Eleven genetic variants of seven important candidate genes involved in manifestation of type 2 diabetes mellitus

1 Introduction

Diabetes especially type 2 diabetes mellitus (T2DM) is securing the rank under five around the world and leading cause of death cardiac disease and cancer. From our previous reports which revealed that South east Asia including India has 78.3 million diabetics which are expected to increase up to 140.2 million till 2040 (Bid et al., 2008; Banerjee and Saxena, 2012a,2012b; Saxena et al., 2012a,2012b; IDF, 2015). The complex interactions and associations among lymphoid cells are usually mediated by a group of proteins which are collectively designated as cytokines. They include immune modulating agents like interleukins and inteferons (Banerjee and Saxena, 2012a,2012b). Apart from this the major sub group of cytokines, the chemokines which are of low molecular weight cytokines that alter the behaviour of leukocyte. Traditionally the cytokines have been classified into pro- and anti-inflammatory. They are antagonistic in behaviour for each other i.e. proinflammatory effector functions are inhibited by anti-inflammatory cytokines. These inflammatory mediators have been proposed to be involved directly or indirectly in causing T2DM and their manifestation. As Asian Indians have more prone and have a greater susceptibility to T2DM, they are a good site of population for carrying out such genetic studies (Bid et al., 2009a,2009b,2010). Polymorphisms (SNPs), mainly those within the regulatory regions of cytokines genes often have to be involved at expression levels and can be disease modifiers. Individuality in the inheritance of this polymorphic genes lead to change in their immune responses. This might change the severity as well as the duration of inflammation, thereby leading to the progression of T2DM (Dinarello, 1996; Wilson et al., 1997; Bidwell et al., 1999). All genes of IL-1 family are polymorphic and most of these polymorphisms have been shown to be involved in either the susceptibility or the severity of inflammatory conditions and diseases (Haukim et al., 2002). The pro-inflammatory cytokine IL-18, a member of IL-1 family is playing a crucial role in the cascade of inflammation. It is highly expressed in atherosclerotic plaques and plays an important role in the destabilization of plaques (Mallat et al., 2002). A polymorphic variation in IL-6 gene might increases risk for T2DM development and was already reported (Vozarova et al., 2003; Illig et al., 2004; Saxena et al., 2014). While, the predominance nature of IL-10 genotype in T2DM is mainly due to its protective nature against the inflammation and encouraging humoral responses which delay the activation of inflammatory cytotoxic reactions involved in destruction of pancreatic β-cell. IL-10 is involved to down regulate the secretion of mainly pro-inflammatory cytokines secreted from monocytes/macrophages (Sankaran et al., 1999). Moreover, VDR gene is also a novel candidate gene and contributes to the T2DM susceptibility (Saxena et al., 2018). VDR is a part of the steroid/thyroid hormone receptor family and is involved in regulating the normal functions of the pancreas especially the secretion of insulin. They play the major role in regulating the β-cell insulin secretion (Saxena et al., 2018). It would be quite interesting to know whether the polymorphisms in aforesaid genes taken together would predispose and unwinding the hidden related facts in the T2DM manifestation. In our earlier reports, we have thoroughly went from anthropometric and biochemical parameters in T2DM in association with individual as well as in combination of polymorphisms with cytokine gene (Saxena et al., 2009,2012a,2012b; Saxena et al., 2015). We have investigated eleven genetic polymorphisms of seven important genes viz. IL-10, TNF-α, IL-6, adiponectin, IL-1, IL-18, and Vitamin D-receptor. We have focused to investigate the gene-gene interactions and their role in determining individual susceptibility to T2DM and their manifestation.

2 Material and methods

3 Results

The biochemical parameters showed significant association with FBS, PPBS, TC, TGL, HDL, LDL and VLDL (P < 0.001) when compared with healthy controls (Table 2). All the genotyping data for individual SNPs in T2DM patients (n = 440) and age-sex matched healthy controls (n = 440) have been summarized and the genotypic frequency of rs1800872, rs1800795, rs2241766, rs17846866 and rs1946518 showed highly significant association in our population. While rs1800629 and rs731236 showed significant association in our population. The rs1800797, rs16944, rs2228570 and rs1544410 showed no significant association (Table 3). When we assessed the allele frequencies of these variants we found that rs1800795, rs2241766 and rs1946518 showed highly significant association and rs2241766 increases the chance of having diabetes by twice [OR; 1.955(95% CI;1.595–2.395)]. Only allele of rs1800629 showed significant association in our population (p = 0.010) (Table 4). Further we have gone to analyse the carriage rate allele frequencies of individual alleles and found that ‘C’ allele of rs1800795 and rs1800872, both ‘T’ and ‘G’ allele of rs2241766 and ‘A’ allele of rs1946518 showed highly significant association. While recessive allele of rs1544410 i.e. allele ‘b’ also showed highly significant association (Table 5). Later on we go for gene gene interaction analysis of all studied variants of IL-10-592A/C (rs1800872), TNF-a-308G/A (rs1800629), IL-6-174G/C (rs1800795), IL-6-597G/A (rs1800797), Adiponectin + 45G/T (rs2241766;Q1), Adiponectin + 10211 T/G (rs17846866;Q2), IL-1b-511C/T (rs16944), IL-18-607 A/C (rs1946518), Vitamin D Receptor (VDR) FokIT/C (rs2228570), VDR TaqIC/T (rs731236) and VDR BsmIA/G (rs1544410) and found very interesting results. We found that the allele set

