Small Ubiquitin-Like Modifier 4 (SUMO4) Gene M55V Polymorphism and Type 2 Diabetes Mellitus: A Meta-analysis Including 6,823 Subjects

Background Many studies suggest that the small ubiquitin-like modifier 4 (SUMO4) M55V gene polymorphism (rs237025) may be associated with an increased risk of type 2 diabetes mellitus (T2DM). However, due to other conflicting results, a clear consensus is lacking in the matter. Objective and methods A meta-analysis consisting of 6,823 subjects from 10 studies was conducted to elucidate relationship between the SUMO4 M55V gene polymorphism and T2DM. Depending on the heterogeneity of the data, either a fixed or random-effects model would be used to assess the combined odds ratio (ORs) and their corresponding 95% confidence interval (CI). Results SUMO4 gene M55V polymorphism was significantly associated with T2DM in the whole population under allelic (OR: 1.18, 95% CI: 1.10–1.28, P = 1.63 × 10−5), recessive (OR: 1.59, 95% CI: 1.14–2.23, P = 0.006), dominant (OR: 0.815, 95% CI: 0.737–0.901, P = 6.89 × 10−5), homozygous (OR: 1.415, 95% CI: 1.170–1.710, P = 0.0003), heterozygous (OR: 1.191, 95% CI: 1.072–1.323, P = 0.001), and additive genetic models (OR: 1.184, 95% CI: 1.097–1.279, P = 1.63 × 10−5). In our subgroup analysis, a significant association was found again in the Chinese population, but not in Japanese or Iranian population. Conclusion SUMO4 gene M55V polymorphism may correlate with increased T2DM risk. Chinese carriers of the V allele of the SUMO4 gene M55V polymorphism may be predisposed to developing T2DM.

discovered molecule located in insulin-dependent diabetes mellitus 5 (IDDM5), has been shown to suppress the transcription of NF-κB. SUMO4 also acts as an anti-oxidant, protecting the pancreatic β-cells from oxidative damage and promoting β-cell survival (4). Therefore, under oxidative stress by autoimmune processes, SUMO4 and the DNA damage signaling protein iKB can be mobilized to activate intracellular pathways associated with cell survival (5). The SUMO4 gene is located at position 6q25 in the IDDM5 locus and spans 688 bp. The gene contains a single exon, encoding 95 amino acids (6). The M55V polymorphism is caused by mutation of an adenosine at the 1633d position to a guanine (rs237025, A163G). The conformational change caused by the polymorphism results in increased NF-κB transcriptional activity and, thus, increased expression of NF-κB-dependent genes.
Though researchers have found good evidence for an association between this polymorphism and T2DM susceptibility, the exact relationship is still debated, possibly due to the varying genetic backgrounds of different ethnic groups (7). In this regard, other associations with T2DM, such HMGA1 and TCFTL2 variants, have been found to be heterogeneous across different ethnic groups (8,9).
In 2004, Bohren et al. found the SUMO4 Met55Val gene polymorphism to be associated with an increased susceptibility to type 1 diabetes mellitus (T1DM) in the Caucasian population (6). Around the same time, Guo et al. replicated the result in a Chinese population (4). In 2005, Noso et al. found Val 55 to be significantly more common in T1DM patients than in control subjects in the Japanese population (4). In 2008, Ikegami et al. has performed the genome-wide association (GWA) study and found that the M55V variant was significantly associated with T1DM in the Asian populations, but not Caucasian populations (10). Other studies have suggested the susceptibility loci for T2DM may lie in the area on Chromosome 6 that SUMO4 is located (11)(12)(13)(14), indicating a common genetic basis for both types of diabetes. Considering the possible functional evidence for SUMO4 as a candidate gene for T2DM, Noso et al. initially explored the contribution of the SUMO4 Met55Val locus to T2DM susceptibility and found a significant association in a Japanese population (15 (17).
We conducted the current meta-analysis from 3,223 T2DM patients and 3,600 controls to verify the association of SUMO4 M55V gene polymorphism and T2DM.

