Katja Hedrich

Early-onset parkinsonism associated with PINK1 mutations: frequency, genotypes, and phenotypes.

To the Editor: We read with great interest the recent report by Bonifati et al. demonstrating that the transcript of an allele bearing the c.1366C T (Gln456Stop) mutation in the PINK1 gene associated with early-onset Parkinson disease (PD) is not detectable by PCR in two unrelated families.1 The authors explained this finding by either lack of expression of the mutant allele or by instability due to the mutation or another change in linkage disequilibrium with the c.1366T alteration. They conclude that this variant is an example of a mutant allele that exerts its major pathogenic effect in PD at the mRNA rather than at the protein level.1 We identified a large German PD family with four affected siblings who were all homozygous for the c.1366C T mutation. We also studied 11 of their asymptomatic heterozygous children and 5 mutation-negative family members. To address the question of an effect of this mutation on the mRNA level, we collected fresh blood from all 20 family members and extracted RNA that was reverse transcribed. We first carried out an RT-PCR spanning exons 4–7 including the mutation (Primers Ex4F: CCAAGAGAGGTCCCAAGC; Ex7R: CCTCACCAACTGTCTCACG). We observed no difference in product intensity even in the homozygous mutation carriers, suggesting that the mutated allele is expressed at a similar level and has a similar stability as the wild type allele (figure, A). To separately investigate the mRNA level of the wild type and mutated allele, we designed mutation-specific primers and performed another RT-PCR (Ex7FWT: TCAATCCCTTCTACGGCC, Ex7FMUT: TCAATCCCTTCTACGGCT, Ex8R: CTCCTCAGTCCAGCCTCAT). We demonstrated the specificity of the primers and the expression of both alleles in all heterozygous carriers (figure, B). We showed that the pathogenic effect of the recurrent c.1366C T mutation in our family is not related to lack of expression or instability. Our results rather support the hypothesis that the lack of mutated transcripts in the families described by Bonifati et al. may be caused by another change in linkage disequilibrium with the mutation. Interestingly, the c.1366C T mutation likely has arisen twice independently in our family or bases on an ancient founder since the haplotypes are partly different (figure, C). It remains unclear which haplotypes the reported unrelated Italian families carry.1 Our heterozygous mutation carriers were asymptomatic, whereas Bonifati et al.’s patients had full-blown PD. It remains to be determined whether RNA expression levels of the mutated allele may be correlated with disease status.

Lewy body Parkinson's disease in a large pedigree with 77 Parkin mutation carriers

We report the clinical, genetic, and neuropathological findings of a seven generation–spanning pedigree with 196 individuals, 25 of whom had levodopa‐responsive parkinsonism. Genetic analyses indicated Parkin mutations in 77 subjects. Among the 25 patients, 5 carried compound heterozygous mutations and met criteria for definite Parkinsons disease (PD) according to UK PD Society Brain Bank guidelines; 8 subjects carried only a heterozygous Parkin mutation. The mutational status of five deceased patients was unknown, and seven PD patients had no Parkin mutation. Survival analyses showed a significant difference in the age‐at‐onset distribution between patients with compound heterozygous mutations and the groups of heterozygous carriers and subjects without detectable Parkin mutations. Autopsy of a 73‐year‐old patient, who carried two mutant Parkin alleles (delExon7 + del1072T), showed PD‐type cell loss, reactive gliosis, and α‐synuclein–positive Lewy bodies in the substantia nigra and locus ceruleus. Surviving neurons were reactive with antibodies to the N terminus of Parkin but not the In‐Between‐RING (“IBR”) domain, which had been deleted by both mutations. This large Parkin pedigree represents a unique opportunity to prospectively study the role of heterozygous Parkin mutations as a PD risk factor, to identify additional contributors to the expression of late‐onset PD in heterozygous carriers, and to reexamine the role of Parkin in inclusion formation. Ann Neurol 2005;58:411–422

Distribution, type, and origin of Parkin mutations: Review and case studies

Early‐onset Parkinsons disease (PD) has been associated with different mutations in the Parkin gene (PARK2). To study distribution and type of Parkin mutations, we carried out a comprehensive literature review that demonstrated two prominent types of mutations among 379 unrelated mutation carriers: exon rearrangements involving exon 3, 4, or both, and alterations in exons 2 and 7, suggesting mutational hot spots or founders. To elucidate the origin of 14 recurrent Parkin mutations in our samples, we carried out a detailed haplotype analysis at the PARK2 locus. Thirty‐eight mutation‐positive individuals, available family members, and 62 mutation‐negative individuals were genotyped. We determined allele frequencies and linkage disequilibrium (LD) to evaluate the significance of shared haplotypes. We observed no LD between markers at PARK2. Our data support a common founder for the most frequent Parkin point mutation (924C>T; exon 7) and indicate a mutational hot spot as cause of a common small deletion (255/256delA; exon 2). Furthermore, the most frequent Parkin exon deletion (Ex4del) arose independently in 2 of our subjects. However, it also occurred as the result of a founder mutation in 2 cases that shared identical deletion break points. This study provides evidence for both mutational hot spots and founder mutations as a source of recurrent mutations in Parkin, regardless of the mutation type.

