e-Methods

Subjects

26 patients/subjects were recruited from the St Olav's University Hospital, Trondheim, Norway, the Sagamihara National Hospital, Kanagawa, Japan, the Mayo Clinic Florida (MCF), Jacksonville, Florida, USA, and the Veterans Affairs Puget Sound Health Care System & University of Washington School of Medicine, Seattle, Washington, USA. Recruitment of cases from the St Olav's University Hospital and the Sagamihara National Hospital was coordinated by staff at MCF. Participants with positive LRRK2 mutation status were recruited together with non-mutation carriers from participating families to allow for blinding of participants who did not wish to know their carrier status. The data on non-mutation carriers are not included in the present study, but were similar to what was reported in other cohortse1. All subjects have been included in a longitudinal study, dating back to 1997, with cerebrospinal fluid (CSF) samples taken between May 2005 and March 2010 in this study. All individuals provided informed consent and underwent an evaluation that consisted of a structured interview, neurological examination, laboratory tests, and neuropsychological assessments. All Parkinson disease (PD) patients met the UK PD Society Brain Bank clinical diagnostic criteria for PDe2, except that having "more than one affected relative" was not considered an exclusion criterion. Demographic and clinical information for all subjects is listed in Table e-1. The mutation status of the subjects is also shown in Table e-1: 11 carried the G2019S mutation in the LRRK2 gene, 4 carried the I2020T mutatione3, 5 carried the N1437H mutatione4, 5 carried the R1441C mutatione5, and 1 carried the R1441H mutation. Notably, among 11 subjects with G2019S mutations, 2 were homozygotes; no differences in clinical phenotypes were observed between the homozygote and heterozygote carrierse6. The study was approved by the Institutional Review Boards of all participating institutions.

Positron emission tomography

Within one year of CSF sample collection, all subjects traveled to the Pacific Parkinson’s Research Centre affiliated with the University of British Columbia, Vancouver, BC, Canada for positron emission tomography (PET) scans. For PD patients, all antiparkinsonian medications were stopped at least 12 h before each assessment. Each subject was scanned with three tracers, i.e., 18F-6-fluoro-L-dopa (FD, a marker for uptake and decarboxylation of levodopa as well as the trapping of dopamine in synaptic vesicles), 11C-()--dihydrotetrabenazine (DTBZ, a vesicular monoamine transporter 2 [VMAT2] ligand), and 11C-d-threo-methylphenidate (MP, a dopamine transporter [DAT] ligand), in three-dimensional mode with an ECAT 953B/31 tomograph (CTI/Siemens, Knoxville, TN, USA) or a GE Advance scanner (General Electric, Milwaukee, WI, USA) according to previously described standard procedurese7. The reconstructed resolution and contrast recovery were carefully matched between the two scanners using phantom studies. The methods of image analysis to determine reference tissue input-derived FD uptake rate constant (kocc) and DTBZ and MP tissue input binding potentials (BPND_DTBZ and BPND_MP) have been described in detail elsewheree7.

CSF samples and Luminex assays

CSF samples were obtained by lumbar puncture as previously describede8. Similar CSF collection and quality control procedures (such as use of polypropylene collection and storage tubes, and rapid aliquotting and freezing of CSF samples) were followed at the four participating centers (the St Olav's University Hospital, the Sagamihara National Hospital, the Veterans Affairs Puget Sound Health Care System & University of Washington School of Medicine, and the University of British Columbia) to minimize potential site variations. The hemoglobin levels in CSF samples were chosen as an index of the degree of red blood cell contamination of CSF and were measured as previously described8. CSF A42, t-tau, and p-tau levels of all cases were measured at the University of Washington School of Medicine using the INNO-BIA AlzBio3 kit (Innogenetics, Gent, Belgium) as previously describede1. All samples were run in a single plate, with multiple quality control procedures in place as describede1.

Statistical analysis

All analyses were performed with PASW Statistics 18.0 (SPSS Inc, Chicago, IL, USA). Because the PET data produced a skewed distribution and a Gaussian distribution could not be induced through transformation of the variables, non-parametric correlation methods (Kendall rank correlation) were used to assess the relationship between the PET measurements and the CSF biomarkers. In these non-parametric correlation analyses, subjects are ranked and the impact on the level of statistical significance from outliers is minimal. Values with P< 0.05 were regarded as significant in this exploratory study given the small sample size.

e-References

e1.Shi M, Bradner J, Hancock AM, et al. Cerebrospinal Fluid Biomarkers for Parkinson Disease Diagnosis and Progression. Ann Neurol 2011;69:570-580; published online October 528, 2010 (doi:2010.1002/ana.22311).

e2.Gibb WR, Lees AJ. The relevance of the Lewy body to the pathogenesis of idiopathic Parkinson's disease. J Neurol Neurosurg Psychiatry 1988;51:745-752.

e3.Hasegawa K, Stoessl AJ, Yokoyama T, Kowa H, Wszolek ZK, Yagishita S. Familial parkinsonism: study of original Sagamihara PARK8 (I2020T) kindred with variable clinicopathologic outcomes. Parkinsonism Relat Disord 2009;15:300-306.

e4.Aasly JO, Vilarino-Guell C, Dachsel JC, et al. Novel pathogenic LRRK2 p.Asn1437His substitution in familial Parkinson's disease. Mov Disord 2010;25:2156-2163.

e5.Wszolek ZK, Pfeiffer RF, Tsuboi Y, et al. Autosomal dominant parkinsonism associated with variable synuclein and tau pathology. Neurology 2004;62:1619-1622.

e6.Ishihara L, Warren L, Gibson R, et al. Clinical features of Parkinson disease patients with homozygous leucine-rich repeat kinase 2 G2019S mutations. Arch Neurol 2006;63:1250-1254.

e7.Nandhagopal R, Mak E, Schulzer M, et al. Progression of dopaminergic dysfunction in a LRRK2 kindred: a multitracer PET study. Neurology 2008;71:1790-1795.

e8.Hong Z, Shi M, Chung KA, et al. DJ-1 and alpha-synuclein in human cerebrospinal fluid as biomarkers of Parkinson's disease. Brain 2010;133:713-726.

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