The complex relationship between adenosine, obesity and Type 2 Diabetes (T2D) prompted us to investigate a possible role of genetic variability within adenosine deaminase (ADA) gene in the susceptibility to T2D in obese subjects.Â  With enzymatic activity ADA contributes to the regulation of adenosine concentration in body fluids and as ecto- enzyme modulates signaling events involving adenosine receptors on cell surface.Three polymorphic sites within the ADA gene, ADA1 (exon 1), ADA2 (intron 2) and ADA6 (exon6) were examined in 299 subjects from the White population of Rome admitted to the Hospital for cardiovascular diseases. Each site shows two alleles: the alleles with higher frequency are ADA1*1, ADA2*1 and ADA6*2.Informed consent was obtained by the patients to participate to the study that was approved by the Council of Department.For the three sites examined the relative risk of T2D in obese subjects (vs non obese) is higher in carriers of the homozygous genotype for the allele with higher frequency. The three loci cooperate to the risk that is low in subjects carrying no homozygous genotype (O.R. 1.25) but it is very high (O.R. 13.240) in subjects carrying the three homozygous genotypes.From a practical point of view, the study of ADA gene may help to detect obese subjects at high risk of T2D who need active preventive measures. The results point to the importance of further studies on the role of genetic variability within ADA gene on the relationship within obesity and T2D

Banci, M.

Bottini, E.

Bottini, F. Gloria

Coppeta, L.

Magrini, A.

Neri, A.

