in vivo 28: 919-924 (2014) Association of SUMO4 M55V and -94Ins/Del Gene Variants with Type-2 Diabetes SEYMA SOZEN1, CEM HOROZOGLU1, ELIF SINEM BIRELLER1, ZEYNEP KARAALI2 and BEDIA CAKMAKOGLU1 1Department of Molecular Medicine, Institute for Experimental Medicine Research, Istanbul University, Istanbul, Turkey; 2Department of Internal Medicine, Haseki Training and Research Hospital, Istanbul, Turkey Abstract. Aim: There are two different types of diabetes suggested a common underlying genetic basis (2, 3). To date, mellitus, type 1 and type 2, with still unclear molecular only some shared susceptibility genes have been reported. mechanisms. In the present study, we aimed to investigate the Ikegami and Ogihara have indicated that type-1 diabetes is role of small ubiquitin-like modifier 4 (SUMO4) M55V and the result of an absolute loss of endogenous insulin due to nuclear factor kappa B1 (NFKB1)-94del/ins in type-2 its autoimmune destruction of insulin-producing cells of the diabetes mellitus. Materials and Methods: We analyzed pancreas, whereas type-2 diabetes occurs due to de-regulated SUMO4 M55V and NFKB1-94del/ins variants in 104 insulin secretion and/or insulin sensitivity and shows patients with type-2 diabetes and 124 healthy controls using variability within each individual (1). The human leukocyte the polymerase chain reaction (PCR) and restriction antigen (HLA) region for insulin-dependent diabetes mellitus fragment length polymorphism (RFLP) techniques. Results: 1 (IDDM1), which is a major susceptibility locus for type-1 The number of SUMO4 M55V MM genotype and M allele diabetes, has also been associated with type-2 diabetes (4, carriers was significantly higher in patients compared to the 5). The IDDM5 locus has been mapped to an approximately control group; however, no efficiency results were found 200-kb interval on chromosome 6q25 (6, 7). This new locus related to NFΚB1-94del/ins polymorphism. Conclusion: It contains two genes, small ubiquitin-like modifier 4 (SUMO4) was found that SUMO4 M55V polymorphism and type-2 and MAPK kinase 7 interacting protein 2 (MAP3K7IP2). diabetes were significantly associated with a possible These may be involved in autoimmunity and inflammation SUMO4 region to type-2 diabetes susceptibility. This through regulation of nuclear factor kappa beta (NF-κB) and preliminary study showed that the distribution of SUMO4 heat shock transcription factor activator 6q25 (6-8). NF-κB is M55V and type-2 diabetes mellitus in Turkish patients may a transcription factor, which plays important roles in form the basis of future research. inflammation, cell survival, angiogenesis and apoptosis. It is located at the signaling pathway terminal of the activated T Diabetes mellitus, a disease described as hyperglycemia cells (9). depending on relative or absolute deficiency of circulating The SUMO4 gene is primarily expressed in the kidney and insulin, comprises of two major forms with distinct immune system (6). Because SUMO4 is located at the sixth pathogeneses, type 1 and type 2. Genetic factors including intron of the mitogen-activated protein (MAP), this location several predisposing genes and environmental effects are indirectly regulates the activation of NFκB in response to involved in both types (1). Although there are distinctive interlukin-10 (IL-10) (10, 11). SUMO4 may regulate the differences in disease pathogenesis, epidemiological data immune response through the substrate inhibitor (IkBα), a show that familial clustering of type 1 and type 2 has 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 Correspondence to: Bedia Cakmakoglu, Department of Molecular frees NFκB to translocate to the nucleus (12). Human SUMO Medicine, Institute for Experimental Medicine Research, Istanbul protein has similar structures to ubiquitine that could University, Vakif Gureba Cad. Capa 34390, Istanbul, Turkey. Tel: conjugate at the same site on IkBα (13). +092 124142000-33305, Fax: +902 125324171, e-mail: [email protected] It has been reported that single nucleotide polymorphisms (SNPs) are related with type-1 diabetes including SUMO4 Key Words: NFKB1, polymorphism, SUMO4, type 2 diabetes. M55V located at the SUMO4/MAP3K7IP2 region in diverse 0258-851X/2014 $2.00+.40 919 in vivo 28: 919-924 (2014) ethnic groups (6, 7). Although there have been reproducible Results association data from Eastern Asian populations (14, 15), results from Caucasian populations showed disparity (6, 7). Demographic data for the study groups are given in Table I. SUMO4 M55V was considered as a causative variant of A statistically significant difference was observed in the IDDM, but with alleles carrying contrary risk (15, 16). A distribution of genotype and allele frequencies in the patient study of 404 patients with type-2 diabetes and 500 controls and control groups for SUMO4 gene polymorphism (p=0,000 in Beijing, China, revealed that