Publication search and inclusion criteria
The included studies should meet the following inclusion criteria: (a) assessment of the association of SUMO4 gene M55V polymorphism with T2DM; (b) T2DM diagnosis and classification meets guidelines proposed by the World Health Organization in 1999: fasting blood sugar no less than 7.0 mmol/l and the 2 h postprandial blood sugar no less than 11.1 mmol/l. Other metabolic conditions, such as acute and chronic complications of diabetes mellitus, T1DM, ketosis, hepatic and renal dysfunction, and other conditions were excluded. (c) Officially published case-control or cohort studies. (d) Genotype in control group follows Hardy-Weinberg equilibrium (HWE).
The following electronic databases were used to conduct a search: China National Knowledge Infrastructure, VIP database, Wanfang database, China Biological Medicine Database, and PubMed. Keywords used for the search were "SUMO4, rs237025, diabetes. " When searching Chinese databases, researchers employed corresponding Chinese terms. Nine publications were retrieved in our initial search of the Pubmed database and four papers met the inclusion criteria for this meta-analysis. Using keyword combination "SUMO4, Met55Val, diabetes" yielded three additional papers, all of which were eligible for our analysis. The eligible studies' characteristics conform to the above inclusion criteria. Finally, "SUMO4, M55V, type 2 diabetes" retrieved another two additional papers. Both papers met inclusion criteria. Additional three Chinese papers were retrieved in the China National Knowledge Infrastructure database by using the keywords combination as "small ubiquitin-like modifier 4, Met55Val, diabetes. " Retrieved studies were published between 2003 and 2017.

Data extraction
Data were extracted by three investigators using a standardized protocol ( Table 1). Two investigators were responsible for identifying duplicate studies while the third acted as the mediator to resolve any disagreements between them. Studies that deviated from the major inclusion criteria were published in duplicate, or provided insufficient data were rejected. Similar data sets published in different articles by a single author group were adopted once in the meta-analysis. Items, such as the first author's name, publication year, ethnicity, matching criteria, genotype number, and total number of cases and controls, are displayed in Table 1.

statistical analysis
Statistical analyses were performed by using Revman 5.0 and STATA 12.0 software (StataCorp, College Station, TX, USA). Six genetic models, allelic (V allele distribution frequency), recessive (VV vs. MV + MM), dominant (MM vs. MV + VV), homozygous (VV vs. MM), heterozygous (MV vs. MM), and additive (total V vs. total M), were used in the current meta-analysis. The association of SUMO4 gene M55V polymorphism and T2DM were compared by using the odds ratios (ORs) corresponding to its 95% confidence intervals (CIs). The heterogeneity between the individual studies was evaluated by using the Chi-square-based Q-test with significance set at P < 0.05 level (18). If heterogeneity was detected in the studies, the random-effects model (DerSimonian and Laird method) would be used to estimate the pooled OR (19). If not, the fixedeffects model (the Mantel-Haenszel method) would be used (20). The combined OR was determined by Z-test with the significance set at P < 0.05 level.
Fisher's exact test was used to assess HWE with the significance set at P < 0.05 level. The potential publication bias was evaluated by the funnel plot. The funnel plot symmetry was assessed by using the Egger's linear regression test on the OR and the significance was set at P < 0.05 level (21).

Pooled analyses
In the subgroup analysis, a significant association was also found in the Chinese population under allelic (OR: 1.  As no significant heterogeneity was detected under allelic, dominant, homozygous, heterozygous, or additive genetic models (Pheterogeneity > 0.05), a fixed-effect model was used to determine the pooled OR. While only under the recessive genetic model, significant heterogeneity was detected (Pheterogeneity < 0.05) and random-effect model was used ( Table 2).

Bias Diagnostics
The publication bias of the individual studies was evaluated by using the funnel plot. No visual evidence for publication bias was evident in the funnel plot under the recessive genetic model (Figure 7). No significant publication bias was detected in this meta-analysis (T = 1.54, P = 0.159) under the recessive genetic model by using Egger's test (Figure 8).

sensitivity analysis
The removal of any one study from the meta-analysis did not change the significant association between SUMO4 gene M55V polymorphism and T2DM under the additive genetic model, suggesting that the results are stable and robust (Figure 9).