DJ-1 (PARK7) mutations are less frequent than Parkin (PARK2) mutations in early-onset Parkinson disease

Background: Mutations in the Parkin gene (PARK2) are the most commonly identified cause of recessively inherited early-onset Parkinson disease (EOPD) but account for only a portion of cases. DJ-1 (PARK7) was recently reported as a second gene associated with recessively inherited PD with a homozygous exon deletion and a homozygous point mutation in two families. Methods: To investigate the frequency of DJ-1 mutations, the authors performed mutational analysis of all six coding exons of DJ-1 in 100 EOPD patients. For the detection of exon rearrangements, the authors developed a quantitative duplex PCR assay. Denaturing high performance liquid chromatography analysis was used to screen for point mutations and small deletions. Further, Parkin analysis was performed as previously described. Results: The authors identified two carriers of single heterozygous loss-of-function DJ-1 mutations, including a heterozygous deletion of exons 5 to 7 and an 11-base pair deletion, removing the invariant donor splice site in intron 5. Interestingly, both DJ-1 mutations identified in this study were found in the heterozygous state only. The authors also detected a polymorphism (R98Q) in 1.5% of the chromosomes in both the patient and control group. In the same patient sample, 17 cases were detected with mutations in the Parkin gene. Conclusions: Mutations in DJ-1 are less frequent than mutations in Parkin in EOPD patients but should be considered as a possible cause of EOPD. The effect of single heterozygous mutations in DJ-1 on the nigrostriatal system, as described for heterozygous changes in Parkin and PARK6, remains to be elucidated.

Evaluation of 50 probands with early-onset Parkinson’s disease for Parkin mutations

BackgroundEarly onset PD has been associated with different mutations in the Parkin gene, including exon deletions and duplications. Methods The authors performed an extensive mutational analysis on 50 probands with onset of PD at younger than 50 years of age. Thirteen probands were ascertained from a registry of familial PD and 37 probands by age at onset at younger than 50 years, blind to family history. Mutational analysis was undertaken on the probands and available family members and included conventional techniques (single strand conformation polymorphism analysis and sequencing) and a newly developed method of quantitative duplex PCR to detect alterations of gene dosage (exon deletions and duplications) in Parkin. Results Using this new technique, the authors detected eight alterations of gene dosage in the probands, whereas 12 mutations were found by conventional methods among the probands and another different mutation in an affected family member. In total, the authors identified compound heterozygous mutations in 14%, heterozygous mutations in 12%, and no Parkin mutation in 74% of the 50 probands. We expanded the occurrence of Parkin mutations to another ethnic group (African-American). Conclusion The authors systematically screened all 12 Parkin exons by quantitative PCR and conventional methods in 50 probands. Eight mutations were newly reported, 2 of which are localized in exon 1, and 38% of the mutations were gene dosage alterations. These results underline the need to screen all exons and to undertake gene dosage studies. Furthermore, this study reveals a frequency of heterozygous mutation carriers that may signify a unique mode of inheritance and expression of the Parkin gene.

Role of parkin mutations in 111 community‐based patients with early‐onset parkinsonism

Early‐onset parkinsonism is frequently reported in connection with mutations in the parkin gene. In this study, we present the results of extensive genetic screening for parkin mutations in 111 community‐derived early‐onset parkinsonism patients (age of onset <50 years) from Germany with an overall mutation rate of 9.0%. Gene dosage alterations represented 67% of the mutations found, underlining the importance of quantitative analyses of parkin. In summary, parkin mutations accounted for a low but significant percentage of early‐onset parkinsonism patients in a community‐derived sample.

