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4.4 STUDIES IN FAMILIAL PD AND MULTIPLE SYSTEM ATROPHY
In collaboration with the Parkinson’s Disease Society Brain Research Centre, 1 was
supervised by Dr S Daniel (SED), and examined the substantia nigra in a series of 20
cases of pathologically proven familial PD. In addition, cortical sections from 10 cases
of pathologically proven MSA (4 OPCA, 6 SND) were examined in order to evaluate
the specificity of neuropathological features in familial PD and the corresponding expression of a-synuclein. The average age of onset of familial PD was 64 (range 44-77 years) and in the MSA cases it was 55 (48-67). Details of family history of PD in most cases were by history and the criteria for inclusion as “a family history” was the presence of PD in a closely affected relative (Parent/sibling/cousin/aunt/uncle). Six cases of MSA were classified histologically as striato-nigral degeneration (SND) and four as olivoponto cerebellar atrophy (OPCA). Alpha-synuclein staining of Lewy bodies was positive in all the cases of familial PD. The GCls in all 10 cases of MSA
were also positive for a-synuclein protein as originally described elsewhere (Tu et al.,
1998). All cases were screened for the two coding mutations G209A and G8 8C in
SNCA in 30 cases by restriction digestion of PCR products as described above. The
coding region of 4 of the cases of pathologically proven MSA were sequenced by another investigator (SD) as part of this project but no pathological mutations were detected (see appendix 2 for sequencing methodology). Figure 4.3 below shows a- synuclein staining of Lewy bodies and Lewy neurites in sections of brain from a PD patient in this study. Methodology for immunostaining is described in appendix 4.3, as this was done by SED and Dr Ann Kingsbury of the PDS Brain Bank.
Fig. 4.3: Alpha-synuclein staining of a) Lewy bodies, and b) Lewy neurites found in human PD brain. Peroxidase detection of a rabbit polyclonal anti-synuclein antibody forms a brown deposit at the site of positive antibody binding.
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4.5 CLINICO-GENETIC STUDIES OF THE PARKIN GENE IN A LARGE EU SERIES
Two studies (study 1 and study 2) were perfomed with the author (myself) as a clinical
collaborator to investigate the numerical importance of Parkin (Kitada et al, 1998a) in
a central European population. Study 1 was an initial study performed as soon as Parkin
was cloned (Kitada et a l, 1998a). Study 2 was performed later on a larger familial
series of patient and also included sporadic and juvenile-onset cases (Luecking et a l,
2000).
4.5.1 Study lithe first study of Parkin in an EU series with PD
The study described below is briefly summarized and the sequencing results included for discussion purposes, as it was a clinical collaborative study involving all the EU partners (including myself in the UK). Sequencing of the Parkin gene in this study,
however, was done solely by Dr N Abbas, INSERM, Paris (Abbas et a l, 1999). From
the GSPD collection of affected sibling pairs, thirty-eight families were selected according to the following criteria: i) presence of parkinsonism with good response to L-dopa (>30% improvement) in at least 2 siblings, and absence of excluding criteria
such as extensor plantar reflexes, ophthalmoplegia, early (after 2 years of disease
evolution) dementia or autonomic failure; ii) onset < 45 years in at least one of sibling; iii) inheritance compatible with autosomal recessive transmission (several patients in a single generation with or without known consanguinity). The families originated from France (n=12), Italy (n=10), Germany (n=7). Great Britain (n=4), Algeria (n=l), Morocco (n=l). The Netherlands (n=l), Portugal (n=l), Vietnam (n=l). Four families from Algeria, France, Italy and Portugal were excluded from sequence analysis because they were found to carry homozygous deletions of either exons 3 or 8-9 (18) or exon 4 (family IT-005).
The patients and unaffected relatives were examined by the designated GSPD investigator in each country (myself in the UK) according to a standardized protocol using the agreed inclusion and exclusion criteria detailed in section 2.2. All patients were videotaped and the clinical data were centralized. Blood samples were taken with informed consent from the patients and their first degree relatives. In order to determine the frequency and diversity of mutations in the Parkin gene as a cause for the AR-JP phenotype in Europe, the 12 coding exons of the Parkin gene were amplified in 35
families with autosomal recessive early onset Parkinsonism (see appendix 2.2 and 4.2). Patients were only sequenced if screening for homozygous deletions in Parkin proved negative. Only the results of the UK families found to be positive for a mutation in the Parkin gene are shown in detail below. Figure 4.5 below shows pedigrees for all UK families with mutations in Parkin from both studies. A clinical summary amd composite figure of the mutations detected in Parkin in the whole of the EU series of which this family was a part (table 4.10, figure 4.9) is given for discussion purposes only below as well as.
4.5.2 Clinical phenotype of first UK kindred (UK 086) described with a Parkin mutation
11:1 This case was first seen aged 60 years of age with a one year history of gait
disturbance, left- sided bradykinesia, rigidity and loss of arm swing and micrographia.
Since the age of 53 she had been taking Procyclidine for “incoordination & tremor”
from her GP. A CT head scan at the time was reported as normal as was a
caeruloplasmin level (to exclude Wilson’s disease, a rare but important differential diagnosis of young-onset parkinsonism). At the time of diagnosis of “idiopathic PD” she was started on Sinemet LS. Eighteen months later she began to complain of on-off fluctuations. She developed marked dyskinesias 30 months after her original diagnosis. Surgery for the severe dyskinesias has been declined. Clinically her reflexes were normal.
11:2 The brother of individual II. 1 developed an asymmetrical right-sided resting and
postural tremor at the age of 42. Ten years later, when first clinically examined by
myself, he had developed bilateral signs and described a 2 year history of a shuffling
gait and freezing when walking. His illness had always been exquisitely L-dopa
responsive (Madopar 125mg TDS) and characterised by early onset marked fluctuations and a non-painful foot dystonia. Clinically his reflexes were normal.
1:1 The father (not clinically examined) was said to be asymptomatic as was the mother
4.5.3 Results o f Parkin sequencing in UK 086
A pedigree for this family (UK 086) and details of the mutation are given in Figure 4.4.
F ig u re 4.4: Pedigree of ‘Parkin’ family UK086, the first UK family to show linkage to PARK2. Sequencing of the Parkin gene in this family revealed a heterozygous
Gln34Arg change in affected family members as shown.