Norman Kock

Alterations in the common fragile site gene Parkin in ovarian and other cancers.

The cloning and characterization of the common fragile site (CFS) FRA6E (6q26) identified Parkin, the gene involved in the pathogenesis of many cases of juvenile, early-onset and, rarely, late-onset Parkinsons disease, as the third large gene to be localized within a large CFS. Initial analyses of Parkin indicated that in addition to playing a role in Parkinsons disease, it might also be involved in the development and/or progression of ovarian cancer. These analyses also indicated striking similarities among the large CFS-locus genes: fragile histidine triad gene (FHIT; 3p14.2), WW domain-containing oxidoreductase gene (WWOX; 16q23), and Parkin (6q26). Analyses of FHIT and WWOX in a variety of different cancer types have identified the presence of alternative transcripts with whole exon deletions. Interestingly, various whole exon duplications and deletions have been identified for Parkin in juvenile and early-onset Parkinsons patients. Therefore, we performed mutational/exon rearrangement analysis of Parkin in ovarian cancer cell lines and primary tumors. Four (66.7%) cell lines and four (18.2%) primary tumors were identified as being heterozygous for the duplication or deletion of a Parkin exon. Additionally, three of 23 (13.0%) nonovarian tumor-derived cell lines were also identified as having a duplication or deletion of one or more Parkin exons. Analysis of Parkin protein expression with antibodies revealed that most of the ovarian cancer cell lines and primary tumors had diminished or absent Parkin expression. While functional analyses have not yet been performed for Parkin, these data suggest that like FHIT and WWOX, Parkin may represent a tumor suppressor gene.

Effect of endogenous mutant and wild-type PINK1 on Parkin in fibroblasts from Parkinson disease patients

Mutations in the PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine-threonine kinase, and Parkin, an E3 ubiquitin ligase, are associated with autosomal-recessive forms of Parkinson disease (PD). Both are involved in the maintenance of mitochondrial integrity and protection from multiple stressors. Recently, Parkin was demonstrated to be recruited to impaired mitochondria in a PINK1-dependent manner, where it triggers mitophagy. Using primary human dermal fibroblasts originating from PD patients with various PINK1 mutations, we showed at the endogenous level that (i) PINK1 regulates the stress-induced decrease of endogenous Parkin; (ii) mitochondrially localized PINK1 mediates the stress-induced mitochondrial translocation of Parkin; (iii) endogenous PINK1 is stabilized on depolarized mitochondria; and (iv) mitochondrial accumulation of full-length PINK1 is sufficient but not necessary for the stress-induced loss of Parkin signal and its mitochondrial translocation. Furthermore, we showed that different stressors, depolarizing or non-depolarizing, led to the same effect on detectable Parkin levels and its mitochondrial targeting. Although this effect on Parkin was independent of the mitochondrial membrane potential, we demonstrate a differential effect of depolarizing versus non-depolarizing stressors on endogenous levels of PINK1. Our study shows the necessity to introduce an environmental factor, i.e. stress, to visualize the differences in the interaction of PINK1 and Parkin in mutants versus controls. Establishing human fibroblasts as a suitable model for studying this interaction, we extend data from animal and other cellular models and provide experimental evidence for the generally held notion of PD as a condition with a combined genetic and environmental etiology.

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.

Differential effects of PINK1 nonsense and missense mutations on mitochondrial function and morphology

Mutations of the PINK1 gene are a cause of autosomal recessive Parkinsons disease (PD). PINK1 encodes a mitochondrial kinase of unknown function which is widely expressed in both neuronal and non-neuronal cells. We have studied fibroblast cultures from four family members harbouring the homozygous p.Q456X mutation in PINK1, three of their wild-type relatives, one individual with the homozygous p.V170G mutation and five independent controls. Results showed bioenergetic abnormalities involving decreased activities of complexes I and IV along with increased activities of complexes II and III in the missense p.V170G mutant. There were increased basal levels of mitochondrial superoxide dismutase in these cells and an exaggerated increase of reduced glutathione in response to paraquat-induced free radical formation. Furthermore, swollen and enlarged mitochondria were observed in this sample. In the p.Q456X nonsense mutants, the respiratory chain enzymes were unaffected, but ATP levels were significantly decreased. These results confirm that mutations of PINK1 cause abnormal mitochondrial morphology, bioenergetic function and oxidative metabolism in human tissues but suggest that the biochemical consequences may vary between mutations.

ε-Sarcoglycan mutations found in combination with other dystonia gene mutations

Myoclonus‐dystonia is a movement disorder associated with mutations in the ε‐sarcoglycan gene (SGCE) in most families and in the DRD2 and DYT1 genes in two single families. In both of the latter families, we also found a mutation of SGCE. The molecular mechanisms through which the detected mutations may contribute to myoclonus‐dystonia remain to be determined.

