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Enrichment of Rare Variants in E3 Ubiquitin Ligase Genes in Early Onset Parkinson’S Disease

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Enrichment of Rare Variants in E3 Ubiquitin Ligase Genes in Early Onset Parkinson’S Disease Enrichment of Rare Variants in E3 Ubiquitin Ligase Genes in Early Onset Parkinson’s Disease Xiaojing Gu Sichuan University West China Hospital Yanbing Hou Sichuan University West China Hospital Yongping Chen Sichuan University West China Hospital Ruwei Ou Sichuan University West China Hospital Bei Cao Sichuan University West China Hospital Qianqian Wei Sichuan University West China Hospital Lingyu Zhang Sichuan University West China Hospital Wei Song Sichuan University West China Hospital Bi Zhao Sichuan University West China Hospital Ying Wu Sichuan University West China Hospital Chunyu Li Sichuan University West China Hospital Huifang Shang ( [email protected] ) West China Hospital of Sichuan University https://orcid.org/0000-0003-0947-1151 Research Keywords: E3 ubiquitin ligase, Early onset Parkinson’s disease, burden, association Posted Date: March 3rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-262042/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/14 Abstract Background Dysfunction of the ubiquitination proteasome system (UPS) is important in the pathogenesis of Parkinson’s disease (PD). Patients with early onset PD (EOPD) are more susceptible to genetic factors. We systematically examined the overlaps between E3 ubiquitin ligase genes and EOPD. Methods A total of 695 EOPD patients were sequenced with whole exome sequencing. Aggregate burden for rare variants (Minor allele frequency <0.001 and <0.0001) in a total of 44 E3 ubiquitin ligase genes causing disorders involved in the nervous system were analyzed. Results There was signicant enrichment of the rare and rare damaging variants in the E3 ubiquitin ligase genes in EOPD patients. Detailly, at the gene-based level, the strongest associations were found in HERC1, IRF2BPL, KMT2D, RAPSN, RLIM, RNF168 and RNF216. Conclusion Our ndings highlight the importance of the UPS mechanism in the pathogenesis of PD from the genetic perspective. Moreover, our study also expanded the susceptible gene spectrum for PD. Background Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by cardinal motor symptoms including bradykinesia, rigidity, tremor and postural instability, as well as various non-motor symptoms such as cognitive decline, autonomic dysfunction and psychiatric problems[1]. PD is considered to be a complex disorder caused by genetic factors, environmental factors and aging together. So far, approximately 30 genes have been found to be causative genes for PD but can only explain a small proportion of the etiology of PD[2], which highlights the importance of discovering the remaining responsible genes for PD as well as exploring the underlying mechanisms. The pathological hallmark of PD is the loss of dopamine neurons and the abnormal accumulation of α-synuclein protein in form of Lewy bodies[3]. Although the pathological mechanisms of PD remain largely unknown, protein degradation and quality control were implicated to be dysregulated in PD and ultimately leading to the abnormal protein deposition[4]. The ubiquitination proteasome system (UPS) is one of the important pathways for protein clearance[4]. Pathologically, ubiquitin, the critical modier that tagged proteins for degradation, has been found in Lewy bodies[5]. Genetically, several PD causative genes have also been found to be involved in UPS, including Parkin, PINK1, UCHL1 and FBXO7[6]. Therefore, these evidences strongly implicated altered ubiquitin signaling and disrupted protein quality control involved in PD. Ubiquitination is a biological process requiring the involvement of several integral components known as ubiquitin ligases. The initiation of protein ubiquitination typically requires an ATP-dependent enzymatic cascade that is initiated with the priming of a ubiquitin onto a ubiquitin activating enzyme (E1) and the transfer to a ubiquitin Page 2/14 conjugating enzyme (E2). Ubiquitin is then covalently attached to a lysine residue on the target protein by an E3 ubiquitin ligase (E3) and this process can be repeated to create a series of ubiquitin chains[7]. There are only 2 E1 and 30–50 E2 genes, while there