“AGGATTTCFTB”, “CAGATTTCFTB”, “CGCATTTCFTB”, “CGGAGGTAFTB”, “CGGAGTTAFTB”, “CGGATGTCFTB”, “CGGATTTCFTB”, “AGGATTTAFTB”, “CGCATTTAFTB”, “CGGATGTAFTB”, “CGGATTCCFTB” and ”CGGATTTAFTB” showed significant association in the manifestation of the disease (Table 6). Allele set of “CGGAGGTAFTB”, “CGGAGTTAFTB” and “CGGATGTCFTB” increase the chance of diabetes upto 1.6 times; the OR are 1.436 [95% CI; 0.796 ∼ 2.587], 1.610 [95% CI; 0.890 ∼ 2.912] and 1.466 [95% CI;0.791 ∼ 2.717] respectively. This shows that these set of allele from IL to 10-592A/C (rs1800872), TNF-a-308G/A (rs1800629), IL-6-174G/C (rs1800795), IL-6-597G/A (rs1800797), Adiponectin + 45G/T (rs2241766;Q1), Adiponectin + 10211 T/G (rs17846866;Q2), IL-1b-511C/T (rs16944), IL-18-607 A/C (rs1946518), Vitamin D Receptor (VDR) FokIT/C (rs2228570), VDR TaqIC/T (rs731236) and VDR BsmIA/G (rs1544410) increased the risk of the disease upto 1.6 times. Linkage disequilibrium (D’) and Correlation coefficient (r2) of IL-10-592A/C (rs1800872), TNF-a-308G/A (rs1800629), IL-6-174G/C (rs1800795), IL-6-597G/A (rs1800797), Adiponectin + 45G/T (rs2241766;Q1), Adiponectin + 10211 T/G (rs17846866;Q2), IL-1b-511C/T (rs16944), IL-18-607 A/C (rs1946518), Vitamin D Receptor (VDR) FokIT/C (rs2228570), VDR TaqIC/T (rs731236) and VDR BsmIA/G (rs1544410) eleven variants with T2DM in North Indian population is shown in (Fig. 1).

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Fig. 1

(A) Linkage Disequilibrium (D’) and (B) Correlation coefficient (r²) of all eleven studied gene variants.

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4 Discussion

Diabetes is basically a condition with an involvement of pro-inflammatory state having an enhanced level of circulating cytokines and suggests a causal role for inflammation in its etiology. Previous reports documented that hyperglycemia is associated with T2DM and acutely enhanced level of peripheral cytokines viz. IL-1, TNF-α and IL-6 (Tsiavou et al., 2004). A significant association of IL-4 gene variants have also been reported with increased risk of T2DM along with other related complications in north Indians (Achyut et al., 2007). However, as per our previous reports in IL-1Ra gene variants we have reported four different alleles in north Indians with T2DM. Most of the T2DM individuals were homozygous genotype I as per our previous report² and its association with coronary artery disease (CAD) individuals with and without T2DM (Marculescu et al., 2002). It was documented that these genetic variants increases the risk of having T2DM upto 3.4 times. Other IL-1 gene variants viz. IL-1 α-889, IL-1 β + 3954 (rs 1143634) and IL-1 β-511) in T2DM as well as periodontitis were also reported (Struch et al., 2008; Lopez et al., 2009). One of the case-control study of IL-1β gene variant found significant association in genotypic frequencies (P < 0.0001) but no association was observed in allele frequencies of −511C and −511 T. While a new sub member of IL-1 family, the IL-18 gene variant have been vastly studied by other groups in T2DM associated complications. One of the renowned research group i.e. Opstad et al. (2011) studied three IL-18 gene variants +183 A/G, −137 G/C and −607C/A in T2DM individuals and reported that only IL-18 + 183 A/G was significantly associated with T2DM manifestation. However, in the population of china only −607C/A gene variant was found to be associated with T2DM (Huang et al., 2010). Controversial results were reported by European researchers on IL-18 gene variants with T2DM and other metabolic diseases and disorders (Thompson et al., 2007; Rafiq et al., 2008; He et al., 2010). In contrast to the previous reports we found that in north Indians, −607C/A gene variants showed significant genotypic association (P = 0.002) and most of the individuals were heterozygous. Moreover, lots of controversies on the results on IL6-174 G/C and -597 A/G gene variants with T2DM or insulin resistance. Apart from this, ‘G’ allele of -174 G/C SNP was found to be associated with increased T2DM risk in Americans and Spanish Caucasians but not in the Finnish Diabetes Prevention Study (DPS) (Kubaszek et al., 2003a,2003b).

