Clinical and genetic association of insulin growth factor-2 gene in diabetes mellitus among south indian population.
Diabetes mellitus is a global health problem with a tremendous impact on morbidity and premature mortality worldwide. It affects 366 million people worldwide (6.4% of the world's adult population aged between 20-79 years). This number will be increased to 552 million by 2030.  Diabetes, a disease associated with altered glucose homeostasis, is common in India. The international diabetes federation estimates that around 61.3 million people in India had diabetes in 2011, and projects that by 2030, this will go up to 101.2 million.  T2DM is recognized as chronic persistent hyperglycemia resulting from pancreatic dysfunction or insulin resistance, and this disease is assuming epidemic proportions. 
Type 2 diabetes mellitus (T2DM) has been recognized as a heterogeneous group of metabolic and multifactorial disorders affecting the adult population. India ranks second in the world in diabetes prevalence, just after China.  Genetic and environmental factors play important roles in the progression of the disease.  Both longitudinal and cross-sectional studies have demonstrated that T2DM is influenced by several behavioral as well as lifestyle factors.  Clinical and epidemiological studies have indicated that obesity is a major risk factor for T2DM, associated with an increased risk of developing insulin resistance and impaired glucose tolerance. Impaired insulin secretion and insulin resistance; the two main pathophysiological mechanisms leading to T2DM have a significant genetic component. 
Insulin-like growth factors (IGFs) are regulators of processes such as growth and metabolism. IGF-1 and IGF-2 contribute to pancreatic [beta]-cell growth and development by regulating [beta]-cell replication, renewal, and apoptosis.  Deregulation of balance between P-cell renewal and apoptosis due to alterations in IGF levels is potentially of great importance in the development of glucose intolerance, a major characteristic of diabetes. In addition, insulin-dependent glucose homeostasis may be affected by IGFs as they act via the insulin signaling pathway.  Defects in the IGF/insulin-signaling pathway affects birth weight and fat metabolism in both domestic animals and humans, which are known risk factor for development of type 2 diabetes (T2D).  IGF-2 polymorphisms have been associated with weight gain; body mass, obesity, and adiposity.  Hence, the aim of this study was to evaluate the association of IGF-2 Apa1 polymorphism in T2D patients.
METHODS AND MATERIALS Sampling
Patients for the study were selected from the Princess Durru shehvar children's and General Hospital, Hyderabad. 3 ml of venous blood for DNA isolation was collected. Demographs was obtained along with information regarding clinical and family history in a well-designed pro forma. The salting out technique was used successfully to isolate large quantities of DNA from blood. DNA was extracted from peripheral blood lymphocytes using 300 pl of whole blood. The isolated DNA was amplified using polymerase chain reaction (PCR). PCR is an enzyme catalyzed biochemical reaction in which small amount of a specific DNA fragment is amplified into large amount of linear double strand DNA using gene specific oligonucleotide primers.
Isolation of DNA and Genotype Analysis
Genomic DNA was isolated from the peripheral blood of subjects using salting out method. The DNA was stored at -20[degrees]C until processing. Genotyping for the IGF-2 Apal gene polymorphism (rs680) was performed by PCR with the use of specific published primers. Forward primer: 5'-CTT GGACTTT GAAGT CAAATT GG-3'; Reverse primer: 5'GGTCGTGCCAATT ACATTTCA-3' synthesized from Sigma-Aldrich Chemical Pvt. Limited (Bangalore, India), followed by restriction fragment length polymorphism analysis. A three-step PCR was performed using XP thermal cycler. Briefly, the PCR conditions included an initial denaturation at 94[degrees]C for 5 min, followed by 30 cycles of denaturation at 94[degrees]C for 30 s, annealing at 55[degrees]C for 30 s and extension at 68[degrees]C for 45 s, final extension at 68[degrees]C for 5 min. The 292 bp amplified PCR product was digested with Apal enzyme at 37[degrees]C for 2 h and electrophoresed on 2% agarose gel with ethidium bromide. Bands of 229 bp were observed in case of GG genotype, 292 bp and 229 bp in AG genotype and an undigested 292 bp band in AA genotype. Restriction enzyme digested PCR products were imaged and analyzed by documentation in UVI Tech gel documentation system (UVI Tech Ltd. Cambridge, United Kingdom).
Genotype and allele frequencies were calculated. The groups were compared using the [chi square] test to analyze the statistical significance of the difference in allelic distribution of various polymorphisms in patients and controls. Values of P < 0.05 were considered statistically significant. Odds ratio was performed using MedCalc for Windows, version 18.104.22.168 (MedCalc Software, Mariakerke, Belgium).
