Jennifer Hussey

Lewy bodies and parkinsonism in families with parkin mutations

Previous work has established that compound mutations and homozygous loss of function of the parkin gene cause early‐onset, autosomal recessive parkinsonism. Classically, this disease has been associated with loss of dopaminergic neurons in the substantia nigra pars compacta and locus ceruleus, without Lewy body pathology. We have sequenced the parkin gene of 38 patients with early‐onset Parkinsons disease (<41 years). Two probands with mutations were followed up. Clinical evaluation of their families was performed, blinded to both genetic and pathological findings. Chromosome 6q25.2‐27 haplotype analysis was carried out independently of the trait; parkin gene expression was examined at both the RNA and protein levels. Haplotype analysis of these families revealed a common chromosome 6, with a novel 40 bp exon 3 deletion that cosegregated with disease. In the proband of the smaller kindred, an exon 7 R275W substitution was identified in addition to the exon 3 deletion; RNA analysis demonstrated that the mutations were on alternate transcripts. However, Lewy body pathology typical of idiopathic Parkinsons disease was found at autopsy in the proband from the smaller kindred. These data suggest that compound heterozygous parkin mutations and loss of parkin protein may lead to early‐onset parkinsonism with Lewy body pathology, while a hemizygous mutation may confer increased susceptibility to typical Parkinsons disease.

Sensitization of Neuronal Cells to Oxidative Stress with Mutated Human α‐Synuclein

Abstract: Linkage of α‐synuclein (α‐SN) mutations tofamilial Parkinsons disease (PD) and presence of α‐SN as a majorconstituent of Lawy body in both sporadic and familial PD implicate α‐SNabnormality in PD pathogenesis. Here we demonstrate that overexpression ofwild‐type or mutant α‐SN does not cause any deleterious effect on thegrowth or continued propagation of transfected human cells, but overproductionof mutant α‐SN heightens their sensitivity to menadione‐inducedoxidative injury. Such enhanced vulnerability is more pronounced in neuronaltransfectants than in their nonneuronal counterparts and is associated withincreased production of reactive oxygen species. The data suggest that mutatedα‐SN, especially with an alanine‐to‐proline substitution at residue 30,sensitizes neuronal cells to oxidative damage.

SCA2 may present as levodopa-responsive parkinsonism

Some kindreds with familial parkinsonism exhibit genetic anticipation, suggesting possible involvement of trinucleotide repeat expansion. Recent reports have shown trinucleotide repeat expansions in the spinocerebellar ataxia 2 (SCA2) gene in patients with levodopa‐responsive parkinsonism. We tested 136 unrelated patients with familial parkinsonism for SCA2 mutations. Two probands had borderline mutations; the rest were normal. (≤31 repeats is normal, 32–35 is borderline, ≥36 is pathogenic). The expanded allele segregated with neurological signs in one kindred. The absence of borderline mutations in the normal population, and the co‐segregation of the expanded allele with neurological signs in one kindred suggest that SCA2 mutations may be responsible for a subset of familial parkinsonism.

SCA-2 presenting as parkinsonism in an Alberta family Clinical, genetic, and PET findings

The authors describe an Alberta family with levodopa-responsive parkinsonism without cerebellar abnormalities. Genetic testing showed expanded repeats for SCA-2; other mutations for parkinsonism were excluded. The expanded allele shows interruption of the CAG repeat with CAA. PET in two affected members showed reduced fluorodopa uptake in striatum and normal raclopride binding. Families with autosomal dominant, levodopa-responsive parkinsonism should be tested for the SCA-2 mutation.

Ethnic differences in the expression of neurodegenerative disease: Machado-Joseph disease in Africans and Caucasians

We describe several families of African origin with SCA3/Machado‐Joseph disease gene expansions. In these cases, the phenotype ranges from ataxia with parkinsonian signs to a syndrome clinically almost indistinguishable from idiopathic, L‐dopa–responsive Parkinsons disease. In contrast, these parkinsonian phenotypes are rare in those of European descent. Haplotype analysis shows that these African families do not share a common founder, thus a cis‐acting element in the promoter is unlikely to be responsible these unusual presentations. We suggest that trans‐acting factors are responsible for the variable phenotype and discuss the implications of diseases showing racially different expressivities.

Transfected synphilin-1 forms cytoplasmic inclusions in HEK293 cells ☆

The discovery of mutations in the gene for alpha-synuclein in familial Parkinsons disease (PD) has led to an increased interest in this pre-synaptic protein. Synphilin-1, a potential synuclein-binding protein, was cloned using yeast two-hybrid assays. The function of synphilin-1 is currently unknown, although it has been reported to be present along with alpha-synuclein in Lewy bodies in PD. In the present study, we monitored synphilin-1 aggregation directly using fusion proteins of synphilin-1 and green fluorescent protein (EGFP). Transfection of synphilin-EGFP fusion proteins formed cytoplasmic inclusions in HEK293 cells. Although these inclusions overlapped with the distribution of alpha-synuclein, they were unlike Lewy bodies in that they were not eosinophilic, and instead were membrane-bound, lipid-rich cytoplasmic inclusions.

