and an age at onset of45 years or younger in at least one of the affected siblings.Dopa-responsive dystonia(DRD) is a rare genetic disease. DRD could becharacterised by parkinsonian features in some patients which can not bedistinguished from AREP clinically. Mutations ofparkin, PINK1, DJ-1和ATP13A2 can cause AREP. 12 DJ-1 mutations including heterozygousdeletion were discovered in patients with early-onset PD. In our previouswork, probands from 15 AREP families and 4 DRD families werescreened for DJ-1 mutations by direct sequencing. A heterozygousmutation(A39S) was confirmed in one family. Gene duplications ordeletions can not be detected by DNA direct sequencing. In order to make clear the frequency of D J-1 mutations in our group of AREP andDRD patients, it is of great importance to Screen for DJ-1 gene Mutationby gene dosage analysis combining direct sequencing.ObjectiveTo establish a platform of gene rearrangement detection usingreal-time PCR. 21 index patients from AREP families and 6 indexpatients from DRD families were screened for mutations of DJ-1 gene byreal-time PCR and direct sequencing.MethodsDuplications or deletions of DJ-1 gene were detected using real-timequantitative PCR; point mutations and small deletion/duplicationmutations were detected by direct sequencing. ALB gene was used as aninternal standard. Initial copy number of DJ-1 gene and ALB gene werecalculated according to the standard curve. The relative copy number ofDJ-1 gene equals to the ratio of DJ-1 and ALB gene copy number.Results21 index patients from AREP families and 6 index patients fromDRD families were screened for gene rearrangement of DJ-1 gene. Therelative copy number of DJ-1 gene exons ranged from 0.8 to 1.2 whichwas thought to be normal. None of duplication or deletion mutation ofDJ-1 gene was found in the group of patients. On the basis of ourprevious work, 6 index patients from AREP families and 2 index patients from DRD families were screened for point mutations of DJ-1 gene bydirect sequencing. A homozygous T29C substitution in exon2 of the DJ-1gene that resulted in a leucine -to- proline missense substitution (L10P)was found in one patient from an AREP family. 3 single nucleotidepolymorphisms(SNP) were also found, including IVS4+30t→g,IVS4+45g→a and IVS4+46g→a.ConclusionsA platform of gene rearrangement detection using real-time PCR hasbeen established. None of duplication or deletion mutation of DJ-1 genewas found in the group of patients. A homozygous missense substitution(L10P) was found. The frequency of DJ-1 gene mutation in the group ofpatients is 9.5％. 3 SNPs were also found, including IVS4+30t→g,IVS4+45g→a and IVS4+46g→a.