Molecular Genetic Studies of Diabetic Subjects

Abstract

Diabetes mellitus is a complex metabolic disorder characterized by chronically high blood glucose levels. It has several types based on underlying etiology and of these, Type 2 Diabetes Mellitus (T2DM or T2D) is the most prevalent which affect 90-95% of all diabetic patients. Its worldwide prevalence is increasing at an alarming rate owing to abundant food supply and physical inactivity. It results due to a complex interaction of genetics and environment. Genetics being the significant player in T2D development is the focus of the current study. A number of genes have recently been identified to associate with T2D through Genome-Wide Association Studies. However, such genetic variants are required to be replicated for their association in different ethnic populations. The genetic screening of T2D associated genes for replication is usually carried out using high-throughput genotyping assays like TaqMan assay which are costly for low-income countries where diabetes is quite prevalent. Therefore, low-cost genotyping methods are desired for genetic screening in such populations. Hence, the main aim of this study is to develop polymerase chain reaction (PCR) based assays and implement them for genetic screening of diabetes in Pakistani patients. As one of the modified forms of PCR called ARMS-PCR (Amplification Refractory Mutation System-PCR) is considered as the cost effective method for genotyping, so in this study efforts were made to successfully develop ARMS-PCR assays for six single nucleotide polymorphism (SNPs) in the four most commonly diabetes associated genes (e.g. TCF7L2: Transcription factor 7-like 2 ; PPARγ: Peroxisome proliferator-activated receptor gamma; KCNJ11: Potassium channel, inwardly rectifying family J, member 11; and FTO: Fat mass and obesity). Subsequently, the specificity of these assays was validated by the gold standard DNA sequencing showing complete accuracy of the assays. Furthermore, these indigenously developed ARMS-PCR assays were applied for genetic screening of human samples (Healthy controls = 233 and Type 2 Diabetics = 211) collected during this study. Genotyping results were statistically analyzed for their association with T2D and its related biochemical parameters. From these analyses, interestingly it was found that the FTO gene variants were significantly associated with T2D (Allelic Odds Ratio, OR=1.54, at 95% Confidence Interval, CI=1.15-2.05, p=0.004 for rs8050136, and OR=1.56, 95% CI= 1.16-2.08, p=0.003 for rs9939609). Since, FTO is generally considered as an obesity related gene but our results showed its association with T2D independent of BMI (Body Mass Index, a physical measure of obesity). Further from the analyses of clinical and biochemical parameters it was found that the SNP genotypes of FTO showed significant effect on blood glucose level (p= 0.018 for rs8050136 and p=0.015 for rs9939609). On the other hand, the genotypes of PPARγ showed significant effect on the cholesterol level (p=0.03), whereas the genotypes of TCF7L2 showed significant effect on triglyceride level (p=0.037 for rs7903146 and p=0.025 for rs12255372). As TCF7L2 is one of the highly associated genes with T2D diabetes in several populations, yet its molecular actions are still largely unexplored. Hence, a study was carried out to investigate the molecular interaction of TCF7L2 with another important gene, Menin, which is implicated in pancreatic beta cell biology. The expression of these genes was manipulated by siRNA and pCMV vector and effects on the expression of other genes were studied by qPCR and western blotting in cell culture and isolated human pancreatic islets. It was found that the expression of TCF7L2 increased by siRNAs against Menin suggesting that their expression is oppositely regulated in the CM cell lines (a model of human pancreatic beta-cells). Similarly siRNA against TCF7L2 decreased the transcription of Menin but its protein expression increased which lead us to hypothesize that the translation of Menin may be regulated independent of its transcription. Conversely, over-expression of TCF7L2 by using adenoviral vector, pCMV-TCF7L2 increased Menin transcription but its protein expression decreased, thus further supporting the hypothesis. In vitro experiments on human pancreatic islets showed that inhibition of Menin increased TCF7L2 expression along with increasing insulin secretion as measured by the stimulatory index of human islets. Overall, it was inferred from these experiments that both these proteins have a close interaction with each other to regulate different cellular processes which may be of importance to beta-cell biology. In summary, the output from this thesis not only provides a pilot data on the genetic association of T2D for the Pakistani population from Faisalabad region but also gives the development of low-cost genetic assays for screening of common diabetogenes. The low-cost PCR based assays will help to extend the genetic association data in resource limited set up and this genetic association data will be further helpful in devising the region-wise genetic model for T2D prediction. Moreover, it also provides preliminary data for functional genetic studies of a commonly associated genetic factor with diabetes, which could be further investigated as a potential therapeutic target. Hence, this study can contribute in prediction, prevention and treatment of diabetes especially in low-income countries.