Atotal of 200 subjects with 100 T2DM patients and 100 healthy volunteers were enrolled in this study. The demographic characteristics of risk factor variables for T2DM patients and control subjects are presented in Table 1. The mean age of the T2DM patients was 61.7 [+ or -] 8.8 years with a mean body mass index (BMI) of 24.0 [+ or -] 3.3 kg/[m.sup.2]. The average age of the control subjects was 63.2 [+ or -] 5.4 years with a mean BMI of 23.8 [+ or -] 2.2 kg/[m.sup.2]. In this study, we found 65% of patients and only 35% in controls were smokers. 30% of patients was alcoholic and only 5% of controls were alcoholic.
The mean and standard deviation values of total cholesterol was (197 [+ or -] 25) and (135 [+ or -] 10), LDL (130 [+ or -] 17) and (112 [+ or -] 13), high-density lipoprotein (HDL) (37 [+ or -] 10) and (40 [+ or -] 8), VLDL (22 [+ or -] 10) and (20 [+ or -] 6), and triglyceride (175 [+ or -] 15) and (138 [+ or -] 10) among T2DM patients and controls, respectively, as shown in Table 2. There was a significant difference in the levels of total cholesterol, HDL-C, and triglycerides between the T2DM patients and controls (P < 0.05) while as VLDL was not associated.
Genotyping for Apa1 polymorphism revealed AA genotypes in 48% and 70% of T2DM and controls, respectively. AC was present in 42% and 28% in T2DM and control group, respectively. CC was seen in 10% of T2DM cases and 2% of the controls. The AA genotype was significantly associated with T2DM (OR, 0.39; 95% CI: 0.22 to 0.70, P 0.001). AC genotype was found more in patients than controls and was associated with T2DM (OR, 1.86; 95% CI: 1.03 to 3.35, P 0.03) and CC genotype was also associated with T2DM (OR, 5.45; 95% CI: 1.16 to 25.52, P 0.03). Frequency of A allele was 0.69 in T2DM and 0.84 in controls (OR, 0.42; 95% CI: 0.26 to 0.68) while as the frequency of C allele was 0.31 in T2DM patients and 0.16 in controls (OR, 2.35; 95% CI: 1.45 to 3.82). Both the alleles were significantly associated (P = 0.005) with T2DM patients when compared to controls (Table 3).
This study was conducted to explore the association between T2DM patients and healthy controls by investigating single nucleotide polymorphism, demographs, and clinical factors among South Indian population. In this study, we found 65% of patients and only 35% in controls were smokers. 30% of patients was alcoholic and only 5% of controls were alcoholic. There was a significant difference in the levels of total cholesterol, HDL-C, and triglycerides between the T2DM patients and controls (P < 0.05) while as VLDL was not associated. We hypothesized that IGF-2 gene was associated with T2DM patients when compared to controls. The AA genotype was significantly associated with T2DM (OR, 0.39; 95% CI: 0.22 to 0.70, P = 0.001). AC genotype was found more in patients than controls and was associated with T2DM (OR, 1.86; 95% CI: 1.03 to 3.35, P = 0.03) and CC genotype was also associated with T2DM (OR, 5.45; 95% CI: 1.16 to 25.52, P = 0.03). Frequency of A allele was 0.69 in T2DM and 0.84 in controls (OR, 0.42; 95% CI: 0.26 to 0.68) while as the frequency of C allele was 0.31 in T2DM patients and 0.16 in controls (OR, 2.35; 95% CI: 1.45 to 3.82). Both the alleles were significantly associated (P = 0.005) with T2DM patients when compared to controls (Table 3).
There is considerable evidence that IGF-2 regulates cell growth, differentiation, and metabolism.  Quantitatively IGF-2 is the predominant circulating IGF present in adults at a concentration of ~700 ng/ml,  three times that of IGF-1. Concentrations are similar in both genders. In common with insulin and IGF-1, binding of IGF-2 to the IGF-1R activates a receptor tyrosine kinase (RTK) associated with the b-subunit leading to an intracellular response. [13-15] Autophosphorylation of the b-subunit by the RTK recruits insulin receptor substrates (IRS) 1-4. Phosphatidylinositol 3-kinase then binds to IRS1 via its regulatory subunit and is activated, in turn activating Akt (protein kinase B). This has a number of intracellular effects, which ultimately promote cell survival and mitogenesis.
