After obtaining informed consent, 132 PD patients underwent a standardized neurological examination by a movement disorder specialist. The diagnosis of PD was based on the UK Brain Bank diagnostic criteria (family history was not used as an exclusion criterion) and those published by Gelb et. al. [2
]. Family history was considered positive if parkinsonism was reported in at least a first- or second-degree relative. Collection of these 132 patients was performed at the Movement Disorder Clinics of both the University of Coimbra Hospital and the Lisbon Hospital Center – Center Region EPE in Lisbon, in a consecutive manner, all patients consent to participate. This cohort is identical to that previously described by us except for the inclusion of 4 additional PD patients [15
]. From this series of 132 subjects we have selected 66 unrelated patients to include only those with a positive family history for parkinsonism, or early-onset disease (age at onset <50 years of age). The remaining 66 patients failed to meet either of these criteria, were related to a proband already included or had previously been found to carry the LRRK2
c.6055G > A; p.G2019S mutation (n = 11). This selection led to the inclusion of 39 patients with positive family history and 46 patients with early-onset PD; 19 patients presented with both an early-onset phenotype and a positive family history, thus the net number of patients from both inclusion groups is 66 (Table ).
Features of patients studied, where more than one affected member was identified in a family only the presenting affected member (proband) is included.
Additionally we have included a control group comprised of 126 healthy subjects as previously described [15
]. Briefly, this control group consisted primarily of spouses accompanying patients to the clinic (~80%); the remaining controls were recruited from non-neurology outpatient clinics, after observation by the movement disorders specialist. This series presented a mean age of 60.5 ± 23.1 years. Apart from the spouses of the patients, no other familiarity with movement disorders patients was found. All individuals are Caucasian and of apparent Portuguese ancestry.
Genomic DNA was extracted from peripheral blood using standard methods. We screened the genes SNCA, PRKN, PINK1 and LRRK2 for sequence variants and, with the exception of LRRK2, for genomic copy number variants. The reference sequence used for the PRKN gene throughout this paper is based on the accession number NM_004562 and codon counting starts from the first ATG.
, all exons were polymerase chain reaction-amplified and sequenced in both directions using BigDye chemistry (Applied Biosystems, Foster City, CA) on an ABI 3100 Genetic Analyzer as previously described [7
]. While for the LRRK2
gene, only exon 41 was screened for mutations, using conditions previously described [15
Gene dosage analysis was performed using the ABI 7900 Sequence Detection System. Exons 1,2, 4–9 and 11–12 of PRKN and exons 1 and 2 of SNCA, as well as the complete coding region of PINK1 were individually co-amplified with β-globin, which served as an endogenous reference gene. Each plate contained six replicates of every genomic DNA sample, control DNA, and a no-template water control. The cycle in the log phase of PCR amplification at which a significant fluorescence threshold was reached (Ct) was used to quantify each exon relative to β-globin. The dosage of each exon relative to β-globin and normalized to control DNA was determined using the 2-ΔΔCt method (Applied Biosystems, Foster City, CA).