Polygenic risk of P arkinson disease is correlated with disease age at onset

1The identification of rare highly penetrant mutations in genes causing familial and early onset Parkinson disease (PD)1, 2, 3, 4, 5 has considerably improved our understanding of disease pathogenesis. Recently, our understanding of idiopathic PD has been enhanced by genome‐wide association (GWA) studies6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 that have collectively identified PD risk variants at >18 loci.6, 7 Despite their high levels of significance, these 18 loci are thought to account for only a very small amount (3–5%) of the expected heritability of PD.17 GWA study data sets can be used to determine a polygenic contribution of common single nucleotide polymorphisms (SNPs) that show disease association but fail to meet the significance threshold for genome‐wide significance (p > 5 × 10−8) to PD. Recent studies confirm that when weak effect loci are also considered17 there is a substantial increase in the estimated heritability detected in PD GWA studies (24%), and this strongly implies that a large proportion of genetic signal must lie below the genome‐wide significance thresholds set in the primary analyses.

2Polygenic score analysis tests whether the alleles of small effect that GWA studies are underpowered to detect confer an aggregate risk and whether the same sets of risk alleles are shared between cohorts/data sets.18 We have investigated the polygenic contribution to PD by assessing whether score alleles identified in a GWA study from the United Kingdom are significantly enriched in cases from 3 independent GWA studies.

3The age at onset (AAO) of PD has a relatively high heritability19 and has been previously shown to be associated with a small number of common variants,14, 20 some of which are strongly associated with disease.14 Using polygenic score analysis, we have considered PD according to a liability threshold model. Conceptually, liability is a quantitative measure that represents all risk factors that determine whether an individual will develop a disease. The liability threshold model assumes that individuals with a total liability greater than or equal to a fixed threshold will develop the disease. Mendelian forms of PD are caused by rare highly penetrant mutations at a single disease locus and are also typically associated with a young AAO. In line with a liability threshold model, a rare highly penetrant mutation is likely to contribute to a large proportion of an individual's disease liability. We propose that if an individual's common risk allele polygenic score is related to their disease liability we might expect patients carrying the highest load of common risk alleles to develop PD at the youngest ages. Conversely, if a large proportion of these cases are monogenic in nature, one might expect an attenuation of any relationship between polygenic score and AAO in the very young, supporting the notion that early onset PD has a substantial monogenic component.21, 22, 23

4Our study provides compelling evidence for a polygenic contribution to PD, and that an individual's polygenic score is correlated with age at disease onset. Importantly, this indicates that a liability threshold model is relevant to PD pathogenesis and that early onset forms of the illness are not limited to Mendelian subtypes.

5This study used data obtained from the meta‐analysis of 5 PD GWA studies (5,333 PD cases and 12,298 controls) of which 259,577 SNPs passed study‐specific quality controls in all studies.6 The summary statistics for each marker in the PD data set were obtained using fixed effect inverse variance weighted meta‐analyses with METAL software (http://www.sph.umich.edu/csg/abecasis/metal/). The AAO and individual genotypes were available for a total of 4,111 PD cases. For these patients, AAO was systematically determined at the time of inclusion by a retrospective interview and is defined as the age at which PD was first diagnosed.

6We followed the approach previously described by the International Schizophrenia Consortium.18 Essentially this involved the selection of a set of SNPs that were in relative linkage equilibrium (r 2 < 0.25) and the generation of additive polygenic risk scores using SNPs with increasingly liberal probability values in a GWA study discovery data set, which were then tested for enrichment within an independent test sample.