Previously, it was shown that polymorphisms in the IGF-1 and IGF-2 genes are associated with features of the metabolic syndrome. [16,17] Gene variants in the IGF-2 gene were found to be associated with IGF-2 levels and BMI. [18,19] Gomes et al  (2006) studied the association between IGF-2 Apa1 polymorphism and the BMI, however, did not find any significant association. A previous study of IGF-2R as a susceptibility gene for T2DM has similarly implicated an insertion/deletion variant in the 3'UTR region of IGF-2R.  The insertion/deletion polymorphism is likely to result in the change in IGF-2R expression. 
The major limitations of this study were small sample size was and only a single single-nucleotide polymorphisms was selected from IGF-2 gene.
In conclusion, this study showed that IGF-2 Apa1 polymorphism is associated with T2DM in south Indians and it can be used as a biomarker for identifying individuals at a high risk of developing T2DM. Limited use of smoking and alcohol may reduce the incidence of T2DM.
We acknowledge Princess Durru Shehvar children's and General Hospital, for providing samples; we would also like to thank patients, volunteers who participated in the study.
[1.] International Diabetes Federation. Diabetes Atlas. 5th ed. Brussels, Belgium: IDF; 2011.
[2.] Vilvanathan S, Gurusamy U, Mukta V, Das AK, Chandrasekaran A. Allele and genotype frequency of a genetic variant in ataxia telangiectasia mutated gene affecting glycemic response to metformin in South Indian population. Indian J Endocrinol Metab. 2014; 18(6):850-4.
[3.] Mayans S, Lackovic K, Lindgren P, Ruikka K, Agren A, Eliasson M, et al. TCF7L2 polymorphisms are associated with type 2 diabetes in northern Sweden. Eur J Hum Genet. 2007; 15(3):342-6.
[4.] Li YY, Gong G, Geng HY, Yang ZJ, Zhou CW, Xu J, et al. CAPN10 SNP43 G>A gene polymorphism and type 2 diabetes mellitus in the Asian population: A meta-analysis of 9353 participants. Endocr J. 2015; 62(2):183-94.
[5.] Hussain H, Ramachandran V, Ravi S, Sajan T, Ehambaram K, Gurramkonda VB, et al. TCF7L2 rs7903146 polymorphism and diabetic nephropathy association is not independent of type 2 diabetes - a study in a south Indian population and meta-analysis. Endokrynol Pol. 2014; 65(4):298-305.
[6.] Khan IA, Jahan P, Hasan Q, Rao P. Angiotensin-converting enzyme gene insertion/deletion polymorphism studies in Asain Indian pregnant women biochemically identifies gestational diabetes mellitus. J Renin Angiotensin Aldosterone Syst. 2013; 15(4):566-71.
[7.] 't Hart LM, Fritsche A, Rietveld I, Dekker JM, Nijpels G, Machicao F, et al. Genetic factors and insulin secretion: Gene variants in the IGF genes. Diabetes. 2004; 53 Suppl 1:S26-30.
[8.] Kim JJ, Accili D. Signalling through IGF-I and insulin receptors: Where is the specificity? Growth Horm IGF Res. 2002; 12(2):84-90.
[9.] Hales CN, Barker DJ. The thrifty phenotype hypothesis. Br Med Bull. 2001; 60:5-20.
[10.] Sandhu MS, Gibson JM, Heald AH, Dunger DB, Wareham NJ. Low circulating IGF-II concentrations predict weight gain and obesity in humans. Diabetes. 2003; 52(6):1403-8.
[11.] O'Dell SD, Day IN. Molecules in focus Insulin-like growth factor II (IGF-II). Int J Biochem Cell Biol. 1998; 30(7):767-71.
[12.] Humbel RE. Insulin-like growth factors I and II. Eur J Biochem. 1990; 190(3):445-62.
[13.] Belfiore A, Malaguarnera R. Insulin receptor and cancer. Endocr Relat Cancer. 2011; 18(4):R125-47.
[14.] Braun S, Bitton-Worms K, LeRoith D. The link between the metabolic syndrome and cancer. Int J Biol Sci. 2011; 7(7):1003-15.
[15.] Leroith D, Scheinman EJ, Bitton-Worms K. The role of insulin and insulin-like growth factors in the increased risk of cancer in diabetes. Rambam Maimonides Med J. 2011; 2(2):e0043.
[16.] Vaessen N, Heutink P, Janssen JA, Witteman JC, Testers L, Hofman A, et al. A polymorphism in the gene for IGF-I: Functional properties and risk for type 2 diabetes and myocardial infarction. Diabetes. 2001; 50(3):637-42.
[17.] Ukkola O, Sun G, Bouchard C. Insulin-like growth factor 2 (IGF2) and IGF-binding protein 1 (IGFBP1) gene variants are associated with overfeeding-induced metabolic changes. Diabetologia. 2001; 44(12):2231-6.