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4 Journal of Clinical Medicine Research Updates, 2016, 3, 4-7    E-ISSN: 2413-7324/16  © 2016 Cosmos Scholars Publishing House Genetic Variability Within ADA Gene and Susceptibility to Type 2 Diabetes in Obese Subjects F. Gloria-Bottini1,*, M. Banci2, A. Neri1, L. Coppeta1, A. Magrini1 and E. Bottini1 1Department Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy 2Department of Cardiology, Valmontone Hospital, Italy Abstract: The complex relationship between adenosine, obesity and Type 2 Diabetes (T2D) prompted us to investigate a possible role of genetic variability within adenosine deaminase (ADA) gene in the susceptibility to T2D in obese subjects.  With enzymatic activity ADA contributes to the regulation of adenosine concentration in body fluids and as ecto- enzyme modulates signaling events involving adenosine receptors on cell surface. Three polymorphic sites within the ADA gene, ADA1 (exon 1), ADA2 (intron 2) and ADA6 (exon6) were examined in 299 subjects from the White population of Rome admitted to the Hospital for cardiovascular diseases. Each site shows two alleles: the alleles with higher frequency are ADA1*1, ADA2*1 and ADA6*2. Informed consent was obtained by the patients to participate to the study that was approved by the Council of Department.  For the three sites examined the relative risk of T2D in obese subjects (vs non obese) is higher in carriers of the homozygous genotype for the allele with higher frequency. The three loci cooperate to the risk that is low in subjects carrying no homozygous genotype (O.R. 1.25) but it is very high (O.R. 13.240) in subjects carrying the three homozygous genotypes. From a practical point of view, the study of ADA gene may help to detect obese subjects at high risk of T2D who need active preventive measures. The results point to the importance of further studies on the role of genetic variability within ADA gene on the relationship within obesity and T2D. Keywords: Obesity, T2D, ADA gene, Enzyme polymorphism, Heterozygote advantage. INTRODUCTION Several experimental observations suggest a complex relationship among adenosine, obesity and type 2 diabetes. An increase of adenosine A1 receptor activity is associated with obesity in T2D [1-3] and we have found an association between adenosine deaminase locus 1 (ADA1) genetic polymorphism and Body Mass Index (BMI) in T2D [4]. Adenosine may increase the sensitivity to insulin in adipose tissue [5-7] and in obese subjects a decrease of A1 receptors number has been reported [8]. Adenosine deaminase (ADA) controls the irreversible deamination of adenosine to inosine contributing to the regulation of adenosine concentration in body fluids. The biochemical genetic polymorphism of ADA was discovered in 1968 by Harris group [9] who identified by starch gel electrophoresis three common genotypes determined by the presence of two codominant alleles ADA1*1 and ADA1*2 at an autosomal locus. ADA1*1 is more enzymatically active as compared to ADA1*2.   *Address correspondence to this author at the Department of Biomedicine and Prevention, University of Tor Vergata, Via Montpellier, 1 00133 Rome, Italy; Tel: +39+6 30889514; Fax: +39+672596028;  E-mail: bottini@uniroma2.it The structure of ADA gene has been elucidated by Viginton et al. in 1986 [10]. The gene is localized on chromosome 20q13.11 and is composed by 12 exons spanning about 32 Kb of DNA. Several polymorphic loci have been identified within exonic and intronic regions of the gene [11]. The possible relationship between this genetic variability and human diseases is at present unknown. Besides the enzymatic function in deamination of adenosine, ADA acts also as ecto-enzyme contributing to the degradation of extra cellular adenosine and interacting with the anchoring proteins CD26 and A1R facilitates signaling events [12, 13]. In the present note we have studied the role of ADA genetic variability in the relationship between obesity and T2D in a sample of subjects admitted to the Hospital for cardiovascular diseases. The high susceptibility to T2D in obese subjects is well known: many obese subjects, however, do not become diabetic while non obese subjects may become diabetic suggesting that genetic factors have an important role in the relationship between obesity and diabetes. Indeed we have recently reported an effect of p53 codon 72 polymorphism on the susceptibility to Type 2 Diabetes in overweight subjects [14]. Genetic Variability within ADA Gene and Susceptibility to Type 2 Diabetes Journal of Clinical Medicine Research Updates, 2016, Vol. 3    5 We have examined three single nucleotide polymorphisms (SNPs) within the ADA gene: ADA1 (Taq 1 site nt 4050-4053) on exon 1 corresponding to the biochemical genetic polymorphism described by the group of Harris in 1968; ADA2  (Pst 1 site nt 19465-19470) localized on intron 2 and ADA6 (MluNI site nt 31230-31235) localized on exon 6. MATERIAL AND METHODS We have studied 299 subjects from the White population of Rome admitted consecutively to the Hospital for cardiovascular diseases. These patients have been considered also in previous studies [14]. Verbal consent was obtained by the patients to participate to the study that was approved by the council of department. The data have been collected a few years ago, before the establishment of an Ethical Committee. All procedures followed were in accordance with the ethical standards and with the Helsinki Declaration of 1964 and its later amendments. Genotypes of ADA1, ADA2 and ADA6 were determined by DNA analysis as previously described [15]. Statistical analyses were performed by commercial software (SPSS) [16]. The number of subjects is not exactly the same in all tables because of random loss of some genotype determination among the loci considered. Eta(η) is a measure of the strength of association between two variables. Eta squared (η2) represents the proportion of variance of dependent variable explained by the independent variable. RESULTS Table 1 shows some demographic data on the subjects studied. Table 1: Demographic Data on the Subjects Studied  Proportion Mean ±S.D. Females 47.4%  Age (years)  66.7±11.6 BMI  27.4±5.2  Table 2 shows the relative risk to become diabetic in obese subjects vs non obese subjects in relation to homozygosity