DIABETES MELLITUS: A COMPLEX METABOLIC DISORDER.EXPLORING THE DISEASE THROUGH FRUCTOSAMINE 3-KINASE GENE ANALYSIS

Diabetes Mellitus is the most prevalent metabolic disorder characterized by chronic hyperglycemia due to primary defects in insulin secretion and/or insulin function. In the last few decades the notion of diabetes has widened, ascertain that the present subdivision into type 1 (T1DM) and type 2 (T2DM) diabetes is a gross oversimplification. Both forms of diabetes seem to result from a complex interplay between genes and environment. Advance technologies have revolutionized the search for genetic influences on complex traits. Genomewide association studies (GWAS) and GWAS meta-analyses have been the most efficient way to identify new T2DM genes. However, despite these advances, the overall effect attributed to these loci is low and their contribution is of little clinical usefulness compared to evaluation of classical risk factors such as body mass index (BMI), age and family history. Therefore, evaluation of glycemic control remain the primary target for diabetes treatments. However, understanding and utilization of single gene effects on specific traits that conglomerate into a complex phenotype is currently the best way to understand the genetic basis at functional level and could results in a possible advantage for disease prevention and management. In this light, to explore the complexity of this disorder and to give some hints in the pathogenesis of diabetes, we have followed a candidate gene approach by studying the Fructosamine 3-kinase (FN3K) gene, whose product is implicated in non-enzymatic glycation of proteins, in an Italian cohort of diabetic individuals. Glycation has long been considered irreversible. Thus, the identification of an enzyme, FN3K, able to reverse this process by decomposing fructosamine 3-phosphate to 3-deoxyglucosone, inorganic phosphate and an amine, opened the perspective that fructosamines could be physiologically removed by proteins, suggesting a protective role in the development of diabetic complications and other pathologies characterized by high fructosamines/AGEs levels. First aim of the present study was to accomplish the analysis of the FN3K gene in a well clinically characterized group of Italian individuals with diabetes (35 T1DM and 35 T2DM) belonging to ADAG study and 33 healthy subjects, by analyzing its promoter region. Then, the FN3K gene (promoter region and all six exons with corresponding intron/exon boundaries) were analyzed in additional 80 T2DM subjects, followed since long time in diabetic clinic. The molecular screening revealed the presence of 15 different genetic variants. Four of them represented new mutations: the c.2 T>A (p.M1?) in the translation starting codon; the c.465 G>A (p.P115=) located in a consensus sequence for the splicing site; the c.559 C>T (p.R187*) leading to the formation of a truncated protein; the missense mutation c.716 A>G (p.Y239C) in exon 6. Presence of these variants were excluded from control group using DHPLC analysis. Other 11 variants identified were polymorphisms; of them 3 were new: the IVS2-27 A>G in intron 2; the c.421 C>T and c.429delATCGGAG in the promoter region. The remaining 8 were polymorphisms already described: c. -232 A>T and c.-385 A>G in the promoter region; c.187 A>C (p.R63=) in exon 2; c.900 C>G (p.S300=) and c.906 C>T (p.G302=) in exon 6; polymorphisms IVS+26 G>A, IVS+31 A>T and IVS4-9-11delTTG were present in non coding regions of the FN3K gene (intron 2, intron 2 and intron 4, respectively). An RNA expression study was performed on new variants (c.2 T>A; c.465 G>A; c.559 C>T and c.716 A>G) in order to confirm the hypothesis on pathogenicity of these variants found at genomic level. Furthermore, mutations c.559 C>T (p.R187*) and c.716 A>G (p.Y239C) were analyzed in a familial context. The RNA analysis didnft confirm the result predicted by in silico modelling, suggesting that these variants might exert a pathological function with other mechanisms or interacting with other polymorphisms present in non coding regions of the FN3K gene where they may produce a subtle difference in regulation expression. A second aim was to find a correlation between genotypes and some clinical parameters typical of diabetes, to offer a better comprehension of glycemic control and its predictive role in the middle and log period. A genotype composed by the combination of 3 polymorphisms (c.-385 A>G; c.