Statistical analysis

Deviations of SNP genotypes from Hardy Weinberg equilibri- um (HWE) in controls were assessed using chi-square tests, while linkage disequilibrium (LD) between SNPs in controls was assessed using r2 values. In evaluation of the primary study aim, we used logistic regression models adjusted for gender to examine whether the proportion of carriers of the minor allele for each SNP varied across age in controls, where age was considered as a continuous variable. After examination of genotype frequencies in the overall group of controls, we chose to consider each SNP under a dominant model (i.e. presence or absence of the minor allele) owing to the relatively small number of homozygotes of the minor allele for many of the SNCA SNPs. We also divided age into categories, and estimated the proportion of control carriers of the minor allele in each age category. When utilizing the overall case- control series, single SNP associations with PD were evaluated using logistic regression models adjusted for age and gender under a dominant model. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated. Owing to the fact that the primary analysis comparing the frequency of SNCA variants across age is exploratory in nature in that this has not been evaluated before, no adjustment for multiple testing was made, and p-values of 0.05 or less were considered as statistically significant. All statistical analyses were performed using R statistical software (version 2.11.9; R Foundation for Statistical Computing, Vienna, Austria).

We speculated that variation in age-related disease genes such as SNCA may not only affect morbidity, but also influence particular aspects of successful aging. More specifically, in addition to its role in PD, we hypothesized that variation in SNCA may be associated with neurological disease in general and longevity. Therefore, the primary aim of this exploratory study was to evaluate the association between common SNCA single nucleotide polymorphisms (SNPs) and survival without neurological disease.

Methods and Subjects

Ethics statement

The Mayo Clinic institutional review board approved all work. All participants provided written informed consent.

Subjects

aging as measured by survival without neurological disease, we utilized a series of 769 controls without neurological disease seen at Mayo Clinic Florida. Unrelated controls were deemed to be free of neurological disease by a neurologist, and did not have a family history of neurological disease. Mean age in the controls was 79611 SD years (Range: 33–99 years), and 385 (50%) were male. All controls were white and of European ancestry. Controls were mostly unrelated spouses/caregivers of neurological patients, and a subset of these controls are included in our aging sibpair study [20]. Utilizing this group of controls, we evaluated the association of SNCA SNPs with age, where since the probability of living longer without neurological disease increases as age without neurological disease increases, age in these controls is a measure of survival without neurological disease.

Results

There was no evidence of a departure from HWE for any of the SNPs (all P.0.05). LD between SNPs is summarized in Figure 1; there was moderate to strong LD between a number of SNCA SNPs, with many r2 values of 0.30 or greater.

allele in controls is displayed in Table 1 for seven different age categories: ,60 years, 60–69 years, 70–74 years, 75–79 years, 80– 84 years, 85–89 years, and $90 years. These age categories were chosen with the goal of displaying trends in as detailed a manner as possible, while maximizing the number of controls in each category. As seen in Table 1, there was a significant decline in the proportion of carriers of the minor allele of rs10014396 as age increased (P= 0.021), where 30% of controls younger than 60 were carriers, compared to 14% of those 90 years of age and older. Results were similar for rs3775439, which was in strong LD with rs10014396 (r2 = 0.84); the proportion of carriers of the minor allele decreased from 32% in controls younger than 60 to 19% in those 90 or older (P= 0.025). For all remaining SNCA SNPs, the proportion of carriers of the minor allele did not noticeably increase or decrease as age increased in controls (all P$0.19).

We supplemented the healthy control series with 426 PD cases also seen at Mayo Clinic Florida, in order to examine associations of SNCA SNPs with PD [21]. Mean age of PD cases was 72611 SD years (Range: 30–92 years), mean age at PD onset was 62612 SD years (Range: 26–85 years), and 233 (55%) were male. All PD cases were white and of European ancestry, and were examined and observed longitudinally by a neurologist and diagnosed with PD according to published criteria [22]. There are no known related samples within or between the diagnosis groups. All subjects self-identified ethnicity.

In Table 2 we present single SNP associations with PD in the entire series of 426 PD cases and 769 controls. Of particular interest, rs10014396 and rs3775439, both of which were associated with survival without neurological disease in controls, were not associated with PD (ORs 0.92 & 0.95, P$0.58). Significant protective associations in relation to PD were observed for rs1372519 (OR: 0.76, P= 0.034) and rs1372520 (OR: 0.76, P= 0.039). For the remaining SNCA SNPs, a number of significant

Genetic analysis

DNA was extracted from whole blood samples using standard protocols. Conserved regions (conservation score .200) were identified across SNCA (coding regions 610 Kb) using the phastConst software embedded in UCSC Genome Browser (http://genome.ucsc.edu), based on the NCBI March 2006 assembly [23]. The conserved SNP approach was used to highlight