[18.] O'Dell SD, Miller GJ, Cooper JA, Hindmarsh PC, Pringle PJ, Ford H, et al. Apal polymorphism in insulin-like growth factor II (IGF2) gene and weight in middle-aged males. Int J Obes Relat Metab Disord. 1997; 21(9):822-5.
[19.] Gaunt TR, Cooper JA, Miller GJ, Day IN, Dell SD. Positive associations between single nucleotide polymorphisms in the IGF2 gene region and body mass index in adult males. Hum Mol Genet. 2001; 10(14):1491-501.
[20.] Gomes MV, Soares MR, Pasqualim-Neto A, Marcondes CR, Lobo RB, Ramos ES. Association between birth weight, body mass index and IGF2/ApaI polymorphism. Growth Horm IGF Res. 2005; 15(5):360-2.
[21.] Villuendas G, Botella-Carretero JI, L'opez-Bermejo A, Gubern C, Ricart W, Fernandez-Real JM, et al. The ACAAinsertion/deletion polymorphism at the 3' UTR of the IGF-II receptor gene is associated with type 2 diabetes and surrogate markers of insulin resistance. Eur J Endocrinol. 2006; 155(2):331-6.
[22.] Lv K, Guo Y, Zhang Y, Wang K, Jia Y, Sun S. Allele-specific targeting of hsa-miR-657 to human IGF2R creates a potential mechanism underlying the association of ACAA insertion/ deletion polymorphism with type 2 diabetes. Biochem Biophys Res Commun. 2008; 374(1):101-5.
Syed Mudassir Hussaini (1), Wasey Ali Yadullahi Mir (2), Mohammed Abdul Tawwab (3), Adil Wajid (1)
(1) Department of General Medicine, Princess Durru Shehvar Children's and General Hospital, Hyderabad, Telangana, India, (2) Department of General Medicine, Revive Hospital, Siddipet, Telangana, India, (3) Deparment of Pharmacy, Shadan College of Pharmacy, Hyderabad, Telangana, India
Correspondence to: Syed Mudassir Hussaini, E-mail: email@example.com
Received: November 30, 2016; Accepted: December 21, 2016
Table 1: Demographic details of T2D patients and controls of this study Parameters T2D (n=100) Controls (n=100) Age (years) 61.7 [+ or -] 8.8 63.2 [+ or -] 5.4 * (P=0.092) Male 65 35 Female 45 55 Smoker 60 40 Alcoholic 30 5 Nonalcoholic 70 95 BMI 24.0 [+ or -] 3.3 23.8 [+ or -] 2.2 * (P=0.504) Age and BMI is shown in mean and standard deviation, BMI: Body mass index, T2DM: Type 2 diabetes mellitus Table 2: Shows the mean levels of lipid profile in the cases of T2D and controls Lipid profile Subjects with Subjects without P T2DM (n=100) T2DM (n=100) Mean total 197 [+ or -] 25 135 [+ or - ] 10 0.001 cholesterol (mg/dl) Mean LDL 130 [+ or -] 17 112 [+ or - ] 13 0.001 cholesterol (mg/dl) Mean HDL 37 [+ or -] 10 40 [+ or - ] 8 0.02 cholesterol (mg/dl) Mean VLDL 22 [+ or -] 10 20 [+ or - ] 6 0.08 cholesterol (mg/dl) Mean TG 175 [+ or -] 15 138 [+ or - ] 10 0.001 Data is shown as Mean, Standard deviation and P- value, HDL: High-density lipoprotein, VLDL: Very low density lipoprotein, T2DM: Type 2 diabetes mellitus Table 3: Genotype and allele frequency of IGF2 gene in T2DM patients and controls Genotype T2D Controls Odds 95% CI P (n=100) (n=100) ratio AA 48 (48) 70 (70) 0.39 0.22-0.70 0.001 AC 42 (42) 28 (28) 1.86 1.03-3.35 0.03 CC 10 (10) 2 (2) 5.45 1.16-25.52 0.03 Allele A 138 (0.69) 168 (0.84) 0.42 0.26-0.68 0.005 C 62 (0.31) 32 (0.16) 2.35 1.45-3.82 0.005 IGF2: Insulin-like growth factor 2, T2DM: Type 2 diabetes mellitus
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|Title Annotation:||Research Article|
|Author:||Hussaini, Syed Mudassir; Mir, Wasey Ali Yadullahi; Tawwab, Mohammed Abdul; Wajid, Adil|
|Publication:||International Journal of Medical Science and Public Health|
|Date:||May 1, 2017|
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