Clinical, 18F-dopa PET, and genetic analysis of an ethnic Chinese kindred with early-onset parkinsonism and parkin gene mutations.

We report on clinical 18F‐labeled 6‐fluorodopa (18F‐dopa) positron emission tomography (PET) and molecular genetic analyses of an ethnic Chinese family in which three siblings presented with early‐onset Parkinsons disease. As described in some parkin patients, neither sleep benefit nor diurnal fluctuation was noted. Interestingly, depression, anxiety, and obsessive–compulsive disorders were manifest. The 18F‐dopa PET scans showed bilateral presynaptic dopaminergic dysfunction without marked lateralization. Molecular genetic analysis showed identical chromosome 6 haplotypes inherited by affected subjects, with alternate allelic deletions of parkin exons 3 and 4. Furthermore, mRNA analyses identified aberrantly spliced parkin transcripts, suggesting that unusual parkin protein isoforms may be expressed in the brain and retain some function.

Parkin-proven disease Common founders but divergent phenotypes

Objective: To compare and contrast clinical and genetic findings in six probands with parkinsonism with a parkin exon 3 438- to 477-bp deletion (Ex3Δ40) to search for evidence of a common founder. Method: Clinical review, parkin gene sequencing, dosage studies, and high-resolution genotype/haplotype analysis were performed. Results: All subjects had two or more signs consistent with a diagnosis of possible or probable PD with age at onset younger than 45 years (mean ± SD 29.3 ± 10.2 years, range 16 to 42 years). Affected individuals were either homozygotes, compound heterozygotes, or Ex3Δ40 carriers with one normal parkin allele. Haplotype analysis revealed both Ex3Δ40 and Ex7 924 C→T (R275W) mutations originated from common founders, the former most probably of Irish descent. Although three cases had Ex7 924 C→T (R275W) and Ex3Δ40 mutations, their clinical presentation and mode of inheritance were variable. Conclusion: Parkin mutations on common parkin haplotypes provide testable hypotheses of parkin function in genetically defined parkinsonism.

Accurate determination of ataxin-2 polyglutamine expansion in patients with intermediate-range repeats.

Spinocerebellar ataxia, type 2 (SCA2), results from an expansion of a stretch of polyglutamine repeats within the coding sequence of the ataxin-2 gene (ATX2), localized to chromosome 12q23-24. Recent studies have widened the clinical phenotype, notably for individuals with repeats of intermediate size, from 32 to 35 glutamine residues. This narrow range necessitates precise determination of repeat size. Diagnostic laboratories most often perform direct genotyping of ATX2 from polymerase chain-amplified patient DNA with subsequent sizing utilizing slab gel polyacrylamide gel electrophoresis (PAGE) or capillary electrophoresis. Using cloning and sequencing methods, we have constructed a ladder of ATX2 alleles of known size and sequence composition. This freely available size ladder will facilitate future quantification of expansions of the ATX2 locus.

A multi-incident, Old-Order Amish family with PD

BackgroundPD is largely a sporadic condition of unknown etiology, but specific inherited mutations are a cause of PD. ObjectiveTo describe a large, multi-incident Amish pedigree with PD. MethodsCase ascertainment, calculation of population prevalence, and calculation of kinship coefficients (a measure of relatedness between two individuals) for affecteds and subjects in a large kindred with PD were conducted. Sequencing of genes with known mutations sufficient to cause PD and marker-by-marker haplotype analysis in chromosomal regions flanking previously described genes with known mutations were performed. ResultsThe authors have examined 113 members of this pedigree and classified 67 as normal (no evidence of PD), 17 as clinically definite PD, 6 as clinically probable PD, and 23 as clinically possible PD. The mean age at onset of the clinically definite subjects was 56.7 years. The phenotype in this family is typical of idiopathic PD, including rest tremor, rigidity, bradykinesia, postural instability, and response to levodopa. In addition, dementia occurred in six of the clinically definite subjects, and many subjects experienced levodopa-related motor complications including wearing off and dopa-induced dyskinesias. In the index Amish community, a minimum prevalence of PD in the population 40 years and older of 552/100,000 was calculated. The mean kinship coefficient in the subjects with PD and those with PD by history (0.036) was higher (p = 0.007) than in a group of age-matched normal Amish control subjects (0.016), providing evidence that PD is inherited in this family. Sequence analysis did not detect any mutations in known PD genes. No single haplotype cosegregates with the disease in any of the chromosomal regions previously found to be linked to PD, and no marker in these regions exhibits increased homozygosity among definite PD cases. ConclusionsPD in this community is more common than in the general population, and this increased prevalence may be due in part to a novel gene(s).