PINK1 , Parkin , and DJ-1 mutations in Italian patients with early-onset parkinsonism

Recessively inherited early-onset parkinsonism (EOP) has been associated with mutations in the Parkin, DJ-1, and PINK1 genes. We studied the prevalence of mutations in all three genes in 65 Italian patients (mean age of onset: 43.2±5.4 years, 62 sporadic, three familial), selected by age at onset equal or younger than 51 years. Clinical features were compatible with idiopathic Parkinsons disease in all cases. To detect small sequence alterations in Parkin, DJ-1, and PINK1, we performed a conventional mutational analysis (SSCP/dHPLC/sequencing) of all coding exons of these genes. To test for the presence of exon rearrangements in PINK1, we established a new quantitative duplex PCR assay. Gene dosage alterations in Parkin and DJ-1 were excluded using previously reported protocols. Five patients (8%; one woman/four men; mean age at onset: 38.2±9.7 (range 25–49) years) carried mutations in one of the genes studied: three cases had novel PINK1 mutations, one of which occurred twice (homozygous c.1602_1603insCAA; heterozygous c.1602_1603insCAA; heterozygous c.836G>A), and two patients had known Parkin mutations (heterozygous c.734A>T and c.924C>T; heterozygous c.924C>T). Family history was negative for all mutation carriers, but one with a history of tremor. Additionally, we detected one novel polymorphism (c.344A>T) and four novel PINK1 changes of unknown pathogenic significance (−21G/A; IVS1+97A/G; IVS3+38_40delTTT; c.852C>T), but no exon rearrangements. No mutations were found in the DJ-1 gene. The number of mutation carriers in both the Parkin and the PINK1 gene in our cohort is low but comparable, suggesting that PINK1 has to be considered in EOP.

High mutation rate in dopa-responsive dystonia: detection with comprehensive GCHI screening.

Mutations in GTP cyclohydrolase I (GCHI) are found in 50 to 60% of cases with dopa-responsive dystonia (DRD). Heterozygous GCHI exon deletions, undetectable by sequencing, have recently been described in three DRD families. We tested 23 individuals with DRD for the different mutation types by conventional and quantitative PCR analyses and found mutations, including two large exon deletions, in 87%. The authors attribute this high mutation rate to rigorous inclusion criteria and comprehensive mutational analysis.

Mutations in DYT1 Extension of the phenotypic and mutational spectrum

Background: Most cases of early-onset primary torsion dystonia (PTD) are caused by the same three–base pair (bp) (GAG) deletion in the DYT1 gene. Exon rearrangements are a common mutation type in other genes and have not yet been tested for in DYT1. Several lines of evidence suggest a relationship of the DYT1 gene with Parkinson disease (PD). Objective: To investigate the frequency and type of DYT1 mutations and explore the associated phenotypes in a mixed movement disorders patient cohort and in controls. Methods: The authors screened 197 patients with dystonia (generalized: n = 5; focal/segmental: n = 126; myoclonus-dystonia: n = 34; neuroleptic-induced: n = 32), 435 with PD, and 42 with various other movement disorders, along with 812 healthy controls, for small deletions in exon 5 of DYT1 and tested for exon rearrangements by quantitative, duplex PCR in 51 GAG deletion-negative dystonia cases. Results: The GAG deletion was detected in five patients: three with early-onset PTD, one with generalized jerky or clonic dystonia, and one with generalized dystonia and additional features (developmental delay, pyramidal syndrome). A novel out-of-frame four-bp deletion (934_937delAGAG) in exon 5 of the DYT1 gene was found in a putatively healthy blood donor. No exon rearrangements were identified in DYT1. Conclusions: In this mixed patient sample, the GAG deletion was rare and in two out of five cases associated with an unusual phenotype. In addition, a novel DYT1 truncating mutation of unknown clinical relevance was found in a putatively unaffected individual. DYT1 exon rearrangements, however, do not seem to be associated with PTD.

The striatal dopaminergic deficit is dependent on the number of mutant alleles in a family with mutations in the parkin gene: evidence for enzymatic parkin function in humans

Autosomal recessive parkinsonism associated with mutations in the parkin gene represents a monogenic form of hereditary parkinsonism. We performed [(18)F]6-fluorodopa (FDOPA) positron emission tomography as a measurement of the nigrostriatal dopaminergic system as well as extensive haplotype analysis of the PARK 2 gene locus in 14 subjects with parkin mutations. In parkin subjects, the reduction of striatal FDOPA uptake increased with the number of mutated alleles and was also slightly obvious in asymptomatic parkin gene carriers in the heterozygous state. The abnormal FDOPA uptake pattern in parkin patients did not significantly differ from that of sporadic Parkinsons disease. Our data are in agreement with an enzymatic dysfunction of the genes translational product, which has been shown to promote protein degradation as an ubiquitin-protein ligase. Thus, parkinsonism in parkin gene carriers may be related to abnormal nigral protein accumulation in the presence of a suprathreshold enzyme dysfunction.