Abstract
Aim: There are two different types of diabetes mellitus, type 1 and type 2, with still unclear molecular mechanisms. In the present study, we aimed to investigate the role of small ubiquitin-like modifier 4 (SUMO4) M55V and nuclear factor kappa B1 (NFKB1)-94del/ins in type-2 diabetes mellitus. Materials and Methods: We analyzed SUMO4 M55V and NFKB1-94del/ins variants in 104 patients with type-2 diabetes and 124 healthy controls using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques. Results: The number of SUMO4 M55V MM genotype and M allele carriers was significantly higher in patients compared to the control group; however, no efficiency results were found related to NFKB1-94del/ins polymorphism. Conclusion: It was found that SUMO4 M55V polymorphism and type-2 diabetes were significantly associated with a possible SUMO4 region to type-2 diabetes susceptibility. This preliminary study showed that the distribution of SUMO4 M55V and type-2 diabetes mellitus in Turkish patients may form the basis of future research.
Diabetes mellitus, a disease described as hyperglycemia depending on relative or absolute deficiency of circulating insulin, comprises of two major forms with distinct pathogeneses, type 1 and type 2. Genetic factors including several predisposing genes and environmental effects are involved in both types (1). Although there are distinctive differences in disease pathogenesis, epidemiological data show that familial clustering of type 1 and type 2 has suggested a common underlying genetic basis (2, 3). To date, only some shared susceptibility genes have been reported. Ikegami and Ogihara have indicated that type-1 diabetes is the result of an absolute loss of endogenous insulin due to its autoimmune destruction of insulin-producing cells of the pancreas, whereas type-2 diabetes occurs due to de-regulated insulin secretion and/or insulin sensitivity and shows variability within each individual (1). The human leukocyte antigen (HLA) region for insulin-dependent diabetes mellitus 1 (IDDM1), which is a major susceptibility locus for type-1 diabetes, has also been associated with type-2 diabetes (4, 5). The IDDM5 locus has been mapped to an approximately 200-kb interval on chromosome 6q25 (6, 7). This new locus contains two genes, small ubiquitin-like modifier 4 (SUMO4) and MAPK kinase 7 interacting protein 2 (MAP3K7IP2). These may be involved in autoimmunity and inflammation through regulation of nuclear factor kappa beta (NF-κB) and heat shock transcription factor activator 6q25 (6-8). NF-κB is a transcription factor, which plays important roles in inflammation, cell survival, angiogenesis and apoptosis. It is located at the signaling pathway terminal of the activated T cells (9).
The SUMO4 gene is primarily expressed in the kidney and immune system (6). Because SUMO4 is located at the sixth intron of the mitogen-activated protein (MAP), this location indirectly regulates the activation of NFκB in response to interlukin-10 (IL-10) (10, 11). SUMO4 may regulate the immune response through the substrate inhibitor (IkBα), a suppressor of NFκB, followed by activation of NFκB (7). SUMO4 is an opposite regulator of NFκB. Upstream of NFκB in the signaling pathway, ubiquitination of IkB is the premise of proteasome-mediated degradation of IkBα that frees NFκB to translocate to the nucleus (12). Human SUMO protein has similar structures to ubiquitine that could conjugate at the same site on IkBα (13).
It has been reported that single nucleotide polymorphisms (SNPs) are related with type-1 diabetes including SUMO4 M55V located at the SUMO4/MAP3K7IP2 region in diverse ethnic groups (6, 7). Although there have been reproducible association data from Eastern Asian populations (14, 15), results from Caucasian populations showed disparity (6, 7).
SUMO4 M55V was considered as a causative variant of IDDM, but with alleles carrying contrary risk (15, 16). A study of 404 patients with type-2 diabetes and 500 controls in Beijing, China, revealed that carriers of the G allele of SUMO4 M55V displayed greater risk for type-2 diabetes (16). Moreover, G allele frequency was markedly increased in 403 patients with type-2 diabetes and diabetic nephropathy in a study from Taiwan (17). Noso et al. obtained significant results from 355 Japanese patients on the relationship between type-2 diabetes with SUMO4 (18).
A gene expression study through microarray showed that SUMO4 was involved in the pathogenesis of type-2 diabetes and the authors nominated SUMO4 as a new therapeutic target (19). Nevertheless, in Western populations no association has been found between this variant and type-2 diabetes (13, 20, 14).
