Association between +45T>G adiponectin polymorphism gene and type 2 diabetes mellitus and metabolic syndrome in a Venezuelan population

Subjects

A total of 90 adult individuals, of which 46 had MS-DM2 and 44 were healthy controls, were enrolled in this study, which was carried out from January 2015 to January 2016. Participants were recruited through a mixed strategy. Some individuals were contacted by phone while in remote locations we made contact with the community leaders in order to verify the size and organization of each neighborhood since public records prove inaccurate. After this, we proceeded to draw sectors and blocks, and then, together with the community leaders, contact the members of the selected houses directly. These individuals participated in the Metabolic Syndrome Prevalence Project¹¹ of the Metabolic Endocrine Research Center "Dr. Félix Gómez" (CIEM) of the Faculty of Medicine of University of Zulia performed from 2007 to 2008. The present study size was the same as the previous one. The participants signed an informed consent form, where they were explained everything related to the project and the handling of their samples and tests. This research was approved by the CIEM bioethics committee and complies with the Helsinki declaration. The studied subjects were chosen since they met the predetermined inclusion criteria and with the absence of selected exclusion criteria, depicted in Table 1. In order to make the diagnosis of MS and DM2, 2009 Consensus⁶ and ADA²² criteria was used respectively.

Anthropometry, body composition and biochemistry analysis

The following variables were measured in the subjects:

Genotyping of the 45 T>G polymorphism of the ADIPOQ gene

Genomic DNA extraction. A total of 5 ml peripheral blood (with EDTA as an anticoagulant) was obtained by venipuncture. For DNA extraction, the combined DNA-Salting out extraction technique was used, as described previously²³, of the Molecular Genetics laboratory of the Medical Genetics Unit of the Faculty of Medicine was used.

PCR amplification of 45 T>G polymorphism of the ADIPOQ gene. A 372 bp DNA fragment covering the region of interest was amplified by PCR, using this set of oligonucleotide primers: F5'-GAATGAGACTCTGCTGAGATGG-3' and R5'- TATCATGTGAGGAGTGCTTGGATG-3’ to amplify the region of interest (Figure 1). The amplification conditions were the following: 95°C for 6 min, followed by 35 cycles of 95°C for 1 min, 45 sec at 56°C and 45 sec at 72°C and a final extension at 72°C for 5 min²⁴. For the amplification reactions, taq DNA polymerase from Promega was used.

Figure 1. The 372 bp DNA fragment covering the region of interest amplified by PCR.

The region of interest was amplified using this set of oligonucleotide primers: F5'-GAATGAGACTCTGCTGAGATGG-3' and R5'- TATCATGTGAGGAGTGCTTGGATG-3’.

RFLP for the detection of the 45 T>G polymorphism of the ADIPOQ gene. The amplified fragment of 372bp was subjected to restriction analysis with the SmaI enzyme. A non-digested fragment (molecular weight, 372 bp) corresponded to the homozygous T/T genotype. The homozygous G/G genotype was evidenced by the presence of two fragments, one 219 bp and the other 153 bp. The product of the digestion of the heterozygous T/G genotype was evidenced by the presence of three fragments (372 bp, 219 bp and 153 bp), as shown in Figure 1.

Statistical analysis

The statistical package SPSS for Windows, version 21.0 was used. Allelic and genotypic frequencies were calculated for the total population evaluated and for the MS-DM2 and control groups. The χ² test was used to compare frequencies and observed and expected values to evaluate the Hardy-Weinberg equilibrium (HWE). Means and medians were determined for biochemical, clinical and anthropometric characteristics accordingly. The normal distribution of the variables was verified by the Kolmorgov-Smirnov test. The comparison of the parametric variables with normal distribution was performed using Student's t-test, when 2 groups were compared and the Mann and Whitney U-test in those variables that had a non-normal distribution. A p-value <0.05 was considered statistically significant. Likewise, a binary logistic regression was carried out in order to obtain the odd ratio adjusted to a confidence interval of 95%, to estimate the relative risk of the variable under study. A p-value <0.05 was considered statistically significant.

Study population characteristics

In this study, the +45 T>G polymorphism of the ADIPOQ gene was associated with the development of MS and DM2 in adult individuals from Maracaibo City. No prior information was found on the association of that polymorphism of the ADIPOQ gene with individuals diagnosed with MS and DM2. Complete raw data are available on OSF²⁵.

Clinical, biochemical and anthropometric characteristics of both groups (MS +DM2 and control patients) are shown in Table 2. Statistically significant differences were found when comparing the means and medians in almost all studied characteristics, with the exception of height and usCRP. Most of the parameters of MS and DM2 individuals were found to be higher than in healthy controls, with exception of HDL-c levels. In line with the findings of the present study, DM2 patients in Brazil, India and Russia^18,26,27 showed similar results in clinical characteristics, such as BMI, HOMA-IR, fasting glucose, TC, TG, HDL-c, LDL-c, systolic blood pressure (SBP) and diastolic blood pressure (DBP). Moreover, Sahli et al.²⁸ reported statistically significant differences in BMI, fasting glucose, TC, TG, HDL-c, LDL-c, DBP, and SBP in a Tunisian population. However, a DM2 Chinese Han population²⁹, showed no significant difference when comparing height and usCRP in diabetic and healthy control groups. These differences could be related to the heterogeneity of the populations, due to the fact that they may have totally different lifestyles, including feeding, physical activity, environmental factors, and customs, among others.