7In this study, we first investigated whether the polygenic score that was based on the PD GWA results of 1 data set were significantly enriched in the cases relative to the controls of another independent PD GWA study. For this analysis, we used 4 natural subsets of the International Parkinson's Disease Genomics Consortium (IPDGC) data where the individual genotypes of both cases (n = 4,294) and controls (n = 10,340) were available. After random pruning for linkage disequilibrium (LD; r 2 < 0.25), there were 59,770 SNPs available for polygenic score analyses. We used our most powerful PD case/control subset as our discovery sample (UK GWA study; 1,705 PD cases/6,200 controls) to select SNPs associated with PD, for each identifying its probability value for tests of allelic association, the effect size, and the allele that was present in the PD group more frequently than in the controls. We termed these the "score" alleles and further categorized them according to whether they met a predetermined significance threshold of association (p < 0.01, 0.05, 0.1, up to 0.5). We next used PLINK v1.07 to calculate the polygenic score for each individual in each of 3 independent case/control cohorts (USA‐1, 876 cases/859 controls; USA‐2, 971 cases/937 controls; Germany‐1; 742 cases/667 controls) as the average number of score alleles they possessed, each weighted by their effect size (B‐coefficient) log of odds ratio (OR) from the PD discovery sample. Logistic regression was then used to test whether the polygenic score distinguished case/control status in the 3 independent studies (USA‐1, USA‐2, and Germany‐1).

8To maximize the quality of the LD pruning, we considered it worthwhile to use the largest and most powerful PD GWA study available to us (5,333 cases and 12,298 controls). LD pruning (using r 2 > 0.25, a physical distance threshold for clumping SNPs of 250kb, and p = 1 as the significance threshold for SNPs, which allowed us to capture all SNPs, even if their association with PD was not significant) identified a set of 104,830 independent SNPs that retained those most significantly associated with the disease.

9The LD‐pruned set of SNPs were subsequently used for polygenic score analysis in the 4,294 PD patients for whom genotype data were available (UK, German, USA‐1, and USA‐2 GWA studies). Of these, AAO data were available in 4,111 patients (mean AAO = 60.9 years, standard deviation = 12.6), for whom we identified the score alleles and calculated their polygenic score. Polygenic scores were then adjusted for the country of origin using linear regression, and the residuals were then normalized by subtracting the mean and dividing by the standard deviation.

10To investigate the relationship between an individual's polygenic score for PD risk alleles and disease AAO, we initially used linear regression analyses of the entire data set. Because clinical estimates of the AAO of PD will inevitably have limited precision in predicting the start of an individual's underlying biological pathology, we also used chi‐square or, where appropriate, Fisher exact tests to compare the polygenic score between individuals with AAO at the extremes ends of the AAO distribution.

11In this study, we investigated whether the polygenic score alleles identified in 1 PD GWA study were significantly enriched in the cases relative to the controls of independent PD data sets. Polygenic score analysis revealed significant evidence for an overall enrichment of the PD score alleles identified in the UK GWA discovery sample14 in the cases of each of 3 independent PD GWA cohorts from the USA (×2) and Germany (Table 1).

12In accordance with the pattern seen in studies of other complex diseases shown to have a polygenic signal,18, 24, 25, 26 restricting our analysis to SNPs that met the lowest association test probability value thresholds (p T) in the discovery sample (p T < 10−4, p T < 10−3, p T < 0.01, p T < 0.05) did not identify a systematic significant inflation in the polygenic scores of the PD cases of the replication samples (p > 0.05). Rather, our most significant evidence was observed when SNPs with p T ≤ 0.5 in the UK sample were included where probability values for a significant inflation in the polygenic scores ranged between 4.42 × 10−4 and 8.22 × 10−5 (see Table 1). For all significant associations the B‐coefficients were positive, indicating that the higher polygenic score in the UK discovery sample corresponds to the higher score in each of the 3 independent replication samples and provides evidence for a polygenic contribution to the development of PD.

13To investigate a potential relationship between an individual's polygenic score and their AAO, we initially used linear regression of all 4,111 PD patients. This revealed nominally significant evidence that AAO was correlated with polygenic score but only when the analysis was restricted to SNPs with p T < 0.01 (Table 2). Closer inspection of the regression analyses revealed that although falling short of nominal significance the B‐coefficients were negative at all p T cutoffs, indicating that our data showed a consistent trend of higher polygenic score corresponding to an earlier AAO of PD.

