Pirkevi et al., Supplement 1
Supplementary data
Subjects
In this study, 78 PD patients [mean age at onset 39±14.13 years (12-69); mean disease duration 10±5.84 years (1-27)] and 63 of their relatives were screened; 47 were males and 31 were females; 52 (32 compatible with dominant and 20 with recessive inheritance) had a positive family history of PD; 26 were isolated cases; 12 index cases had consanguineous parents. Patients were recruited from several referral centers throughout Turkey, and they were evaluated by a neurologist specialized in movement disorders. All patients met the diagnostic criteria of clinically definite PD: at least two of the triad of signs (akinesia, rigidity, resting tremor), >30% improvement with levodopa at least at onset, and absence of exclusion criteria. Dominant inheritance was defined by the presence of at least two affected cases in two successive or two different generations; and families with at least two affected siblings in only one generation were compatible with a recessive pattern of inheritance. The ethics committee of each participating institution approved this study, and all subjects gave an informed consent.
Clinical data of the index patient (PD136)
The 70 year old index case experienced left-sided resting leg tremor at the age of 60. PD diagnosis was made according to UK brain bank criteria. In ten years, she developed an asymmetric bilateral moderate bradykinesia, resting tremor, and rigidity more prominent on her left side. Her UPDRS score is 35, her symptoms still respond to the dopaminergic treatment, but she developed a “wearing off” phenomenon (predictable “off”) and has non-disturbing mild and inconsistent dyskinesias. She has no autonomic involvement, and her cognition is intact. She has minor depression according to DSM-IV. Her SMMT score is 28. She is prescribed L-Dopa/carbidopa/entecapone (500mg/125mg/1000mg)/day and pribedil 150 mg/day.
Screening for the G2019S mutation
DNA extraction from blood leucocytes was performed with MagNA Pure Compact Versatile Nucleic Acid Purification System (Roche Diagnostics, Switzerland). The presence of the G2019S mutation was analyzed in 78 Turkish PD patients and their 63 blood-related relatives by direct sequencing of LRRK2 exon 41, using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA), according to manufacturer’s recommendations. Sequencing products were loaded on an ABI 3730 automated sequencer and analyzed with SeqScape (Version 2.5) software (Applied Biosystems, Foster City, CA).
Haplotype analysis
Using 4 microsatellite markers and 14 SNPs on chromosome 12, spanning 243kb flanking the G2019S mutation [3, 4], the haplotype associated with the G2019S mutation was manually reconstructed in the index patient (PD136) and her clinically unaffected daughter (PD253). In addition to the other two haplotypes already described [3, 4, 6], the haplotype associated with G2019S in Japan was reconstructed with two Japanese patients (1107 and 1108), carrying the G2019S mutation. Genotyping and haplotype analysis were performed as previously described [8].
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