Variants were extracted from 44 ubiquitin E3 ubiquitin ligase genes responsible for monogenic diseases with the nervous system affected (Supplementary Table 1), and filtered into nested categories based on two frequency thresholds (MAF < 0.001 and MAF < 0.0001) and 3 tiers of functional criteria (nonsynonymous, damaging and PTVs).
Among the 44 E3 ubiquitin ligase genes, no rare coding variants were found in MARCH6, TRIM71 and VPS11. The observed variants in above-mentioned 2 categories and 3 tiers are listed in Supplementary Table 2. Following adjustment for multiple comparisons, significant associations were detected for the E3 ubiquitin ligase gene set considering both non-synonymous variants, likely damaging variants and PTVs, when using the both the MAF of 0.001 and 0.0001 (Supplementary Table 2 and Fig. 1A). In general, our results implicated an enrichment of rare and rare damaging variants in E3 ubiquitin ligase gene in Chinese EOPD patients.
To determine which E3 ubiquitin ligase genes/variants may be responsible for the observed association with EOPD risk, we performed exploratory analyses to assess for potential contribution of variants within each gene separately. In these association analyses, combined frequency of rare variants in 6 genes and extra rare variants in 10 genes had the strongest aggregate associations with EOPD (Supplementary Table 3, and Fig. 1B and 1C). Moreover, when taking the predicted function into consideration, 6 genes with rare and extra rare damaging variants were found to be enriched in EOPD patients, those include HECT and RLD domain containing E3 ubiquitin protein ligase family member 1 gene (HERC1), interferon regulatory factor 2-binding protein-like gene (IRF2BPL), histone-lysine N-methyltransferase 2D gene (KMT2D), leucine rich repeat and sterile alpha motif containing 1 gene (LRSAM1), 43 kDa receptor-associated protein of the synapse isoform 2 gene(RAPSN), Ring finger protein, LIM domain interacting gene (RLIM), and ring finger protein 216 gene(RNF216)(Supplementary Table 4, Fig. 1D and 1E).
Furthermore, in the rare variants burden analyses, rare and extra rare variants in 7 genes were found to be enriched in the patients with EOPD, including HERC1, IRF2BPL, KMT2D, LRSAM1, RAPSN, RLIM and RNF216. Moreover, when taking the predicted function into consideration, rare and extra rare damaging variants in HERC1, IRF2BPL, KMT2D, RAPSN, RLIM, RNF168 and RNF216 were found to be enriched in the patients with EOPD (Table 1).
Table 1
Results of the 5 algorithms for the rare variants burden analyses for per gene.
|
Rare variants
|
Rare damaging variants
|
|
MAF
|