14We recognize that imprecision in the clinical estimates of the AAO of PD could adversely affect the power of our regression analysis. We therefore next compared the polygenic scores of patients whose AAO was at the lower 5% (AAO < 40 years, n = 248) with those at the upper 5% (AAO ≥ 80 years, n = 196) of the AAO distribution. This revealed that patients with an AAO < 40 years had a significantly higher polygenic score (mean = 0.14, standard error [SE] = 0.076) than those with an AAO ≥ 80 years (mean = −0.05, SE = 0.059). Looking at the numbers of patients with higher polygenic score categorized by early versus late onset, we consistently observed this pattern (OR > 1) at all p T values and for all polygenic scores cutoffs > 0 (Fig 1). Our most significant result was when we compared patients with polygenic scores > 1.5 (p T = 0.2, p = 0.00014), which revealed that 33 (13%) of patients with a polygenic score > 1.5 had an AAO < 40 years, whereas only 6 (3%) had an AAO ≥ 80 years (OR = 4.8, relative risk [RR] = 4.3). Moreover, our data also revealed a consistent relationship between disease AAO and polygenic score at all p T thresholds.

15Relaxing our AAO threshold by ±5 years at either end of the AAO distribution demonstrated that although we consistently observed the same pattern at all thresholds of AAO, our strongest effects were seen when comparing the patients at the most extreme ends of the AAO distribution (Fig 2). It has previously been reported that the genetic structure in a population can be correlated with age.27 We investigated the possibility of this adversely affecting our results by performing an analogous analysis that compared the distribution of the PD score alleles between the oldest and youngest 5% (corresponding to <50 years and >87 years, respectively) of an independent cohort of Alzheimer disease (AD) patients (3,177 AD cases and 7,277 controls).28 This failed to identify a significant difference in the distribution of the PD score alleles between the 2 age groups (minimum p = 0.49, data not presented). We therefore conclude that the most likely explanation for our results being strongest when comparing PD patients at the most extreme ends of the AAO distribution is a reduction in power due to the inherent imprecision of relating age at diagnosis to a biological AAO and also that our selection of PD score alleles inevitably captures a proportion of SNPs that are not causal.

16Because the 104,830 independent SNPs that we used to identify PD score alleles included those most significantly associated with the disease, it is plausible that our results are being artificially biased by SNPs whose evidence for association is due to or merely a consequence of LD with the very strong association signal of known GWA study hits. To investigate this possibility, we repeated our analysis using identical analysis thresholds but this time excluding all 1,729 SNPs that after LD pruning were present at the 18 genomic regions previously reported to be strongly associated with PD, including the human leukocyte antigen locus.6 Given that each of these regions is likely to span at least 1 true PD susceptibility allele that would now be excluded from our polygenic score analysis, this approach is highly conservative. Nevertheless, this analysis again revealed significant evidence that individuals with higher polygenic scores had on average a lower AAO of PD, with our most significant result indicating the same magnitude of RR and OR between polygenic score and AAO (Fig 3). Moreover, we also obtained analogous results when we used an alternative method of LD pruning that ignored the strength to which SNPs were associated with PD and also excluded SNPs from the 18 associated regions (data not presented). These analyses suggest that our findings are not dependent on either the previously identified susceptibility loci or the SNPs that are falsely associated with PD merely as a consequence of LD with the very strong association signals.

17The molecular genetic data reported in this study provides strong support for a large polygenic contribution to the overall heritable risk of PD. This implies that the genetic architecture of PD includes many common variants of small effect and is likely to be reflected in a large number of susceptibility genes and a complex set of biological pathways relevant to the disease. The PD score alleles identified in this cohort are not significantly enriched (minimum p = 0.14) in an independent GWA study for AD,29 indicating that the polygenic component to PD that we have identified is disease specific. Moreover, to conduct our analysis we had to define a training GWA study and a series of replication data sets. We achieved this by splitting the IPDGC meta‐analysis by its original GWA studies and observed a similar pattern of results when we defined the PD score alleles in a sample from the United Kingdom and tested for enrichment in samples from Germany and the USA (and vice versa). It is therefore unlikely that our observations are an artifact of subtle population substructure present in 1 of our sample cohorts.