Furthermore, nondiabetic individual was found to be associated with IL6-174C/C genotype in higher insulin sensitivity (Fernandez-Real et al., 2000; Kubaszek et al., 2003a,2003b). While -174 G/C was found to be associated with T2DM risk in North Indians and in the metagenomic Indian study (Mukhopadhyaya et al., 2010; Popko et al., 2010; Ferreira et al., 2011). Although TNFα-308 G/A was also a focused gene variant in T2DM of several groups. In Arragonians, TNFα-308 G/A gene variant was found to be associated with T2DM (Vendrell et al., 2003). However, Greece and UK/Irish origin reports showed contended results with other reports from China, Japan and Mexico (, Boraska et al., 2010). Significant associations was reported from Croatian Caucasian, Finnish, Indian, Mexican and Taiwanese individuals on TNFα-308 G/A gene variants with T2DM. No association was observed in genotypic and allelic frequencies. The −592*A allele of IL-10-592 A/C gene variant was reported and found to be increased in T2DM individuals. The low frequency of T2DM in Taiwanese population may be attributed to the involvement of ‘C’ allele of IL-10-592 which is very rare in Caucasians. Many research groups have reviewed the Adiponectin gene variants in various ethnic groups.

The study on urban Asian Sikhs, 5 tag SNPs of Adiponectin gene revealed that it has no relation in T2DM. However, in Whitehall II study, adiponectin was found independently a predictor of diabetes and GWAS showed significant association with the gene loci of Adiponectin. In +45 T/G gene variant, subjects with ‘GG’ and ‘TG’ genotypes were at greater risk of having T2DM in obese Iranians while, both these genotypes were related with gestational T2DM in the women of Malaysia. Our previous report reported for the first time that +10211 T/G polymorphism in intron 1 of Adiponectin gene is related with T2DM individuals in support with previous report in an Asian Indians. Result also in support with Pima Indians and French Caucasians. Linkage disequilibrium is the occurrence of some allele and genetic combinations in a population more or less often than would be expected from a random formation of haplotypes from other alleles depends on their frequencies.

Gene-gene interaction analysis of eight gene variants when taken together in our previous genetic report, CGGATTTC* and CGGAGTCA* set of allele combination was reported to increase the risk upto 7.5 and 4.248 times respectively. Similarly, in this present report we have observed that allele set of “AGGATTTCFTB”, “CAGATTTCFTB”, “CGCATTTCFTB”, “CGGAGGTAFTB”, “CGGAGTTAFTB”, “CGGATGTCFTB”, “CGGATTTCFTB”, “AGGATTTAFTB”, “CGCATTTAFTB”, “CGGATGTAFTB”, “CGGATTCCFTB” and ”CGGATTTAFTB” in IL-10-592A/C (rs1800872), TNF-a-308G/A (rs1800629), IL-6-174G/C (rs1800795), IL-6-597G/A (rs1800797), Adiponectin + 45G/T (rs2241766;Q1), Adiponectin + 10211 T/G (rs17846866;Q2), IL-1b-511C/T (rs16944), IL-18-607 A/C (rs1946518), Vitamin D Receptor (VDR) FokIT/C (rs2228570), VDR TaqIC/T (rs731236) and VDR BsmIA/G (rs1544410) were found to significantly associated and increases the risk of having T2DM.

Moreover, allele set of “CGGAGGTAFTB”, “CGGAGTTAFTB” and “CGGATGTCFTB” increase the chance of diabetes upto 1.6 times in our population. This study reflects that these individuals are more susceptible of having T2DM. The present study will provide a new insight in the development and the manifestation of T2DM. Gene variant studies have shown a considerable level of variation amongst various ethnic groups around the globe. Therefore, it is mandatory to perform such genetic studies so that individuals can be benefitted. Individuals who are at high risk will be able to take prior precautionary measures so, they may avoid or delay the onset of disease. These genetic studies showing gene interaction for the susceptibility of the disease may be used as prognostic markers and alter treatment strategies for T2DM.