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SHORT REPORT J Neurol Neurosurg Psychiatry: first published as on 5 January 2004. Downloaded from DJ-1 mutations in Parkinson’s disease D G Healy, P M Abou-Sleiman, E M Valente, W P Gilks, K Bhatia, N Quinn, A J Lees, N W Wood ......

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tions in two —is necessary for disease.13 This hypothesis Mutations in the DJ-1 have recently been shown to would be supported if Parkinson’s disease pathogenesis cause autosomal recessive Parkinson’s disease. To estimate turned out to involve a pathway where interaction between the prevalence of this mutation, an analysis was undertaken DJ-1 and parkin occurred. It may also explain why affected of 39 index cases of Parkinson’s disease in whom a family parkin heterozygotes usually have clinically unaffected parents. history suggested autosomal recessive inheritance. No DJ-1 mutations were found in these patients, indicating that this METHODS gene is unlikely to be of numerical significance in clinical This study was approved by the joint research ethics practice. The hypothesis was also tested that young onset committee of the Institute of Neurology and the National Parkinson’s disease patients in whom, despite extensive Hospital for Neurology and Neurosurgery. Informed consent analysis, only a single heterozygous parkin mutation was was obtained from all patients. found, might harbour a second mutation in the DJ-1 gene— We selected for study 39 index cases of Parkinson’s disease that is, digenic inheritance. No patient was found with a with probable autosomal recessive inheritance, on the basis single mutation in both DJ-1 and parkin genes, making this that they had at least one other sibling similarly affected with mode of inheritance unlikely. Finally it was confirmed that the disease, without evidence of disease in any other generation. Thirty two of these families were of European PARK6 and PARK7 (DJ-1), despite being phenotypically origin and seven were of Asian origin. We excluded cases similar and mapping to the same small chromosomal region known to have even a single parkin gene mutation, though of 1p36, are caused by mutations in separate genes. seven of these patients had not had parkin gene analysis. Thirteen patients had the additional clinical finding of focal dystonia, but no patient had blepharospasm, a possible marker for DJ-1. Two patients reported depression, but none ollowing the recent publication of a fifth Parkinson’s 1 suffered anxiety disorder, which has also been noted in DJ-1. disease gene, DJ-1, by Bonifati et al, there has been At the time of collection, some of these patients may not have Fmuch interest in ascertaining its biological and clinical had a full psychiatric history recorded. importance. To date, however, there have been no published We also selected four index Parkinson’s disease patients of http://jnnp.bmj.com/ data estimating DJ-1 prevalence in Parkinson’s disease. European ancestry with heterozygous parkin gene missense Mutations in DJ-1 were discovered in two consanguineous point mutations. These heterozygous mutations were deter- families from geographically isolated regions in Europe. This mined by directly sequencing the entire opening reading brings to five the number of genes reported that are directly frame of the gene, as well as the intron–exon boundaries and 2–5 implicated in Mendelian Parkinson’s disease. While the 1 kb upstream of the start of gene transcription, including function of the remains to be elucidated, it has been the parkin promoter. A semiquantitative multiplex polymer- proposed that it may be involved in the cellular response to ase chain reaction (PCR) was used to detect exon rearrange- 1 6 . The gene maps to 1p36 ments (deletions and duplications). Only patients with a on September 28, 2021 by guest. Protected copyright. (PARK7 locus) and comprises eight exons, the first of which single heterozygous mutation were included in this hetero- is alternatively spliced and non-coding. Three other recessive zygous parkin subgroup. 7–9 Parkinson’s disease loci map to this chromosomal region, The original PARK6 family (the Marsala kindred)7 and two including PARK6 (1p36–p35), which appears to have a other families, which this group has previously mapped to broadly similar phenotype. This has led to suggestions that 1p36–35 by linkage analysis, were chosen for analysis by PARK6 and PARK7 are allelic, either because of chromosomal direct sequencing, to determine whether PARK6 was caused rearrangements or because of erroneous linkage mapping. by D-1 mutations. We therefore sought to confirm that PARK6 was not caused by DJ-1 mutations. Genetic analysis There has also been considerable recent interest in the DNA was extracted from peripheral lymphocytes according to increasingly large number of young onset or familial cases of standard protocols. For sequence analysis, coding exons were Parkinson’s disease, in whom only one pathogenic parkin amplified by PCR under the following conditions: denatura- mutation has been found despite extensive analysis.10 11 This tion at 94˚C for 15 minutes, followed by 35 cycles of has led to suggestions that a single mutation in these patients denaturation at 94˚C, annealing at 58–60˚C and elongation may be sufficient to cause the phenotype, either by haplo- at 72˚C for 30 seconds each, and a final elongation step of insufficiency or by a dominant negative effect. 10 minutes at 72˚C. PCR was carried out in a final volume of An alternative hypothesis that we chose to test is that these 50 ml including 2.5 mM MgCl2, 0.2 mM deoxynucleotide patients may also harbour pathogenic mutations in the DJ-1 triphosphate, 0.5 mM forward and reverse primer (as gene and that only the combination of a heterozygous published2), 1 U Taq polymerase, and 10 ng of DNA. mutation in each of these genes resulted in Parkinson’s Bidirectional dideoxy chain terminator sequencing was disease. This digenic mode of inheritance has been described carried out according to the manufacturers’ instructions in other autosomal recessive disorders such as human insulin (BigDye, Applied Biosystems, Warrington, UK) and the resistance, which is bi-allelic digenic,12 and Bardet-Biedl products were electrophoresed on an ABI 3100 automated syndrome, where triallelic inheritance—that is, three muta- DNA sequencer (Applied Biosystems).