carriers of the G allele of χ2=17,56 and p=0,005, χ2=7,84 respectively, Table II). SUMO4 M55V displayed greater risk for type-2 diabetes SUMO4 MV genotype in the control group was statistically (16). Moreover, G allele frequency was markedly increased higher than the patient group and this genotype was four in 403 patients with type-2 diabetes and diabetic nephropathy times more likely to protect against type 2 diabetes (p=0,000, in a study from Taiwan (17). Noso et al. obtained significant χ2=24,47, Odds Ratio (OR)=0,23, %95 Confidence Interval results from 355 Japanese patients on the relationship (CI)=0,12-0,42). SUMO4 MM genotype in the patient group between type-2 diabetes with SUMO4 (18). was also statistically higher than in the control group and this A gene expression study through microarray showed that genotype had 4.23 times higher risk for developing type 2 SUMO4 was involved in the pathogenesis of type-2 diabetes diabetes (p=0,000, χ2=21,08, OR=4,23 95% CI=2,37-8,04). and the authors nominated SUMO4 as a new therapeutic Carriers of SUMO4 V+ (VV + MV) genotype in the control target (19). Nevertheless, in Western populations no group (84,6%) was higher than in the patient group (56,5%) association has been found between this variant and type-2 and was three times more likely to protect against type-2 diabetes (13, 20, 14). diabetes (p=0,000, χ2=21,08, OR=0,23; 95% CI=0,12-0,44; Taking into consideration the functional and genetic Data not shown in table). No statistical difference was evidence, we aimed to screen for the SUMO4 M55V and observed in the distribution of genotype and allele NFKB1-94del/ins gene variants in Turkish patients with type frequencies in the patient and control groups for NFΚB1 2 diabetes mellitus. gene polymorphism (p>0.05) (Table III). Materials and Methods Discussion Subjects. In total, 228 Turkish subjects, 104 patients with type 2 In the present study we screened 104 patients with type-2 diabetes (68 women and 36 men) and 124 healthy control subjects diabetes and 124 healthy controls for SUMO4 M55V and (76 women and 48 men), were enrolled to our study. The median age of patients with type 2 diabetes and control subjects with no NFΚB1-94ins/del variants to investigate the role of genetic clinical diabetes and no family history of diabetes was 51.5±13.3 variations on the risk of type-2 diabetes. Our results showed and 48.4±10.8 years, respectively. a statistically significant difference in the distribution of genotype and allele frequencies for SUMO4 between patients DNA isolation. After obtaining written informed consent from the and controls. SUMO4 MM genotype was higher in the participants and approval from the Istanbul University’s Ethics patient group than in the controls and had 3.48-times higher Committee, blood specimens were collected in tubes containing risk for developing type-2 diabetes. On the other hand ethylenediaminetetraacetic acid (EDTA). DNA was ex¬tracted from peripheral blood lymphocytes using the salting-out procedure (21). persons who carriers of SUMO4 V allele (VV and MV) had three times protection against to type-2 diabetes. Genotyping. Polymorphisms were genotyped using polymerase chain Genome-wide studies have shown the linkage of type-2 reaction (PCR)/restriction fragment length polymorphism (RFLP) diabetes at chromosome 6q where a susceptibility gene for methods. PCR was used to amplify the region of SUMO4 M55V type 1 diabetes (1DDM5) was mapped, in African–American polymorphism and the NFKB1 94ins/del polymorphism (22, 23). (24), Chinese (25) and Finnish populations (26). Genes on PCR-RFLP with Tru1I restriction enzyme (MBI Fermentas, 6q22–26 that influence fasting serum insulin levels have also Lithuania) at 65˚C overnight and Van91I restriction enzyme (MBI been reported (27). In this respect, chromosome 6q may Fermentas, Lithuania) at 65˚C for 2 hours for SUMO4 and NFKB1, respectively, and all the final samples were evaluated using agarose harbor susceptibility genes for type 1 and type 2 diabetes. gel electrophoresis (2%). The obtained bands of 182 bp, 161 and 21 Controversial results were obtained from genotyping studies bp were evaluated as the MM, VV and MV, respectively for of distinct ethnic groups including Asians and Europeans. SUMO4. Accordingly, the bands of 254 bp, 206 and 48 bp were Noso et al. revealed the association between SUMO4 M55V evaluated as WW, DD and WD, respectively for NFKB1. variant and type-1 and type-2 diabetes (19). Studies from Japan have implied a relationship between SUMO4 variant Statistical analysis. The SPSS 11.0 software was used for statistical and type-1 diabetes in the Asian population. Moreover, other analysis (SPPS Inc., Chicago, IL, USA). Chi square test and Fisher test were used to assess the differences of genotype and allele studies have suggested the possibility of a contribution of the frequency between the two groups.