Taking into consideration the functional and genetic evidence, we aimed to screen for the SUMO4 M55V and NFKB1-94del/ins gene variants in Turkish patients with type 2 diabetes mellitus.
Materials and Methods
Subjects. In total, 228 Turkish subjects, 104 patients with type 2 diabetes (68 women and 36 men) and 124 healthy control subjects (76 women and 48 men), were enrolled to our study. The median age of patients with type 2 diabetes and control subjects with no clinical diabetes and no family history of diabetes was 51.5±13.3 and 48.4±10.8 years, respectively.
DNA isolation. After obtaining written informed consent from the participants and approval from the Istanbul University's Ethics Committee, blood specimens were collected in tubes containing ethylenediaminetetraacetic acid (EDTA). DNA was ex¬tracted from peripheral blood lymphocytes using the salting-out procedure (21).
Genotyping. Polymorphisms were genotyped using polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) methods. PCR was used to amplify the region of SUMO4 M55V polymorphism and the NFKB1 94ins/del polymorphism (22, 23).
PCR-RFLP with Tru1I restriction enzyme (MBI Fermentas, Lithuania) at 65°C overnight and Van91I restriction enzyme (MBI Fermentas, Lithuania) at 65°C for 2 hours for SUMO4 and NFKB1, respectively, and all the final samples were evaluated using agarose gel electrophoresis (2%). The obtained bands of 182 bp, 161 and 21 bp were evaluated as the MM, VV and MV, respectively for SUMO4. Accordingly, the bands of 254 bp, 206 and 48 bp were evaluated as WW, DD and WD, respectively for NFKB1.
Statistical analysis. The SPSS 11.0 software was used for statistical analysis (SPPS Inc., Chicago, IL, USA). Chi square test and Fisher test were used to assess the differences of genotype and allele frequency between the two groups. Comparison of intergroup demographic data was determined by using the Student's t-test.
Results
Demographic data for the study groups are given in Table I. A statistically significant difference was observed in the distribution of genotype and allele frequencies in the patient and control groups for SUMO4 gene polymorphism (p=0,000 χ2=17,56 and p=0,005, χ2=7,84 respectively, Table II). SUMO4 MV genotype in the control group was statistically higher than the patient group and this genotype was four times more likely to protect against type 2 diabetes (p=0,000, χ2=24,47, Odds Ratio (OR)=0,23, %95 Confidence Interval (CI)=0,12-0,42). SUMO4 MM genotype in the patient group was also statistically higher than in the control group and this genotype had 4.23 times higher risk for developing type 2 diabetes (p=0,000, χ2=21,08, OR=4,23 95% CI=2,37-8,04). Carriers of SUMO4 V+ (VV + MV) genotype in the control group (84,6%) was higher than in the patient group (56,5%) and was three times more likely to protect against type-2 diabetes (p=0,000, χ2=21,08, OR=0,23; 95% CI=0,12-0,44; Data not shown in table). No statistical difference was observed in the distribution of genotype and allele frequencies in the patient and control groups for NFKB1 gene polymorphism (p>0.05) (Table III).
Discussion
In the present study we screened 104 patients with type-2 diabetes and 124 healthy controls for SUMO4 M55V and NFKB1-94ins/del variants to investigate the role of genetic variations on the risk of type-2 diabetes. Our results showed a statistically significant difference in the distribution of genotype and allele frequencies for SUMO4 between patients and controls. SUMO4 MM genotype was higher in the patient group than in the controls and had 3.48-times higher risk for developing type-2 diabetes. On the other hand persons who carriers of SUMO4 V allele (VV and MV) had three times protection against to type-2 diabetes.
Genome-wide studies have shown the linkage of type-2 diabetes at chromosome 6q where a susceptibility gene for type 1 diabetes (1DDM5) was mapped, in African–American (24), Chinese (25) and Finnish populations (26). Genes on 6q22–26 that influence fasting serum insulin levels have also been reported (27). In this respect, chromosome 6q may harbor susceptibility genes for type 1 and type 2 diabetes. Controversial results were obtained from genotyping studies of distinct ethnic groups including Asians and Europeans. Noso et al. revealed the association between SUMO4 M55V variant and type-1 and type-2 diabetes (19). Studies from Japan have implied a relationship between SUMO4 variant and type-1 diabetes in the Asian population. Moreover, other studies have suggested the possibility of a contribution of the SUMO4/MAP3K7IP2 region to both type-1 and type-2 diabetes (19, 28). In this study, it was shown that the GG allele frequency was higher in patients who were independent of exogenous insulin than that of insulin-dependent patients, suggesting that insulin secretion was adequate in patients with higher G allele frequency; however, insulin resistance may exist. The M55V substitution increased the pathogenesis of chronic inflammation by diminishing SUMO4 inhibition on the activity of NFκB; therefore, it was considered as a risk factor for type-2 diabetes (19).