CARV
|
SSU
|
SUM
|
CMAT
|
BST
|
CARV
|
SSU
|
SUM
|
CMAT
|
BST
|
AFF4
|
< 0.001
|
0.460
|
0.460
|
0.618
|
0.460
|
0.460
|
0.710
|
0.710
|
0.514
|
0.710
|
0.710
|
< 0.0001
|
0.522
|
0.386
|
0.126
|
0.386
|
0.386
|
0.386
|
0.386
|
0.102
|
0.386
|
0.386
|
HACE1
|
< 0.001
|
1.000
|
0.798
|
0.344
|
0.798
|
0.798
|
0.766
|
0.604
|
0.208
|
0.604
|
0.604
|
< 0.0001
|
0.408
|
0.172
|
0.088
|
0.172
|
0.172
|
0.420
|
0.176
|
0.076
|
0.176
|
0.176
|
HECW2
|
< 0.001
|
0.176
|
0.134
|
0.070
|
0.134
|
0.134
|
1.000
|
0.900
|
0.484
|
0.900
|
0.900
|
< 0.0001
|
0.054
|
0.006
|
0.006
|
0.006
|
0.006
|
0.338
|
0.048
|
0.032
|
0.048
|
0.048
|
HERC1
|
< 0.001
|
0.018
|
0.000
|
0.000
|
0.000
|
0.000
|
0.026
|
0.000
|
0.000
|
0.000
|
0.000
|
< 0.0001
|
0.006
|
0.000
|
0.000
|
0.000
|
0.000
|
0.004
|
0.000
|
0.000
|
0.000
|
0.000
|
HERC2
|
< 0.001
|
0.040
|
0.012
|
0.008
|
0.012
|
0.012
|
0.126
|
0.084
|
0.054
|
0.084
|
0.084
|
< 0.0001
|
0.004
|
0.000
|
0.000
|
0.000
|
0.000
|
0.006
|
0.000
|
0.000
|
0.000
|
0.000
|
HUWE1
|
< 0.001
|
0.002
|
0.000
|
0.000
|
0.000
|
0.000
|
0.036
|
0.000
|
0.000
|
0.000
|
0.000
|
|
< 0.0001
|
0.104
|
0.006
|
0.006
|
0.006
|
0.006
|
0.162
|
0.006
|
0.006
|
0.006
|
0.006
|
IRF2BPL
|
< 0.001
|
0.026
|
0.000
|
0.000
|
0.000
|
0.000
|
0.032
|
0.000
|
0.000
|
0.000
|
0.000
|
|
< 0.0001
|
0.024
|
0.000
|
0.000
|
0.000
|
0.000
|
0.032
|
0.000
|
0.000
|
0.000
|
0.000
|
KMT2C
|
< 0.001
|
0.626
|
0.484
|
0.232
|
0.484
|
0.484
|
0.588
|
0.538
|
0.708
|
0.538
|
0.538
|
|
< 0.0001
|
0.006
|
0.000
|
0.000
|
0.000
|
0.000
|
0.024
|
0.002
|
0.002
|
0.002
|
0.002
|
KMT2D
|
< 0.001
|
0.002
|
0.000
|
0.000
|
0.000
|
0.000
|
0.008
|
0.000
|
0.000
|
0.000
|
0.000
|
|
< 0.0001
|
0.002
|
0.000
|
0.000
|
0.000
|
0.000
|
0.004
|
0.000
|
0.000
|
0.000
|
0.000
|
LRSAM1
|
< 0.001
|
0.014
|
0.000
|
0.000
|
0.000
|
0.000
|
0.102
|
0.010
|
0.008
|
0.010
|
0.010
|
|
< 0.0001
|
0.024
|
0.000
|
0.000
|
0.000
|
0.000
|
0.264
|
0.008
|
0.008
|
0.008
|
0.008
|
MID1
|
< 0.001
|
0.206
|
0.002
|
0.002
|
0.002
|
0.002
|
0.290
|
0.118
|
0.054
|
0.118
|
0.118
|
|
< 0.0001
|
0.174
|
0.000
|
0.000
|
0.000
|
0.000
|
0.230
|
0.002
|
0.002
|
0.002
|
0.002
|
MID2
|
< 0.001
|
0.236
|
0.014
|
0.014
|
0.014
|
0.014
|
0.268
|
0.024
|
0.024
|
0.024
|
0.024
|
|
< 0.0001
|
0.388
|
0.002
|
0.002
|
0.002
|
0.002
|
0.394
|
0.000
|
0.000
|
0.000
|
0.000
|
NEDD4L
|
< 0.001
|
0.396
|
0.186
|
0.062
|
0.186
|
0.186
|
0.276
|
0.090
|
0.040
|
0.090
|
0.090
|
|
< 0.0001
|
0.734
|
0.734
|
0.254
|
0.734
|
0.734
|
0.984
|
0.732
|
0.226
|
0.732
|
0.732
|
NHLRC1
|
< 0.001
|
0.206
|
0.062
|
0.038
|
0.062
|
0.062
|
0.724
|
0.724
|
0.296
|
0.724
|
0.724
|
|