18We have also investigated the potential relationship between AAO and polygenic score. To do this we hypothesized that if a person's polygenic score represents a measure of their overall load of PD risk alleles then, in accordance with a liability threshold model, an individual's disease liability should be related to their polygenic score. For established PD risk factors, it is recognized that rare highly penetrant Mendelian variants (eg, homozygous PARK2 mutations) typically lead to a reduced AAO when compared to less penetrant disease mutations (eg, G2019S at LRRK2). As Mendelian mutations are expected to represent a substantial proportion of a carrier's disease liability, we predicted that PD patients who do not carry highly penetrant Mendelian risk mutations but manifest the disease at a younger age would on average carry the highest polygenic load of common PD susceptibility alleles. Our study has identified compelling evidence that supports this hypothesis; patients with an early AAO consistently had a significantly higher polygenic score when compared to those with a late AAO. This indicates that early onset forms of PD are not limited to Mendelian genetic subtypes but can also be contributed to by an accumulation of common polygenic alleles. Moreover, as our study did not include a prescreening to identify highly penetrant mutations, it is possible that a small number of carriers remain in this cohort; our analysis can therefore be considered conservative.

19As might be expected, we observed our strongest enrichment when we compared patients with an AAO at the lower and upper 5% of our sample distribution (OR = 4.8, RR = 4.3), which suggested that a PD patient with a polygenic score > 1.5 is ∼4 times more likely to develop the disease before the age of 40 years than after 80 years of age. Importantly, by adjusting the polygenic scores for the country of origin, we minimized any possible adverse effects of population stratification. Excluding all SNPs spanning genomic regions that harbor known PD susceptibility loci did not adversely affect our findings, implying that the main contribution of the PD polygenic signal identified in this study is from common SNPs that show disease association but fail to meet the probability value threshold for genome‐wide significance.

20Further studies are required if we are to progress from evidence for a polygenic contribution to PD to understanding the specific genetic factors that comprise the polygenic component. Increasing the discovery sample size will allow more loci with increasingly small individual effect sizes to pass the threshold of genome‐wide significance, and should substantially refine the polygenic scores derived here. Moreover, as we have previously shown, using approaches such as gene pathway analyses it is possible to utilize the polygenic signal captured and identify genes or biological systems relevant to PD.30

21It is possible that our findings are being influenced by rare PD susceptibility variants that are in LD with the common alleles analyzed in this study. The ongoing efforts of studies performing exome and whole genome sequencing in large numbers of PD case/control cohorts will allow us to establish the haplotype structure of common and rare alleles, and will allow us to understand which loci are subject to "synthetic association."31 Moreover, as previously demonstrated in other complex diseases,32 future polygenic score analysis of variants identified by exome/genome sequencing is expected to further inform our understanding of the genetic underpinnings of PD. Although it is an important measure, we recognize that clinical estimates of the AAO of PD can often actually reflect the age at diagnosis and as such will inevitably have limited precision in predicting the start of an individual's underlying biological pathology. Applying polygenic score analyses to the results of large sequencing studies of clinically well‐characterized cohorts will help overcome the inherent imprecision of measuring AAO and applying polygenic score analysis to PD score alleles that inevitably include a proportion of SNPs that are not causal.

22Finally, we have used the term PD score allele, as this approach cannot differentiate the minority of true PD risk alleles from variants not associated with the disease. As such, the derived polygenic scores have little value for predicting an individual's risk of developing PD. However, measures of polygenic burden could prove useful in distinguishing PD patients whose disease liability is most likely to carry the largest or smallest genetic component. Identifying these individuals would benefit genetic recall studies and could facilitate a better understanding of how gene–gene and gene–environment interactions increase risk to PD.

23N.M.W., V.E.‐P., and H.R.M. designed the overall study. N.W.W., T.G., A.B., P.H., and J.H. oversaw sample collection and recruitment and supervised all clinical aspects. V.E.‐P. performed all statistical analysis. N.M.W., V.E.‐P., H.R.M., S.L., M.A.N., and A.B.S. interpreted the results. All authors contributed to writing and editing the manuscript.

24H.R.M.: grants, Welsh Assembly Government, Ipsen Fund, MNDA, PSP Association; speaking fees, Teva, UCB; advisory board, Abbvie, Teva, Boerhing‐Ingelheim; webcast fees, GSK; travel expenses, Teva, Medtronic; coapplicant on a patent application related to C9ORF72—Method for diagnosing a neurodegenerative disease (PCT/GB2012/052140). J.H.: consultancy, Eisai Pharma; grant, Biogen Idec Pharma.