NUS1 variants identified in Cohort A
From the 1,542 PD patients and 1,625 healthy controls in our study, we identified 13 variants including 10 rare and 3 low-frequency variants in 98 patients and 78 controls (No common variants were observed). Of these, eight polymorphisms (rs974335534, rs550854234, rs761121795, rs539668656, rs969919569, rs573584810, rs369403261, and rs28362519) have been reported previously and five single-nucleotide polymorphisms (S24S, F30F, A80A, N144K, and L159I) were novel. Among the 13 variants, 10 rare variants (H19H, S24S, F30F, K58N, A80A, R86L, T106T, N144K, L159I, and D163H) were identified in 12 patients and one healthy control. Considering the functional effect of the variants, seven were synonymous and seven were nonsynonymous as shown in Table 1. Pathogenicity predictions indicated that three (R86L, N144K and D163H) of the nonsynonymous variants may be deleterious (Table 1, Table S1).
Burden analyses
In Cohort A, significant associations comparing patients and controls were detected for the NUS1 gene (P = 0.023, adjusted P = 0.046). Further stratification by the variants’ predicted functional properties showed that the association was mainly in the nonsynonymous variants (P = 0.022, adjusted P = 0.046). Stratification by allele frequency showed that the significant association mainly involved the rare variants (P = 0.0026, adjusted P = 0.016). However, when synonymous variants (P = 0.071), deleterious variants (P = 0.20), or low-frequency variants (P = 0.21) were considered for analysis, no significant associations were observed (Table 2). Our burden results further support the view that NUS1 is a risk gene for PD.
Association between low-frequency variants and PD
Additionally, we investigated the association between 3 low-frequency variants (rs550854234, rs539668656, and rs28362519) and PD. To further increase the power to detect associations, we collected the results for rs550854234, rs539668656, and rs28362519 from the Cohort B and combined the two cohorts for joint analysis. Among the three low-frequency variants, we identified two independent variants (rs550854234 and rs539668656, R2<0.2) that were associated with PD (adjusted P < 0.05) (Table 3).The frequency of rs539668656 in PD patients was higher than that of healthy controls even after correction for multiple comparisons (OR = 2.80, 95% CI = 1.36-5.80, P = 0.0054, adjusted P = 0.016). The rs550854234 variant was associated with a decreased PD risk after correction for multiple comparisons (OR = 0.76, 95% CI = 0.60-0.97, P = 0.027, adjusted P = 0.041). However, the rs28362519 variant was not significantly associated with PD (P = 0.48).
The patients with PD from the combined cohort were divided into EOPD and LOPD cohorts (i.e., stratified by AAO; Table 3). In cases of LOPD, we found that rs539668656 was associated with an increased LOPD risk after correction for multiple comparisons (OR = 2.96, 95% CI = 1.22-7.19, P = 0.017, adjusted P = 0.025), and rs550854234 was associated with a decreased LOPD risk after correction for multiple comparisons (OR = 0.66, 95% CI = 0.47-0.92, P = 0.016, adjusted P = 0.025). However, neither of the three variants were significantly associated with EOPD (P > 0.05).
Variant-clinical data association
We also analyzed the clinical features of NUS1-variant carriers and non-carriers (Table 4). We found that in Cohort A, patients with rare variants had onset with PD with 6.16-year earlier than did non-carriers (OR=-6.16, P=0.0087), and had suffered a 6.88 times potential high risk of depression (HAMD[23], OR=6.88, P=0.019) than those without rare variants. In the combined cohort, patients with rs550854234 had decreased risk for depression (HAMD[23], OR=0.51, P=0.035) than the non-carriers, and rs550854234 in patients were positively associated with tremor score (UPDRS Item 20 and 21[24], β=0.86, P=0.027). Patients with rs539668656 had more severe mental/behavioral symptoms and greater emotional impairment (UPDRS-Part I[24], β=1.38, P=0.012) than did non-carriers.
Additionally, we further validated the effects of the NUS1 variants on PD-related phenotype using a more conservative strategy: Permutation test by 10,000 times of random permutations, which does not require assumptions of distribution, also showed significant effects for the NUS1 rare variants on the AAO (EMP2=0.019), the NUS1 rare variants and rs550854234 on the incidence of depression (EMP2=0.0041; EMP2=0.041;respectively), rs550854234 on the tremor level (EMP2=0.022), and rs539668656 on mental/behavioral symptoms and emotional impairment (EMP2= 0.015).