< 0.0001
|
0.202
|
0.006
|
0.006
|
0.006
|
0.006
|
0.412
|
0.412
|
0.134
|
0.412
|
0.412
|
PCGF2
|
< 0.001
|
0.522
|
0.498
|
0.616
|
0.498
|
0.498
|
0.658
|
0.468
|
0.600
|
0.468
|
0.468
|
|
< 0.0001
|
1.000
|
0.722
|
0.262
|
0.738
|
0.722
|
0.984
|
0.732
|
0.226
|
0.732
|
0.732
|
PEX10
|
< 0.001
|
0.236
|
0.082
|
0.052
|
0.082
|
0.082
|
1.000
|
0.780
|
0.264
|
0.780
|
0.780
|
|
< 0.0001
|
0.202
|
0.006
|
0.006
|
0.006
|
0.006
|
0.438
|
0.024
|
0.024
|
0.024
|
0.024
|
PEX12
|
< 0.001
|
0.120
|
0.016
|
0.014
|
0.016
|
0.016
|
0.158
|
0.022
|
0.016
|
0.022
|
0.022
|
|
< 0.0001
|
0.256
|
0.000
|
0.000
|
0.000
|
0.000
|
0.308
|
0.002
|
0.002
|
0.002
|
0.002
|
PEX2
|
< 0.001
|
0.772
|
0.772
|
0.254
|
0.772
|
0.772
|
0.728
|
0.728
|
0.420
|
0.728
|
0.728
|
|
< 0.0001
|
0.280
|
0.038
|
0.038
|
0.038
|
0.038
|
0.422
|
0.422
|
0.120
|
0.422
|
0.422
|
PLAG1
|
< 0.001
|
0.608
|
0.592
|
0.558
|
0.592
|
0.592
|
0.736
|
0.516
|
0.188
|
0.516
|
0.516
|
|
< 0.0001
|
0.486
|
0.016
|
0.016
|
0.016
|
0.016
|
0.486
|
0.016
|
0.016
|
0.016
|
0.016
|
RAG1
|
< 0.001
|
0.488
|
0.320
|
0.142
|
0.320
|
0.320
|
0.238
|
0.148
|
0.070
|
0.148
|
0.148
|
|
< 0.0001
|
0.096
|
0.006
|
0.006
|
0.006
|
0.006
|
0.170
|
0.004
|
0.004
|
0.004
|
0.004
|
RAPSN
|
< 0.001
|
0.010
|
0.000
|
0.000
|
0.000
|
0.000
|
0.012
|
0.000
|
0.000
|
0.000
|
0.000
|
|
< 0.0001
|
0.048
|
0.000
|
0.000
|
0.000
|
0.000
|
0.082
|
0.000
|
0.000
|
0.000
|
0.000
|
RBCK1
|
< 0.001
|
0.718
|
0.718
|
0.558
|
0.718
|
0.718
|
0.656
|
0.652
|
0.226
|
0.652
|
0.652
|
|
< 0.0001
|
0.000
|
0.250
|
0.620
|
0.250
|
0.250
|
0.000
|
0.104
|
0.692
|
0.104
|
0.104
|
RLIM
|
< 0.001
|
0.106
|
0.000
|
0.000
|
0.000
|
0.000
|
0.118
|
0.000
|
0.000
|
0.000
|
0.000
|
|
< 0.0001
|
0.294
|
0.000
|
0.000
|
0.000
|
0.000
|
0.314
|
0.000
|
0.000
|
0.000
|
0.000
|
RNF113A
|
< 0.001
|
0.558
|
0.558
|
0.136
|
0.558
|
0.558
|
0.396
|
0.046
|
0.046
|
0.046
|
0.046
|
|
< 0.0001
|
0.598
|
0.000
|
0.000
|
0.000
|
0.000
|
0.444
|
0.024
|
0.024
|
0.024
|
0.024
|
RNF125
|
< 0.001
|
1.000
|
0.396
|
0.162
|
0.396
|
0.396
|
1.000
|
0.396
|
0.162
|
0.396
|
0.396
|
|
< 0.0001
|
1.000
|
0.044
|
0.000
|
0.044
|
0.044
|
1.000
|
0.044
|
0.044
|
0.044
|
0.044
|
RNF13
|
< 0.001
|
0.772
|
0.588
|
0.520
|
0.588
|
0.588
|
0.456
|
0.408
|
0.670
|
0.408
|
0.408
|
|
< 0.0001
|
0.44
|
0.024
|
0.024
|
0.024
|
0.024
|
0.486
|
0.044
|
0.044
|
0.044
|
0.044
|
RNF168
|
< 0.001
|
0.044
|
0.002
|
0.002
|
0.002
|
0.002
|
0.078
|
0.000
|
0.000
|
0.000
|
0.000
|
|
< 0.0001
|
0.132
|
0.002
|
0.002
|
0.002
|
0.002
|
0.224
|
0.000
|
0.000
|
0.000
|
0.000
|
RNF170
|
< 0.001
|
0.432
|
0.432
|
0.108
|
0.432
|
0.432
|
0.648
|
