Small Ubiquitin-related Modifier (SUMO)3 and (SUMO)4 Gene Polymorphisms in Parkinson’s Disease
Received Date: Mar 21, 2019 / Accepted Date: Jun 04, 2019 / Published Date: Jun 20, 2019
Objective: The ubiquitin/proteosome system is one of the main axes of the pathogenesis of Parkinson’s disease (PD). Small ubiquitin-related modifier (SUMO) proteins are involved in many biochemical events including regulation of transcriptional activity, modulation of signal transduction pathways, and response to cellular stress indicating a role for SUMO in the ubiquitin/proteosome system. In this study, our aim was to examine the prevalence of SUMO gene variants and their clinical associations in PD.
Methods: Fifty-four consecutively recruited PD patients and 74 age-gender matched healthy controls were included. SUMO1, 2, 3 and 4 genes were screened by a next generation sequencing method using blood samples of participants. Single nucleotide polymorphisms (SNPs) with a significantly altered prevalence were determined by Bonferroni correction.
Results: Two SNPs in the SUMO4 gene rs237025 and rs237024 and two SNPs in the SUMO3 gene rs180313 and rs235293 were found to have altered prevalence in PD. Although there was no association among these SNPs and clinical features of the patients, an increased family history of cancer was found in patients with SUMO3 gene variants.
Conclusion: Several SUMO SNPs were identified for the first time in PD patients suggesting that SUMO is involved in the pathophysiology of the disease. rs237025 has also been associated with diabetes mellitus indicating a pathogenic mechanism for SUMO that is shared with other degenerative disorders.
Keywords: Parkinson’s disease; SUMO gene; Sumoylation; Ubiquitin; Genetics
Parkinson’s disease (PD) is the second most common degenerative disorder of the central nervous system worldwide, with an estimated 7-10 million people affected . Dopaminergic neuron loss and Lewy bodies (LBs) are considered as defining pathological characteristics of PD. The accompaniment of neurofilaments, ubiquitin, and β-amyloid in LBs was demonstrated before the main component α-synuclein was identified in 1997 . Later, immunoreactivity for many other proteins, including parkin, synphilin-1 and the small ubiquitin-related modifier (SUMO) was shown in LBs [3-5].
The mammalian SUMO paralogs SUMO-1 and SUMO-2/SUMO-3, although partially redundant, may fulfill different functions as suggested by various studies on substrate specificity, mono-/polySUMOylation, expression, and oxidative stress [6-9]. It is well known that ubiquitin and SUMO share similarities in respect to tertiary structure and conjugation/deconjugation cycles. SUMO has several different isoforms in mammalians and carries a consensus motif, ubiquitin conjugating enzyme 9 (UBC9) as exclusive SUMO-E2 conjugation enzyme . SUMOylation simultaneously discovered by two groups (Matunis and Mahajan) emerged in recent years as a likely candidate mechanism to regulate a plethora of processes within the cell [11,12]. Surprisingly, research in recent years uncovered the existence of mixing units such as the SUMO-targeted ubiquitin ligases (STUbLs) or E3 ligases with dual functions for SUMO and ubiquitin . Notably, SUMOylation and ubiquitination both regulate α-synuclein degradation and aggregation, a hallmark of PD pathology .
Despite its well-established significance in PD pathogenesis, genetic variants of SUMO genes have never been investigated in PD. In this study, our aim was to reveal the presence and prevalence of SUMO gene variants and their associations with clinical features of PD.
Materials and Methods
In this study, 54 PD (34 male, 20 female) patients and 74 age/gender-matched healthy controls (37 male, 37 female) were included. The diagnosis of the patients was based on clinical PD criteria formulated by the Brain Bank of the United Kingdom PD Association . The study was approved by the Institutional Review Board and signed consent was obtained from all participants. Patients with pyramidal or cerebellar system findings, dyspraxia, autonomic dysfunction, and history of head trauma, encephalitis and exposure to toxic substances were excluded. Also, patients with Parkinson plus syndromes, vascular parkinsonism were not included. Age and gender-matched healthy controls without any known systemic, neurological or psychiatric illness were recruited. In the PD group, severity and clinical course of the disease were assessed by Hoehn-Yahr (H&Y) scale and Unified Parkinson’s Disease Rating Scale (UPDRS) [16-18]. All cases were taken on a standard structured interview and neurological examination was performed.