-232 A>T; c.900 C>G) mostly associated with a variation in FN3K enzymatic activity and with HbA1c values was considered. On the bases of glycated hemoglobin values of each patients, the identified genotypes were compared on HbA1c mean values for the different groups. The analysis didnft find out a difference in HbA1c values among different groups. However, two interesting observations came out: the genotype containing the favorite alleles for the 3 polymorphisms (GG at c.-385 A>G; TT at c.-232 A>T; CC at c.900 C>G) seemed to be related to a low concentration of HbA1c; patients with different complications (macro- or micro-vascular) displayed different genotypes. The role of FN3K variants in development or progression of diabetes remain unclear. The lack of an association between polymorphisms identified in the FN3K gene and diabetes explains that probably this enzyme cannot by itself account for the entire deglycation story. It alone cannot be responsible for the susceptibility to this pathology or for the development of its complications. Moreover, recent studies provided experimental evidences for epigenetic mechanisms as plausible means by which environmental factors integrate their effects with genetic variants to mediate T2DM risk. Current research provides insights for the importance of nutrition in terms of health and disease prevention. Studies on the gepigenetic dieth have revealed that the consumption of some foods like soy, curry spices, red grapes, as well as blueberries has beneficial effects on the prevention of diseases. One way of exerting its impact is through modulation of non-coding RNA levels, microRNAs (miRNAs), short (.22 nucleotides) non-coding regulatory elements functioning as translational repressors. miRNAs have an important role in many cellular processes and are proposed as promising pharmaceutical targets in various fields, such as cancer and metabolic diseases. Recent findings have shown that altered circulating miRNA profiles were linked to pathological conditions, thus raising the possibility of their use as promising non-invasive biomarkers for the detection, classification and prognosis of diseases. We had then the opportunity to collaborate with the University of Hull (UK), involved in understanding the effect of soy phytoestrogen in the management of diabetes. The potential health benefits of soy are widely publicized. These legumes contain complex carbohydrates,vegetable protein, soluble fibers, oligosaccharides, minerals, and phytoestrogens, particularly the isoflavones genistein and daidzein. Two hundred men with T2DM and compensated hypogonadism were randomized and administered either 30 g of soy protein with 66 mg of isoflavones per day, or 30 g soy protein alone without isoflavones for 12 weeks. Given the estrogenic effect of phytoestrogens, the hypothesis to test was if there were any effects of soy with and without isoflavones on testosterone, this would be exaggerated in men with low testosterone levels. The primary outcome of this study was the change in testosterone levels. The secondary outcomes were changes in glycemic control and cardiovascular risk markers including insulin resistance, lipid profile, highly sensitive CRP (hsCRP) and endothelial function. miRNA expression profile in peripheral plasma samples of 10 selected subjects under active treatment with soy and isoflavons, was performed to evaluate a possible change in expression level after treatment. Fifty-seven circulating miRNAs differed for about 2 fold between pre- and post-treatment, including 7 up-regulated and 50 suppressed miRNAs. Among them miR-34a-5p, miR-144-3p and miR-19b-3p differed significantly between pre- and post- treatment. In this study, after soy supplementation changes in testosterone levels, in glycemic control and cardiovascular risk markers were observed. Moreover, the number of expressed miRNA seemed to increase after treatment. A consistent and truly significant effect of soy on the miRNAs plasma profile could not be demonstrated, since a validation study is needed to confirm these findings and proceed with further evaluation. However, these preliminary results are encouraging for future investigations on soy isoflavones potential and for miRNA utilization as biomarkers. In conclusion, our findings provide new insights in FN3K genetics and give further attempts on the hypothesis that phenotype might be influenced by allelic heterogeneity and/or mutations at multiple modifier genes. It would be interesting to analyze the FN3K gene in subjects belonging to the soy and testosterone study to evaluate a possible concomitant effect of FN3K genetic variants on the progression of the disease.