DiscUssiOn
In the current meta-analysis, a significant association was detected between SUMO4 gene M55V polymorphism and T2DM under SUMO4 Gene M55V Polymorphism and T2DM Frontiers in Endocrinology | www.frontiersin.org November 2017 | Volume 8 | Article 303    Heterogeneity was reduced in the Chinese and Japanese populations, suggesting that ethnicity was the main source for the heterogeneity. As only one study of the Iranian population was present in our analysis, the heterogeneity detection was not applicable. The SUMO4 gene M55V polymorphism is located in the evolutionarily crucial CUE structural domain and involves the substitution of the highly conserved Met (ATG) for Val (GTG). This substitution could modify site at which PKC phosphorylates the protein. The change in molecular conformation and functional activity reduces its ability to regulate NF-κB, resulting in increased NF-κB activation enhancement and overexpression of NF-κB-dependent gene products. In 2004, Guo et al. found that the M55V substitution resulted in 5.5 times greater NF-κB transcriptional activity and approximately two times greater expression of IL12B (4).
Nuclear factor-κB activation is an important molecule in the inflammatory response (29), upregulation of endothelin (30), and apoptosis (31). NF-κB activation may also promote apoptosis in both pericytes and endothelial cells in the pathogenesis of diabetes retinopathy (32).
In recent years, T2DM has been increasingly associated with mild, but systemic chronic inflammation that is characteristic of obesity and metabolic syndrome. The disruption of the internal equilibrium by factors, such as stress and poor nutrition, activates the inflammatory response as a protective response. Initially, the body is in a peri-inflammatory state, but long-term activation of this state can result in chronic inflammation (33). NF-κB is an important inflammatory factor involved in IR, but also acts as transcription factor for other pro-inflammatory proteins (34). The pivotal studies have also found that the SUMO4 cytosis substrate included the anti-oxidative stress proteins, the regulation proteins for DNA repair and synthesis, protein degradation associated proteins, glycometabolism-associated proteins under stress conditions induced by starvation (35). These findings point toward the functional complexity of SUMO4, which warrants further investigation and discussion.
In 2012, Tang et al. performed a meta-analysis on the relationship between SUMO4 gene M55V polymorphism and T2DM where they found a significant association between them in the Asian population (P < 0.05) (36). However, only six individual studies were included and only four genetic models were adopted in their meta-analysis. In addition, with no subgroup analysis stratified by ethnicity, their conclusion may be more limited to the analysis we offer in this current study. In 2017, Zhang et al. performed a meta-analysis on this subject and came to a similar conclusion (37). However, only five Chinese individual studies published before 2012 were included in Zhang's meta-analysis and five genetic models were merely used. While in the current meta-analysis, six genetic models were used and the publications in 2015 and 2017 were also included. Hence, our study increased the number of studies to seven individual studies in the Chinese population and offers an updated and more comprehensive result.
This study is not without limitations. Many factors can influence plasma concentrations of SUMO4, such as SUMO4 rs237024 and rs600739 polymorphisms, diet, smoking, and hypertension (16). In addition, the current meta-analysis lacks large-scale studies on the subject. This variant has been studied only in Chinese, Japanese, and Iranian subjects. No study in other ethnicities has been found. Furthermore, no data from GWA studies on the region that includes this variant have been found. Much remains to be clarified on how the SUMO4 gene M55V polymorphism affects patient T2DM susceptibility.
In conclusion, we found a significant association between SUMO4 gene M55V polymorphism and T2DM risk in the current meta-analysis. Individuals with the Val allele of SUMO4 gene M55V polymorphism may be more susceptible to T2DM in the Chinese population. This association in Japanese or Iranian population needs to be further verified in the larger samples studies. This conclusion may help researchers formulate an individual treatment strategy to prevent T2DM in the future.
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