Next generation sequencing
128 human genomic DNA samples were sequenced by next generation sequencing method. Exomes regions of SUMO1, SUMO2, SUMO3, SUMO4 genes were sequenced.
DNA was isolated from blood samples by Invitrogen DNA isolation kit (Carlsbad, CA, USA) and stored at -80°C. In PCR operations, amplification was conducted using a total volume of 50 μl comprising: 100 ng genomic DNA; a reaction buffer with magnesium chloride and potassium chloride; 1 U Taq polymerase; 2.5 mM deoxynucleotide; and 10 pmol of forward and reverse primers. Using the primers set forth in Table 1, the following cycles were applied: 95°C for 5 min, 35 cycles at 95°C for 30 sec and at 60°C for 40 sec.
|Primer Set||Forward Primer Series||Reverse Primer Series||Amplicon Length||Amplicon Location|
Table 1: Primers used in PCR studies.
Thermal cycler protocol was applied for 8 min at 68°C and 10 min at 72°C. The obtained PCR products were confirmed with gel electrophoresis. All amplicons obtained after PCR were diluted to be at the same concentration and purified using Agencourt Ampure beads kit (Agencourt Bioscience Corporation, MA, USA). The samples were then sequenced using the Illumina MiSeq platform and chemicals, following the manufacturer’s protocols. With an average of 360000 readings per sample, a total of 4019498 2x 150 bp readings and 5.13 Gb data were obtained.
The readings in the target position (Table 2) from the array data obtained after the scrambling were filtered out. The obtained raw sequence data were truncated considering the quality scores (Trimmomatic v0.27). The corrected crude sequence data was aligned to the human genomic reference sequences (GRCh37, GRCh38) (using the Burrows-Wheeler Aligner). Subsequently, a local realignment was performed Genome Analyzer Tool Kit (GATK IndelRealigner v3.3.0) to align the the indel. After merging and alignment, reading count optimization, base quality recalibration was performed by filtering the repetitive readings using the GATK (v3.3.0) application. In the last step, single point variants and indices were determined using GATK (Unified Genotyper).
|Experiment Group||Experiment Group (%)||Control Group||Control Group (%)|
Table 2: Comparison of Significant Variants in The SUMO Gene Between the Patient and Control Groups.
All candidate variants (small insertions, deletions, single nucleotide changes) obtained as a result of the exon sequencing analysis were first compared to the minor allele frequency (MAF) (MAF ≤ 0.01 variants) using general control databases (ExAC, 1000 Genome) in Table 3. The obtained variants were assessed using Integrative Genomic Viewer (IGV 2.3, Broad Institute) program.
|CHR||REGION||GENE||TYPE||REFERENCE||ALLELE||P VALUE||BONFERRONI||FDR P VALUE CORRECTION||SAMPLE # (CASE)||TOTAL # (CASE)||SAMPLE FREQUENCY (CASE)||SAMPLE # (CONTROL)||TOTAL # (CONTROL)||SAMPLE FREQUENCY (CONTROL)||dbSNP / rs NUMBER||1000G||MAF|
Table 3: The genetic characteristics and statistical results of the identified variants.
Prevalences of SUMO gene variants were compared among patient and healthy control groups by Chi-square test and Bonferroni test. Chi-square, T-test and Mann-Whitney U statistical tests were used for the comparison of clinical and demographic parameters among study groups. p<0.05 was considered statistically significant.