Parkin gene alterations in hepatocellular carcinoma

The Parkin gene is an extremely large gene (1.5 Mb) within the highly unstable FRA6E common fragile site (CFS) region, which is frequently altered in ovarian, breast, and hepatocellular carcinomas. Because Parkin/FRA6E has genomic similarities to FHIT/FRA3B and WWOX/FRA16D, two other large tumor‐suppressor genes that are within CFS regions, we were interested in characterizing Parkin gene alterations and their possible association with cancer. After analyzing 50 cancer‐derived cell lines including 11 hepatocellular carcinoma (HCC) cell lines, we found that one HCC cell line, PLC/PRF/5, had a detectable homozygous deletion encompassing exon 3. Using quantitative duplex PCR and fluorescence in situ hybridization analysis to characterize the copy number changes of Parkin exons in HCC cell lines, we found that 4 of 11 HCC cell lines had heterozygous deletions of Parkin exons and one, Hep3B, had an exon duplication. Parkin protein expression was significantly decreased or absent in all 11 HCC cell lines. Furthermore, more than 50% of HCC primary tumors had decreased Parkin expression compared to that in normal liver tissue. Parkin gene–transfected PLC5 and Hep3B cells grew more slowly than vector‐only transfectants and also showed increased sensitivity to apoptosis induced by cell‐cycle inhibitors. Therefore, we suggest that Parkin may be involved in tumor suppression and that the loss of Parkin contributes to the development of hepatocarcinoma.

Genetic heterogeneity in ten families with myoclonus-dystonia

Background: Myoclonus-dystonia (M-D) is a movement disorder with autosomal dominant inheritance and reduced penetrance but may also occur sporadically. Recently, mutations in the epsilon-sarcoglycan gene (SGCE) were shown to cause M-D. Furthermore, single variants in the dopamine D2 receptor (DRD2) and DYT1 genes were found in combination with SGCE mutations in two M-D families, and another M-D locus was recently mapped to chromosome 18p11 in one family. Methods: The authors clinically and genetically characterised ten consecutive cases with myoclonus-dystonia; seven familial and three sporadic. Twenty nine M-D patients and 40 unaffected family members underwent a standardised clinical examination by a movement disorder specialist. Index cases were screened for mutations in the SGCE, DYT1, and DRD2 genes and for deletions of the SGCE gene. Suitable mutation negative families were tested for linkage to the SGCE region and to chromosome 18p11. Results: Two SGCE mutations were detected among the seven familial but no mutation in the sporadic cases. Haplotype analysis at the new M-D locus was compatible with linkage in two families and excluded in another family, suggesting at least one additional M-D gene. There were no obvious clinical differences between M-D families with and without detected mutations. Conclusion: M-D is genetically heterogeneous with SGCE mutations accounting for the disease in only part of the clinically typical cases.

Mode of Inheritance and Susceptibility Locus for Restless Legs Syndrome, on Chromosome 12q