for ADA1, ADA2 and ADA6 polymorphisms. For all ADA sites the relative risk to become diabetics is higher in homozygous subjects for the more common allele than in carriers of the less frequent allele. Table 2: The Effect of ADA1, ADA2 and ADA6 Genotypes on the Relative Risk of Diabetes in Obese Subjects vs Non Obese Subjects. Odds Ratio: Diabetes vs non Diabetes / BMI >30 vs BMI ≤ 30 ADA1 genotypes  *1/*1 Carriers of ADA1*2 allele N° of subjects 264 35 O.R. 5.81 3.50 95% C.I. 2.93 – 11.56 0.51 – 25.32 η2 0.125 0.066 ADA2 genotypes  *1/*1 Carriers of ADA2*2 allele N° of subjects 187 110 O.R. 8.52 3.28 95% C.I. 3.66 – 20.06 1.13 – 9.59 η2 0.184 0.056 ADA6 genotypes  *2/*2 Carriers of ADA6*1 allele N° of subjects 192 106 O.R. 5.91 4.23 95% C.I. 2.72 – 12.93 1.25 – 14.43 η2 0.137 0.069  Table 3 shows the relationship between the number of homozygous genotypes for the more frequent allele in the three ADA loci and the relative risk to become diabetics in obese subjects vs non obese subjects. Odds ratio and strength of association between obesity and T2D increases with the number of homozygous genotypes. Three-way contingency table analysis by a log linear model shows a statistically significant interaction among diabetes, BMI and ADA suggesting that the association between obesity and diabetes depends on ADA genotype. The analysis also shows a significant cooperative effect of ADA and obesity on the susceptibility to diabetes: the strength of association between ADA and diabetes, however, is lower as compared to the strength of association between obesity and diabetes. We have analyzed the effect of sex and age (≤65 years’ vs > 65 years) on the relationship between genetic variability of ADA gene and susceptibility to diabetes in obese subjects Odds Ratio diabetics vs non diabetics/ BMI>30 vs BMI ≤ 30 is always higher in carriers of homozygous genotypes in the three loci than in other subjects. 6    Journal of Clinical Medicine Research Updates, 2016, Vol. 3 Gloria-Bottini et al. DISCUSSION AND CONCLUSION The data suggest a relationship between genetic variability within the ADA gene and susceptibility to T2D in obese subjects. The susceptibility appears negatively correlated with the number of loci in which is present the variant with low frequency suggesting an heterozygote advantage in the protection of obese subjects from diabetes. The mechanism underlying this advantage has to be elucidated. If confirmed in other clinical settings, our observation could allow to assign a higher risk of diabetes to obese subjects who are homozygous for the major allele in all the three loci of ADA gene. ADA1 is an exonic polymorphism associated with quantitative variation of enzymatic activity. ADA2 is an intronic polymorphism and ADA6 is an exonic polymorphism characterized by a synonymous substitution, which does not change the protein sequence but could influence tissue specific expression. ADA2 and ADA6 could be in linkage disequilibrium with ADA sites directly responsible for the association observed. Indeed the analysis of linkage disequilibrium within the ADA gene performed by Cruciani et al. [17] has shown areas of weak association and areas of strong association between pairs of internal markers. The correlation between obesity and diabetes is stronger in subjects homozygous for the more frequent allele in all the three loci examined than in other subjects. It is possible that the three loci directly or indirectly control different mechanisms involved in the susceptibility to diabetes in obese subjects: the maximum risk is attained when the three mechanisms operate simultaneously. Further investigations on the role of ADA in the susceptibility to T2D in obese subjects would be rewarding. The limitations of our study are represented by the relatively small number of subjects examined and by the fact that it has been carried out on a sample of subjects admitted to the hospital for cardiovascular diseases and this could have introduced some non-identified bias. CONFICLT OF INTEREST All authors declare that they have no conflict of interest. Table 3: The Relationship between Genetic Variability within the ADA Gene and the Relative Risk of Diabetes in Obese vs Non Obese Subjects. For ADA1, ADA2 and ADA6 the More Frequent Alleles are Respectively ADA1*1, ADA2*1 and ADA6*2 and the Homozygous Genotypes are ADA1*1/*1, ADA2*1/*1 and ADA6*2/*2 Number of Homozygous Genotypes for the More Frequent Allele at ADA1, ADA2 and ADA6 Loci   0 1 and 2 3 % proportion of diabetics 16.7% 21.1% 8.9% BMI≤ 30  Total n° 6 128 89 % proportion of diabetics 20.0% 47.4% 56.7% BMI > 30 Total n° 5 38 30 Odds Ratio Analysis O.R.  95% C. I.  1.25 0.033 - 2.470  3.37 1.464 – 7.770  13.24  4.284 – 42.438 Chi square test of independence  Χ2 df P η2  0.412 1 0.777 0.04  8.951 1 0.004 0.07  27.928 1 <10-6 0.26 Three-way contingency table analysis by a log linear model (for this analysis class 0 has been joined with class 1+2) x=ADA; y=diabetes ; z=BMI                                        G               df           p xyz interaction                                                             5.575         1         0.023 xy independence                                                         7.083         2         0.030 yz independence                                                       35.977         2     <<0.0001 Independence of y from x and z                                                              37.180         3     <<0.0001  Genetic Variability within ADA Gene and Susceptibility to Type 2 Diabetes Journal of Clinical Medicine Research Updates, 2016, Vol. 3    7 REFERENCES [1] Xu B, Berkich DA, Crist GH, LaNoue KF. 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Genomics 2003; 82: 20-33. https://doi.org/10.1016/S0888-7543(03)00096-X  Received on 17-12-2016 Accepted on 22-12-2016 Published on 26-12-2016  http://dx.doi.org/10.15379/2413-7324.2016.03.02 © 2016 Gloria-Bottini et al.; Licensee Cosmos Scholars Publishing House. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.  

Genetic Variability Within ADA Gene and Susceptibility to Type 2 Diabetes in Obese Subjects

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Genetic Variability Within ADA Gene and Susceptibility to Type 2 Diabetes in Obese Subjects

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