SUMO gene variants
As a result of the in-depth sequencing, 48 SNPs were detected in the SUMO1, SUMO2, SUMO3 and SUMO4 genes. Significant (p<0.05) SNPs determined by comparing patients and controls were rs237025 and rs237024 in the SUMO4 gene and rs180313 and rs235293 in the SUMO3 gene, respectively. None of the p values remained significant after Bonferroni correction in Table 3. According to these results, 42 of the 54 PD patients were found to display rs237025 and rs237024 variants of the SUMO4 gene. On the other hand, in 8 patients, rs180313 and rs235293 variants were determined in the SUMO3 gene. The SNP and p values in the control group are shown in Table 3.
Comparison of PD patients with and without SUMO variants
PD patients with and without SNPs SUMO genes were compared in terms of clinical and demographic characteristics. The parameters considered in the comparison are summarized in Tables 4A and 4B. There were no statistically significant differences between the groups in terms of age, sex, age at onset, duration of disease, Hoehn-Yahr scores, dementia, depression, and family history of PD. PD patients with rs180313 and rs235293 variants but not rs237025 and rs237024 variants had a significantly higher prevalence of family history of cancer (Table 4B).
|PD patients with rs237025 and rs237024 variants (42)||PD patients without rs237025 and rs237024 variants (12)||p-value|
|Age||62.8 ± 11.8||62.8 ± 17.2||0.497*|
|Age of disease onset||52.6 ± 12.9||54.6 ± 15.3||0.347*|
|Duration of disease||10.1 ± 6.2||8.2 ± 5.5||0.150*|
|Hoehn-Yahr score||1.8 ± 0.6||2.1 ± 0.7||0.094†|
|UPDRS toral-score||64.5 ± 66.2||66.2 ± 32.1||0.443†|
|History of PD in the family||6||3||0.660**|
|History of cancer in the family||7||5||0.148**|
|History of DM type II in the family||9||5||0.299**|
DM: diabetes mellitus; M: male; F: female.
* Student’s t-test; ** chi-square test; † Mann-Whitney U test; †† first symptom of PD.
Table 4A: Distribution of demographic and clinical features of Parkinson's disease (PD) patients according to the presence of rs237025 and rs237024 variants in the SUMO4 gene.
|PD patients with rs180313 and rs235293 variants (8)||PD patients without rs180313 and rs235293 variants (46)||p-value|
|Age||62.0 ± 10.3||62.9 ± 13.5||0.415*|
|Age of disease onset||55.0 ± 9.9||52.7 ± 13.9||0.294*|
|Duration of disease||7.0 ± 4.9||10.2 ± 6.2||0.165*|
|Hoehn-Yahr score||1.8 ± 0.5||1.9 ± 0.7||0.273†|
|UPDRS toral -score||50.4 ± 31.0||67.6 ± 46.2||0.101†|
|History of PD in the family||2||7||0.864**|
|History of cancer in the family||6||6||0.001**|
|History of DM type II in the family||4||10||0.212**|
DM: diabetes mellitus; M: male; F: female.
* Student’s t-test; ** Chi-square test; † Mann-Whitney U test; †† First symptom of PD.
Table 4B: Distribution of demographic and clinical features of Parkinson's disease (PD) patients according to the presence of rs180313 and rs235293 variants in the SUMO3 gene.
PD is a neurodegenerative disease that primarily affects the substantia nigra, and accounts for 80% of all parkinsonism cases. Epidemiological studies have shown that about 1% of the population over 65 years old suffer from this disease. At least 60-80% of dopaminergic cells of substantia nigra need to be lost in order for the findings of PD to appear . Precise mechanisms leading to nigral degeneration, responsible for the clinical manifestations of the disease are as yet not entirely clear and genes associated with increased PD susceptibility still continue to be characterized. Hereditary predisposition, environmental factors, mitochondrial dysfunction and ubiquitin cycling are crucially involved in this process .