Demographic data of the study groups.
A study from Iran showed that the M55V polymorphism of SUMO4 gene was not associated with type-2 diabetes in Iranian subjects, but the author highlighted that this result may have occurred due to the number of samples (29). No association was found with type-1 diabetes in two studies from Western populations that investigated the SUMO4 M55V variant: (30) in Lithuania, 100 type-1 diabetes patients and 90 controls (30) and in Sweden, 673 type-1 diabetes and 535 controls (20). Guo et al. in a genotyping and functional study reported that the M55Vsubstitution resulted in 5.5-times greater NFκB-transcriptional activity and almost 2-times greater expression of IL12B, a NFκB dependent gene in 944 patients with type-1 diabetes from European, American, Italian, French, Spanish, Mexican and Asian populations. A new pathway might be involved in the pathogenesis of type-1 diabetes (7). No association with type-1 diabetes has been indicated in populations from the United Kingdom, the United States of America, Ireland, Finland, Romania and Norway (15). Based on these results, Lin et al. (31) found a significant association of SUMO4 M55V polymorphism with type-1 diabetes in their meta-analysis of 9,190 cases and 10,456 healthy controls (including sixteen case-control studies) in an Asian population and a significant association of SUMO4 M55V polymorphism with type-1 diabetes in a Caucasian population .
Six different studies based on the Asian population were included in this meta-analysis concerning the correlation of M55V and type 2 diabetes (19, 26, 32-36). A significant association of SUMO4 M55V polymorphism with type-2 diabetes was observed but all type-2 diabetes patients were Asians in this study. There are insufficient numbers of studies in Western populations related to type-2 diabetes and SUMO4 M55V. In the literature no consensus has been made on M55V substitution and its relationship with type-1 and type-2 diabetes. Asian and Caucasian populations were mostly investigated in studies. Genetic heterogeneity has to be considered when assessing the results.
Distribution of SUMO4 and allele frequencies in patients and control groups.
Distribution of NFKB1 genotype and allele frequencies in patients and control groups.
Activation of NFκB could be depressed by SUMO, which regulates modification of IκBα (12, 13). In several studies, the suppression of autoimmune responses by down-regulation of the transcription factor NFκB has been found to be related with the SUMO4 function. Abnormal NFκB signaling pathway activation has also been related with type 1 diabetes. IκBα is a crucial factor for SUMO4 in the regulation of the NFκB pathway because it is one of the most important inhibitors for NFκB (37). NFκB, a transcriptional factor for inflammation, is relevant with the pathogenesis of diabetes mellitus. Moreover, the interaction between NFκB and IkBα is crucial for insulin resistance (38). Insulin resistance could be stimulated by the activation of the inflammation signaling pathway. It was concluded that this was a functional polymorphism, which modifies the association between polyunsaturated fatty acids (PUFA) intake and plasma high-density lipoprotein (HDL)-cholesterol in two distinct populations. Furthermore, we genotyped NFKB1-94ins/del polymorphism in Turkish patients as a possible candidate that may be linked to type 2 diabetes.
Conclusion
Based on our Turkish patients' data, we showed that type-2 diabetes was significantly associated supporting the possibility of a contribution of the SUMO4 region to type 2 diabetes susceptibility with SUMO4 M55V polymorphism. It was concluded that SUMO4 affects type-2 independently from NFKB1, because no association with type 2 diabetes and the NFKB1 -94ins/del variant was shown in our study.
Acknowledgements
This work was supported by the Research Fund of Istanbul University. (Project No: 6743). We would like to thank to Mr. David Chapman for English editing.
Footnotes
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Conflicts of Interest
None declared.
- Received May 28, 2014.
- Revision received July 9, 2014.
- Accepted July 11, 2014.
- Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