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...... Long range PCR for the detection of the exon 1–5 deletion Authors’ affiliations described by Bonifati et al was done as previously reported.1 D G Healy, P M Abou-Sleiman, W P Gilks, A J Lees, N W Wood, Department of Molecular Neuroscience, Institute of Neurology, Queen J Neurol Neurosurg Psychiatry: first published as on 5 January 2004. Downloaded from RESULTS Square, London WC1N 3BG, UK In a cohort of 39 index Parkinson’s disease patients with E M Valente, CSS hospital, IRCCS, San Giovanni, Rotondo, CSS-Mendel probable autosomal recessive inheritance, we did not find Institute, Rome, Italy K Bhatia, N Quinn, Sobell Department of Motor Neuroscience and any pathogenic mutation in the DJ-1 gene. Movement Disorders Institute of Neurology, Queen Square, London, UK We also did not find pathogenic DJ-1 mutations in a smaller cohort of four patients in whom, despite extensive Competing interests: none declared genetic analysis, we could only determine a single hetero- Correspondence to: Professor N W Wood, Department of Molecular zygous parkin gene mutation. Neuroscience, Institute of Neurology and National Hospital for Finally no DJ-1 mutations were found in three PARK6 Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK; linked families. [email protected] Received 21 May 2003 DISCUSSION Accepted 21 June 2003 The large cohort of autosomal recessive patients studied indicates that the frequency of pathogenic DJ-1 mutations is low in Parkinson’s disease, especially given that we excluded REFERENCES from analysis any known parkin gene mutations. We also 1 Bonifati V, Rizzu P, Van Barren M, et al. The mutations in the DJ-1 gene associated with autosomal recessive early onset parkinsonism. Science screened all our samples for the 14 kbp deletion reported by 2003;299:256–99. 1 Bonifati et al, and did not find it. This indicates that the 2 Polymeropoulos MH, Lavedan C, Leroy E, et al. Mutation in alpha-synuclein deletion may be confined to the genetically isolated popula- gene identified in families with Parkinson’s disease. Science tion in the Netherlands, where it was originally found.1 1997;276:2045–7. 3 Kitada T, Asakawa S, Hattori N, et al. Mutations in the Parkin gene cause At present the role of DNA diagnostic testing for mutations autosomal recessive juvenile parkinsonism. Nature 1998;392:605–8. in DJ-1 is unclear, but these data suggest that such testing 4 Le WD, Xu P, Jankovic J, et al. Mutations in NR4A2 associated with familial will not yield numerically significant results and therefore, Parkinson disease. Nat Genet 2003;33:85–9. 5 Leroy E, Boyer R, Auburger G, et al. The ubiquitin pathway in Parkinson’s unlike the parkin gene, DJ-1 is unlikely to be of clinical disease. Nature 1998;395:451–2. importance in neurological practice. More work will also be 6 Van Duijn CM, Dekker M, Bonifati V, et al. PARK7, a novel locus for autosomal needed to elucidate the possible role of large genomic recessive early onset parkinsonism on chromosome 1p36. Am J Hum Genet 2001;69:629–34. rearrangements. 7 Valente EM, Bentivogio AR, Dixon PH, et al. Localization of a novel locus for We were unable to prove our hypothesis of digenic autosomal recessive early-onset parkinsonism, PARK6, on human inheritance between parkin and DJ-1 in Parkinson’s disease. chromosome 1p35–p36. Am J Hum Genet 2001;68:895–900. 8 Hicks AA, Petursson H, Jonsson T, et al. A susceptibility gene for late-onset This does not exclude other recessive genes being involved in idiopathic Parkinson’s disease. Ann Neurol 2002;52:549–55. this mode of inheritance of Parkinson’s disease. Indeed our 9 Hampshire DJ, Roberts E, Crow Y, et al. Kufor-Rakeb syndrome, pallido- group also has evidence of young disease onset patients with pyramidal degeneration with supranuclear upgaze paresis and dementia, non-synonymous heterozygous DJ-1 mutations in whom a maps to 1p36. J Med Genet 2001;38:680–2. 10 West AB, Periquet M, Lincoln S, et al. The complex relationship between second pathogenic mutation has not been found (unpub- Parkin mutations and Parkinson’s disease. Hum Mol Genet lished data). Large scale rearrangements, however, have not 2002;11:2787–92. http://jnnp.bmj.com/ as yet been excluded in these patients. Further work is 11 Foroud T, Uniacke SK, Liu L, et al. Heterozygosity for a mutation in the parkin gene leads to later onset Parkinson disease. Neurology needed to establish the role of digenic or oligogenic 2003;60:796–801. inheritance in Parkinson’s disease. 12 Beadles PL, Badano JL, Ross AJ, et al. 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