The Predictive Value Of Single Nucleotide Polymorphism Of The Human Tumor Necrosis Factor-Alpha And Interleukin-10 Genes And Analysis Of Plasma Levels Of These Cytokines In Gestational Diabetes Mellitus And Their Clinical Implications

Abstract

Gestational diabetes mellitus (GDM) is a major public health concern affecting maternal and foetal health. It is defined as carbohydrate intolerance of variable severity first recognised during pregnancy that reflects a risk for adverse outcomes. It is still not known why GDM develops in some pregnant women. The progressive increase of insulin resistance during pregnancy and decrease in insulin secretion may lead to GDM. Recently, factors such as cytokines were found to play a role in insulin resistance.

The possession of specific genetic polymorphisms have been implicated as predisposing factors and if such alterations are operational in this maternal condition, then one could hypothesise that genetic polymorphisms might be useful as a marker to diagnose susceptibility to GDM. Based on this concept, the aim of this study was to investigate the association between single nucleotide polymorphism (SNP) in the human promoter of the tumour necrosis factor-alpha (TNF-α) gene as well as interleukin-10 (IL-10) gene with the development of GDM. The study also involved quantification of plasma levels of TNF-α, IL-10, and insulin to determine associations with gestational age. The SNPs were genotyped using polymerase chain reaction and restriction fragment length polymorphism. Cytokines as well as insulin were quantified using ELISA.

Demographic data derived from the selected population studied showed that pregnant women with GDM were significantly older and of higher parity and BMI than mothers with normal pregnancy (p<0.05). Ethnic distribution did not significantly differ in the two study groups (p>0.05), hence subsequent genetic studies did not attempt to look at ethnic group differences.

Majority (75%) of the GDM mothers had HBA1c < 6.5 %, indicating good glycaemic control. Contrary to what is reported elsewhere, in this study, we found no significant differences in the incidence of antenatal complications as well as pregnancy outcomes between controls and study population. Neonatal and maternal outcomes were almost similar in the two groups. We think that this is due to the tight glycemic control, close monitoring of the GDM mother as well as timely intervention at 38 weeks of gestation that is practised at the tertiary hospital that this study was conducted.

The difference of SNP genotype and allele frequencies observed in TNF-αpromoter gene at position -308 between the two groups were found to be not significant (p>0.05). This finding did not support our hypothesis that a SNP in the TNF- gene can be used as a predictive factor for GDM. Similarly, there were no significant (p>0.05) difference in the genotype and allele frequencies of SNP at positions – 824 and -1082 SNP in the promoter of the IL -10 gene between GDM and control groups. However, we found a significant (p<0.05) difference in the genotype and allele frequencies of a SNP at position -597 in the promoter of the IL-10 gene between the two study groups. Subjects who were carried the mutant alleles at position -597 in the promoter region of the human IL-10 gene were found to be 2.2 times more likely to develop GDM compared to those who do not have.

We also found eight different theoretically possible allele combinations in our study groups. We showed that there were some significant (p<0.05) differences observed in haplotype frequencies between the GDM and control groups. In addition, we also detected two rare haplotypes i.e. ATG and ACA haplotype.

Plasma levels of TNF-α as well as IL-10 were compared in the two study subjects in different stages of pregnancy and six weeks post partum. There were no significant (p>0.05) differences in the levels of these two cytokines observed at different stages of pregnancy as well as between control and GDM groups.

The plasma levels of IL-10 were lower in GDM subjects compared to controls. However, this difference was found to be statistically not significant (P>0.05). This was not an expected finding but we postulate that this pattern was observed may be a reflection of the good glycaemic control in the GDM patients included in this study. It would be interesting to evaluate the IL-10 levels in GDM subjects with poor glycaemic control to see if there could be significant differences in the plasma IL-10 levels.

The plasma levels of insulin increased with increasing gestational age and by third trimester and then decreased, with the GDM patients showing a consistently lower level. However, the difference was once again not statistically significant (P>0.05). Considering that our GDM patients were older, of higher parity and higher BMI, we postulate the impact of apoptosis on the islets cells or alternate impacts that may operate in insulin sensitisation when IL-10 levels didn’t reach that of normal patients. The implications of such findings are far and wide.

The control subjects had significant differences between insulin levels of during pregnancy and post partum but in the GDM subjects, insulin levels at 6 weeks post partum were significantly different from insulin levels at 32 weeks of pregnancy, which may reflect lower insulin response and β-cells defect in GDM subjects.

Although others have looked at other cytokines including adipokines in GDM, on the whole this study is a step towards understanding the role and genetics of cytokines as well as their plasma levels (of TNF-αand IL-10) in pregnancy. Our findings showed that SNP at position -597 was significantly associated with development of GDM and shows potential for use as a predictive factor.

Although we could not establish any cause-effect relationship, as is often the case when biochemical and genetic markers are used to predict disease, we have tried to draw some valuable correlated data for these cytokines which may prove valuable in future studies. Taken together and with regard to other studies, it seems that there is no single underlying factor mediating the pathogenesis of diabetes in pregnancy. Therefore considering other variables in the formula could give us better predictive value. Perhaps a mathematical model needs to be developed that will include epidemiologic factors, plasma biomarkers and polymorphisms of single nucleotide in the affected population.