To the Editor: We read with interest the report by Desautels et al. (2001), who have described a susceptibility locus for restless legs syndrome (RLS), on chromosome 12q, in a family with putative autosomal recessive inheritance of RLS. RLS is a movement disorder characterized by a desire to move the extremities, often associated with motor restlessness, paresthesias/dysesthesias, worsening of symptoms at rest with at least temporary relief by activity, and worsening of symptoms in the evening or night (Walters 1995). A positive family history can be found in >40% of the idiopathic cases. Most reports of familial cases, as well as twin studies, suggest autosomal dominant transmission (Winkelman et al. 2001) with high penetrance (Trenkwalder et al. 1996; Lazzarini et al. 1999; Ondo et al. 2000). To evaluate the role of the described chromosome 12q locus for RLS, we ascertained two large South Tyrolean families (E and LA) with clinically definite RLS. Inheritance followed a classic pattern of autosomal dominant transmission. Pedigrees of the families are shown in figure 1. The diagnosis was established according to the criteria of the International Restless Legs Syndrome Study Group (Walters et al. 1995). Genomic DNA was isolated from 51 family members (family E includes 9 [7 female and 2 male] affected individuals, with mean age at onset 31±7 years; family LA includes 10 [7 female and 3 male] affected individuals, with mean age at onset 37±9 years). Genotyping of the following DNA markers that span the candidate region containing the recently described locus on chromosome 12q was performed on an automated-sequencing machine (Li-Cor): D12S1064 (95.03 cM), D12S1044 (96.54 cM), D12S393 (104.12 cM), and D12S78 (111.87 cM). The marker-map positions are based on the sex-averaged maps from the Center for Medical Genetics, Marshfield Medical Research Foundation. Linkage analysis was conducted using the FASTLINK (Schaffer et al. 1994) and VITESSE programs (O’Connell and Weeks 1995). For each family, we considered both a dominant model and a recessive model for RLS. For the former, we used a conservative, affecteds-only model and a disease-allele frequency of 0.001. For the latter, we adopted the model parameters used by Desautels et al. They also used an affecteds-only model, but they incorporated a disease-allele frequency of 0.25 and a high phenocopy rate of 0.80. Figure 1 Pedigrees of families E and LA, with haplotypes for the four markers that span ∼13 cM on chromosome 12p. The order of markers is indicated to the left of each generation. Individuals affected with RLS are denoted by solid symbols, those with possible ... In both of the families that we studied, linkage was unambiguously excluded using a dominant model (multipoint LOD scores across the region ranged from −2.46 to −6.67 in family LA and from −1.61 to −5.14 in family E). In the larger family (LA), linkage was also excluded using the recessive model (multipoint LOD scores across the region ranged from −2.16 to −4.23). In family E, we obtained no positive evidence for linkage by use of the recessive model (multipoint LOD scores across the region ranged from −0.51 to 0.47). With nine affected individuals, the maximum potential LOD score for this family is at least 2.5. The recessive model suggested by Desautels et al. requires a disease-allele frequency on the order of a common polymorphism. Furthermore, they specify a genotype-specific penetrance value of 0.80 for f0, which represents the probability that homozygous normal individuals (i.e., non–disease-allele carriers) are affected—or, more simply, the phenocopy rate. For relatively common diseases, the population prevalence of the disorder is often used as an estimate of f0. A phenocopy rate of 80% far exceeds the population prevalence (2%–10%) of idiopathic and secondary RLS combined. We reproduced the results from Desautels et al., by analysis of the same family that they studied and by use of their marker data and model parameters. We then specified lower (but still generous) rates of 10% and 20% and obtained lower LOD scores. Specifically, the highest two-point LOD score was 0.35 (s=0.0), at D12S1300, by use of the 20% phenocopy model. Although RLS is not a rare disease, its prevalence in the general population does not reach the proportions suggested by the model advocated for this family. The family studied by Desautels et al. is French Canadian and derives from a population that, at least historically, has been a genetic isolate. Families from such populations may be particularly useful for identifying disease and susceptibility genes for common disorders. Of interest in this family is the fact that two of the four married-in spouses were diagnosed with “probable” RLS and therefore were potentially homozygous disease-allele carriers (although they were considered as having “unknown” disease status in the linkage analysis). This “trilineality” may be the result of increased relatedness in this kindred, given their population history, and could strengthen Desautels et al.s results if further explored and documented. Desautels et al., however, have not discussed this matter in their article. In conclusion, we did not confirm the susceptibility locus for RLS, on chromosome 12q, in either of the families that we studied. We also question certain parameters used in the recessive model by Desautels et al., and we suggest that additional information on family structure may be useful in the search for RLS genes in this French Canadian family.

MDR1 variants and risk of Parkinson disease

The multidrug resistance protein 1 (MDR1 or ABCB1) gene encodes a P-glycoprotein that protects the brain against neurotoxicants. Certain MDR1 genetic variants are known to compromise the function of this transporter and may thus be associated with Parkinson disease (PD). We therefore conducted a large case-control study investigating the potential relationship between MDR1 variants and PD. We determined the frequency of three MDR1 variants in 599 European PD patients and controls and further stratified the population by ethnicity, age at onset, and exposure to pesticides. We detected no relevant association in either the entire sample, or when separately investigating by ethnic origin or age at onset. However, the distribution of c.3435C/T differed significantly between PD patients exposed to pesticides compared to those non-exposed (odds ratio = 4.74; confidence interval = [1.009; 22.306]); p = 0.047), suggesting that common MDR1 variants might influence the risk to develop PD in conjunction with exposure to pesticides.

Myoclonus-dystonia: Detection of novel, recurrent, and de novo SGCE mutations

Symptoms of myoclonus–dystonia (M-D; DYT11) affect mostly proximal muscles of the top half of the body, usually start during childhood or early adolescence, and are often responsive to alcohol.1 Psychiatric abnormalities such as depression have been reported in several families.2,3⇓ M-D follows a pattern of autosomal dominant inheritance but may also occur sporadically. Mutations in the e-sarcoglycan gene ( SGCE ) were shown recently to cause M-D in several families.3–7⇓⇓⇓⇓ The human SGCE gene is maternally imprinted, resulting in reduced penetrance when the mutation is inherited from the mother.6 We investigated four families with typical M-D (6 patients, 13 unaffected). The diagnosis of M-D was established according to the recently modified clinical criteria.1 In all families, symptoms predominantly affected the upper half of the body and were alcohol responsive in three families (not tested in Family C). Features of depression were reported in three index cases (Families B, C, and D). Family history was positive in two cases (Families C and D). Mutational analysis of the SGCE gene was performed by sequence analysis in all index cases (n = 4) and in available family members of three families (n = 15) as described (figure …