are over 600 E3 ubiquitin ligase genes. The diversity of E3 genes have been linked to neurological disorders including neurodegenerative disease, neuro-developmental disorders, and intellectual disability, such as Alzheimer’s disease, PD, multiple sclerosis and Autism spectrum disorder [8]. Considering the important role of ubiquitination in the genetic, mechanical and pathological changes of PD, and early onset PD (EOPD) is more susceptible to genetic factors[9], in the current study, we aimed to systematically examine whether there were overlaps between E3 ubiquitin ligase genes and EOPD with a whole exome sequencing (WES) dataset. Methods Patients A total of 695 EOPD patients (age of onset ≤ 45y) admitted to the Department of Neurology, West China Hospital were recruited into the study. All the PD patients were diagnosed by experienced neurologists based on the established clinical diagnostic criteria for PD[10, 11]. Clinical characteristics items and rating scales for patients were collected by face-to-face interview. Written informed consent was obtained from all participants. The study was approved by the ethics committee of West China hospital, Sichuan University. Sequencing, genes selection, variants ltration DNA samples were collected according to the standard SDS-phenol-chloroform method. WES was applied as addressed in our previous study[12]. Total coverage < 10 and altered frequency < 20% represented poor sequencing quality and were excluded from the analysis. The E3 ubiquitin ligase genes were downloaded from the Epithelial Systems Biology Laboratory (http: hpcwebapps.cit.nih.gov/ ESBL/ Database/ E3-ligases). Then we focused on those 44 genes that can cause monogenic disease with the nervous system involved by searching Online Mendelian Inheritance in Man database (OMIM, https://omim.org/) (Supplementary table 1). Controls were from the gnomAD East Asian population (https://gnomad.broadinstitute.org/). Variants were ltered rstly based on two minor allele frequency (MAF) thresholds: (1) rare<0.001; and (2) extra rare<0.0001. Then, 3 categories of comparison were conducted: (1) non-synonymous; (2) likely damaging;(3) protein-truncating variants (PTVs, frameshift variants, stop-gain variants, stop-loss variants, start-gain variants and start-loss variants). Combined Annotation Dependent Depletion (CADD) integrates predictions from numerous bioinformatic algorithms into a single ‘C-score’ and ranks all possible nucleotide changes in the genome based on potential to disrupt gene/protein function[13]. In accordance with the previous study, we dened a stringent CADD C-score ≥ 12.37 as likely damaging variants, representing the top 2% most damaging of all possible nucleotide changes in the genome—this subset is enriched for known pathogenic alleles[14]. Moreover, the in-frame deletion/insertion variants lead to the change of the protein length and are classied as moderately strong evidence for pathogenicity[15]. Therefore, in- frame deletion/insertion was classied as damaging variants in the current study. Statistical analysis Genetic association analysis was performed with Chi square test or Fisher’s exact test in the whole E3 ubiquitin ligase genes dataset and each gene. And a p value < 0.0012(0.05/44) was considered as statistically signicant after Bonferroni correction. Burden analysis was conducted with 5 algorithms with AssotesteR Package in the per Page 3/14 gene level, including Comprehensive Approach to Analyzing Rare Genetic Variants (CARV), Sum of Squared Score (SSU), Sum Test (SUM), Cumulative Minor Allele Test (CMAT), and Bayesian Score Test (BST)[16]. Moreover, in order to highlight those gene driving associations detected in the gene set, secondary analyses were also performed to evaluate variants in each E3 ubiquitin gene independently. Bonferroni-correction was applied for the multiple comparison and an adjusted p < 0.0012(0.05/44) was considered as statistically signicant. The nal results were considered signicant if at least 3 algorithms were signicant. Results Variants were extracted from 44 ubiquitin E3 ubiquitin ligase genes responsible for monogenic diseases with the nervous system affected (Supplementary Table 1), and ltered 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, signicant 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
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