Frequency of ADIPOQ genotypes

Table 3 depicts the allelic and genotypic frequencies of the +45T>G polymorphism of the ADIPOQ gene, in patients with MS-DM2 and healthy control patients. The T/T genotype was found in greater frequency in the total population, although patients with MS-DM2 showed a greater frequency, as described by Sahli et al.²⁸ in a Tunisian MS population. T/G and G/G genotypes were found in both groups but showed lower frequencies. The healthy controls showed a genotypic frequency of 27% and 5% for the T/G and G/G genotypes, respectively, which indicates that the homozygous T genotype is three times higher in healthy individuals. Other studies generated opposite results^26,27,30,31, since the T/T genotype was found more frequently in healthy control patients than in DM2 ones.

Allelic frequencies

Regarding the alleles, a higher frequency of the T allele was observed over the G allele in both study groups, the T allele being more frequent in the population with MS-DM2, where the Hardy-Weinberg balance χ² = 5.99 is met, because the observed allele frequencies are as expected. Such findings relate to the MS population studied in Tunisia²⁸ but differ with the type 2 diabetes mellitus individuals studied in Brazil, India and Ireland^26,27,31 since the highest frequency of the T allele occurred in the healthy control group Table 4.

Comparison of populations

As shown in Table 4, the population that differ the most from the one characterized in this study, turned out to be the Brazilian one²⁶, although Brazil is near Venezuela, the studied subjects were of Japanese ancestry; this could be the reason of such genetic differences although the lifestyles and customs are similar in both countries.

Association of anthropomorphic characteristics with ADIPOQ status

Table 5 shows that there was a significant difference among waist circumference, fasting glucose, total cholesterol, triacylglycerides, LDL-c, DBP and SBP, for the T/T and T/G + G/G genotypes regarding the total population. Those that had the presence of the G allele of the polymorphism exhibited lower values except for the usCRP in which those subjects that carried the T allele had lower levels in this parameter.

On the other hand, the control population (without MS-DM2) showed statistically significant difference regarding the DBP. In the MS-DM2, a significant difference was only observed in fasting insulin levels between groups. In addition, it was demonstrated that in these groups, the subjects who presented the G allele showed lower mean values than of those without this allele (homozygous T), except for the usCRP levels, which were higher in individuals with the G allele. Potapov et al.¹⁸ obtained contradictory results in their study in a DM2 Russian population, since the fasting glucose levels showed higher values for subjects carrying the G allele, and in control subjects, a statistically significant difference was only found in SBP, which was higher in those individuals who carried the G allele.

Previous studies

Several studies have reported the behavior of several anthropometric, clinical and biochemical characteristics in their total populations concerning the +45 T>G polymorphism, finding opposite data that the one found during this study. In a non-diabetic female population in Greece¹⁶, fasting insulin and HOMA-IR levels were significantly lower in carriers of the rare +45G allele (T/G+G/G), which was also evidenced in Italian individuals with or without DM2³², in non-diabetic individuals in Taiwan³³, there were only significant differences in the BMI, with higher values for T allele carriers, unlike Punjab individuals³⁴ who those that carried the G allele displayed higher BMI and WC values.

According to Menzaghi et al.³⁵, the biology of these associations with anthropometric, clinical and biochemical characteristics is still unclear, for example, it is mentioned in their research that studies in animal models show that adiponectin is a potent insulin activator and modulator, in addition to regulating energy homeostasis and glucose tolerance. This relationship has also been found between the adiponectin levels in rodents and humans, which suggests that the +45 T>G ADIPOQ polymorphism can exert its action by diminishing or enhancing the expression of adiponectin, which can increase or decrease body weight and insulin resistance. In other populations, this variant may be associated with DM2, obesity and MS, whereas in other association studies, insulin resistance and DM2 are a consequence of the presence of the T allele.

Additionally, a binary logistic regression model was used to determine the influence of the +45 T/G polymorphism of the ADIPOQ gene on MS-DM2, where the odds ratio showed no association of this polymorphism as a risk factor predisposing to MS-DM2. The presence of the rare allele is considered a protection factor (Table 6). These findings oppose those previously reported by Stumvoll et al.³⁶ who studied non-diabetic Germans and recognized that the +45G allele was associated with higher IR indexes. This led to lower insulin sensitivity compared to T allele carriers while those subjects who carried the polymorphism could present a higher risk of obesity. The same phenomenon was described in diabetics and non-diabetics in Northern India³⁴; however, no association between +45 T/G polymorphism and the development of DM2 was found in Koreans individuals with and without DM2³⁷. The G allele of the +45 T>G polymorphism of the ADIPOQ gene has been associated with endocrine-metabolic pathology in Chinese individuals with and without MS in a similar population, whereas in Tunisia no association of polymorphism with MS was found^28,38.

Although there is a lack of consistency among studies, there is evidence that the 45G allele of the ADIPOQ gene plays a protective role in MS and DM2, whereas the 45T allele behaves as a risk allele for the development of obesity and insulin resistance, which are both related to the development of MS and DM2^32,33,39, consistent with the findings in this study.

The +45 T>G polymorphism of the ADIPOQ gene is a silent mutation without its own biological effect, since it is a GGT to GGG substitution, which translates to Gly15Gly. Adiponectin’s role in insulin sensitivity and BMI, as well as its role in both MS and DM2 pathologies are associated with genetic alterations linked to the polymorphism-related disequilibrium. mRNA G allele transcripts vary, proving higher than those of T allele of heterozygous individuals. This suggests that promoter gene polymorphisms or other adiponectin gene regulatory elements could be involved in the decrease or increase of the expression of the studied adipokine, being able to affect the mRNA allowing the stability of the splice sites in the same, affecting the amount of circulating proteins^28,30. It has also been observed that haplotypes that are in ligation unbalance with one or more functional variants can affect the levels of adiponectin in plasma, producing for example a change in the secondary structure of DNA, having a major influence on transcription, processing or translation; however, this is hypothetical, since the exact genetic mechanisms responsible for the expression of the specific allele have not been elucidated yet³².