0.448
|
0.080
|
0.448
|
0.448
|
|
< 0.0001
|
0.704
|
0.074
|
0.074
|
0.074
|
0.074
|
1.000
|
0.044
|
0.044
|
0.044
|
0.044
|
RNF216
|
< 0.001
|
0.326
|
0.000
|
0.000
|
0.000
|
0.000
|
0.368
|
0.000
|
0.000
|
0.000
|
0.000
|
|
< 0.0001
|
0.368
|
0.000
|
0.000
|
0.000
|
0.000
|
0.426
|
0.000
|
0.000
|
0.000
|
0.000
|
RSPRY1
|
< 0.001
|
0.380
|
0.114
|
0.048
|
0.114
|
0.114
|
0.198
|
0.042
|
0.042
|
0.042
|
0.042
|
|
< 0.0001
|
0.272
|
0.010
|
0.010
|
0.010
|
0.010
|
0.458
|
0.066
|
0.066
|
0.066
|
0.066
|
STUB1
|
< 0.001
|
0.452
|
0.250
|
0.606
|
0.250
|
0.250
|
0.572
|
0.440
|
0.534
|
0.440
|
0.440
|
|
< 0.0001
|
0.438
|
0.064
|
0.064
|
0.064
|
0.064
|
0.644
|
0.638
|
0.130
|
0.638
|
0.638
|
TRAF7
|
< 0.001
|
0.660
|
0.660
|
0.260
|
0.660
|
0.660
|
0.678
|
0.510
|
0.208
|
0.510
|
0.510
|
|
< 0.0001
|
0.132
|
0.002
|
0.002
|
0.002
|
0.002
|
0.142
|
0.000
|
0.000
|
0.000
|
0.000
|
TRAIP
|
< 0.001
|
0.446
|
0.190
|
0.068
|
0.190
|
0.190
|
0.230
|
0.088
|
0.042
|
0.088
|
0.088
|
|
< 0.0001
|
0.230
|
0.002
|
0.002
|
0.002
|
0.002
|
0.290
|
0.002
|
0.002
|
0.002
|
0.002
|
TRIM2
|
< 0.001
|
0.578
|
0.578
|
0.190
|
0.578
|
0.578
|
0.584
|
0.584
|
0.182
|
0.584
|
0.584
|
|
< 0.0001
|
0.384
|
0.006
|
0.006
|
0.006
|
0.006
|
0.400
|
0.002
|
0.002
|
0.002
|
0.002
|
TRIM32
|
< 0.001
|
0.096
|
0.012
|
0.010
|
0.012
|
0.012
|
0.106
|
0.008
|
0.008
|
0.008
|
0.008
|
|
< 0.0001
|
0.134
|
0.002
|
0.002
|
0.002
|
0.002
|
0.280
|
0.000
|
0.000
|
0.000
|
0.000
|
TRIM36
|
< 0.001
|
0.668
|
0.534
|
0.242
|
0.534
|
0.534
|
0.420
|
0.264
|
0.102
|
0.264
|
0.264
|
|
< 0.0001
|
0.110
|
0.006
|
0.006
|
0.006
|
0.006
|
0.046
|
0.002
|
0.002
|
0.002
|
0.002
|
TRIM37
|
< 0.001
|
0.558
|
0.458
|
0.162
|
0.458
|
0.458
|
0.648
|
0.544
|
0.208
|
0.544
|
0.544
|
|
< 0.0001
|
0.186
|
0.052
|
0.022
|
0.052
|
0.052
|
0.224
|
0.090
|
0.044
|
0.090
|
0.090
|
TRIP12
|
< 0.001
|
0.540
|
0.540
|
0.196
|
0.540
|
0.540
|
0.542
|
0.542
|
0.188
|
0.542
|
0.542
|
|
< 0.0001
|
0.168
|
0.012
|
0.012
|
0.012
|
0.012
|
0.170
|
0.012
|
0.012
|
0.012
|
0.012
|
UBE3A
|
< 0.001
|
0.462
|
0.248
|
0.592
|
0.248
|
0.248
|
0.572
|
0.440
|
0.534
|
0.440
|
0.440
|
|
< 0.0001
|
0.634
|
0.634
|
0.314
|
0.634
|
0.634
|
0.634
|
0.634
|
0.314
|
0.634
|
0.634
|
UBE3B
|
< 0.001
|
0.040
|
0.064
|
0.960
|
0.064
|
0.064
|
0.120
|
0.158
|
0.862
|
0.158
|
0.158
|
|
< 0.0001
|
1.000
|
0.768
|
0.300
|
0.768
|
0.768
|
0.794
|
0.590
|
0.246
|
0.590
|
0.590
|
UBR1
|
< 0.001
|
0.590
|
0.396
|
0.200
|
0.396
|
0.396
|
0.556
|
0.456
|
0.168
|
0.456
|
0.456
|
|
< 0.0001
|
0.120
|
0.004
|
0.004
|
0.004
|
0.004
|
0.058
|
0.000
|
0.000
|
0.000
|
0.000
|
Padjust=0.05/41 = 0.0012 |