In this context, significance of SUMO and sumoylation in neurodegenerative disorders has been recently scrutinized. Although PD cases are mostly sporadic, several genes have also been associated with familial types of disease. α-synuclein, DJ-1 and parkin are three of these genes and are target proteins for SUMO, indicating the role of this molecule in the molecular mechanisms of PD pathogenesis [21,22]. SUMO proteins bind to large number of cellular targets. It modulates protein-protein and protein-DNA interactions, modifies intracellular localizations of proteins and protects cells from ubiquitin-induced degradation [11,12]. Sumoylation is functionally a more varied modifier than ubiquitination and unlike ubiquitination, proteins do not directly target the proteasome. Instead, sumoylation blocks proteosomal degradation by competing with ubiquitination for a common lysine residue and substrate samples are used for this process [5,23]. SUMO and ubiquitin also have a variety of functional and structural properties that play a role in the regulation and coordination of different stages of DNA damage recognition and repair, regulation of replication and replication stress, protection of genomic stability, and various other cellular events [14,23].
In our study, two significant SNPs were determined in the SUMO4 gene (rs237025 and rs237024) and two additional SNPs were found in the SUMO3 gene (rs180313 and rs235293). To our knowledge, three of the four significant SNPs have not been previously reported and rs237025 has only been linked to diabetes mellitus, another degenerative disease. None of the identified SNPs appear to be associated with severity or clinical features. Nevertheless, increased prevalence of these SNPs in PD patients might be due to their involvement in the pathogenesis of PD. The rs237025 variant of SUMO4 causes a missense mutation leading methionine to convert to valine. SUMO4 is known to contribute to enhanced cell survival through suppression of inflammation. Moreover, the rs237025 variant of SUMO4 has been suggested to promote intracellular inflammation pathways through NF-κB activation and thus negatively influence cell survival . It is well known that different degenerative disorders might share common molecular mechanisms and due to its multifunctionality, SUMO might be one of these common mechanism factors.
The rs237024 SNP is in the 3’UTR region of the SUMO4 gene and rs180313 and rs235293 genes are found in the intronic area of the SUMO3 gene. Nevertheless, it is well known that mutations and SNPs occurring in non-coding regions may have significant molecular and clinical consequences . Exact mechanisms by which these SNPs contribute to PD pathogenesis need to be further studied.
A notable finding in our study was the association between SUMO3 rs180313 and rs235293 variants and family history of cancer. To our knowledge, SNPs of SUMO genes have not been associated with increased cancer risk. However, SNPs of SUMO-conjugating enzyme UBC9 and E3 SUMO-protein ligase protein inhibitor of activated STAT 3 (PIAS3) genes have been shown to contribute to increased risk of breast cancer . Nevertheless, since only a limited number of patients displayed these variants, our results need to be confirmed by future studies.
In conclusion, in this study, we have determined certain SNPs in SUMO genes for the first time in PD patients, have found a link between SUMO4 and a neurological disease for the first time and thus provided further evidence for involvement of sumoylation in the pathophysiology of the disease. The mechanisms by which identified SUMO3 and SUMO4 SNPs contribute to the pathogenesis of PD need to be further studied by functional experiments. Our results also indicate that sumoylation molecules may be potential targets for novel therapeutics of PD. This notion requires a better understanding of biochemical activators of SUMO genes, which have been vastly understudied.
We are grateful to the patients and families for their participation and to the anonymous reviewers for their valuable advice.
The present work was supported by the Research Fund of Istanbul University.
Compliance with Ethical Standards
All procedures performed in studies involving human participants were in accordance with the ethical standards of Istanbul University, Istanbul Faculty of Medicine, Clinical Research Ethical Committee (Project Number 2011/672-528) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Citation: Kucukali CI, Salman B, Yuceer H, Ulusoy C, Abaci N, et al. (2019) Small Ubiquitin-related Modifier (SUMO)3 and (SUMO)4 Gene Polymorphisms in Parkinson’s Disease. J Neurol Disord 7: 407. DOI: 10.4172/2329-6895.1000407
Copyright: © 2019 Kucukali CI, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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