1.0 Introduction. 1.1 Diabetes mellitus (DM)

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1 1.0 Introduction Type 2 Diabetes (T2D) is a complex disorder which involves complex interplay between genetic and environmental factors and it is characterised by dysfunction in insulin secretion combined with insulin resistance (Stumvoll et al., 2005). T2D has developed into a major health problem with its rapidly growing prevalence worldwide (Bonnefond et al., 2010). In past three decades, the status of T2D has changed from being considered as a mild preventable disorder of elderly to one of the major causes of early morbidity and mortality (Gregg et al., 2007). The magnitude of T2D epidemic is further amplified because of its macrovascular (CVDs and strokes) and microvascular (retinopathy, nephropathy and neuropathy) complications (Vinik, 2012). This explosive increase in prevalence of T2D can be attributed clearly to pandemic of obesity (Wing et al., 2011). In addition to this, the modern way of sedentary living, high caloric diet, improved socio economic status and globalisation has created new physiological conditions, particularly affecting the level of expression of genes involved in fuel metabolism. Most likely, the genetic variants of these genes may have evolved to adapt to new physiological conditions posed by the changing environment. The nature and prevalence of polymorphisms may vary among different populations depending upon the environment and inherited genetic pool. Genotyping the polymorphisms in these genes may explain the difference in disease susceptibilities among populations worldwide (Bougneres, 2002). Thus, understanding the molecular genetic basis of these complex traits will continue to be a growing concern among geneticists dealing with T2D and obesity, the main fuel disorders of the era. 1.1 Diabetes mellitus (DM) DM has been defined as the group of metabolic disorders characterised by chronic hyperglycaemia which occurs due to defects in insulin secretion, insulin action or both (American diabetes Association, 2011). 1

2 Traditionally DM has been classified into two major types: Type 1 Diabetes (T1D) and Type 2 Diabetes (T2D). T1D is an autoimmune form of DM characterised with absolute insulin deficiency due to autoimmune destruction of pancreatic β-cells. It has an early age of onset and affects only 5-10% of the cases reported with DM. Due to absolute deficiency of insulin T1D was previously known as Insulin Dependent Diabetes Mellitus (IDDM) (American Diabetes Association, 2011). T2D is a complex polygenic disorder marked by prominent symptoms of insulin resistance in peripheral tissues, insulin deficiency and defects in insulin secretion leading to elevated blood glucose levels. It accounts for 90-95% of the reported DM cases and is commonly known as the disease of the elderly due to late age of onset. T2D was also previously known as Non Insulin Dependent Diabetes Mellitus (NIDDM) (American Diabetes Association, 2011). 1.2 Symptoms of T2D Patients with T2D frequently experience following symptoms: Frequent thirst Excessive urination Blurry vision Irritability and skin rash Numbness in the hands or feet Frequent skin, bladder or gum infections Problems in healing of wounds Extreme fatigue ( 2

3 1.3 Complications of T2D The complications of T2D have been divided into microvascular complications and macrovascular complications. Microvascular complications of T2D include: diabetic retinopathy, diabetic neuropathy, diabetic nephropathy and diabetic foot. Macrovascular complications of T2D include: coronary artery disease (CAD), coronary vascular disease (CVD) and cerebral circulation complications like stroke ( 1.4 Defining and characterising T2D T2D is a complex multifactorial disorder involving delicate balance between multiple genetic components and environmental factors (Ahdi et al., 2012). Together with genetic factors, environmental factors also play a key role in development of T2D. The adaptation towards comfortable, sedentary and westernised lifestyle has lead to rapid increase in global prevalence of T2D (Ahlqvist et al., 2010). It has been predicted that by 2025, India will harbour 20% of the total T2D cases reported all over the world (Shaw et al., 2010). This rapid increase in the prevalence of T2D among Indians can be attributed to factors like high BMI, weight gain and hypertension as compared to other ethnic groups. Despite of having lower obesity profile as indicated by BMI range, Indians have greater waist circumference and WHR leading to prominent central obesity which is a know factor for T2D. Moreover, Indians have also been evidenced to have greater total abdominal and visceral fat leading to increased insulin resistance, metabolic syndrome and coronary heart disease (Misra, 2003). A recent study by (Bhardwaj et al., 2011) also confirmed the high prevalence of central adiposity among urban Asian Indians of North India than the urban population of South India. Besides central obesity, hypertension is also a major environmental risk factor for T2D among 3

4 Indians (Mohan et al., 2007). As the prevalence of T2D and obesity is increasing dramatically worldwide, so is the prevalence of hypertension. Increase in prevalence of hypertension can be observed individually but most significantly in conjugation with one another (Allcock and Sowers, 2010). The serious matter of concern is that, both hypertension and T2D affect same major organs of vascular system. Diseases like left ventricular hypertrophy and coronary artery disease (CAD) are more frequent in hypertensive diabetics than in patients with hypertension or T2D alone. The concomitant presence of hypertension and T2D accelerates development of diabetic retionopathy, stroke and decrease renal function (Grossman and Messerli, 2008). This drastic rise in the incidence of obesity, hypertension and T2D has occurred recently due to fat rich diet, stress and physical inactivity, but at the same time our genes have not changed. The adaptation to changing environmental conditions occurs genetically. This can be explained by the fact that genetic selection favoured the genotypes producing more energy with lesser food (Neel, 1962). In this modern era, individuals with these genotypes are genetically more susceptible for developing obesity and T2D. Therefore, these genes are good candidates for association studies in T2D. Besides environmental factors, genetic factors also play dominant role in the aetiology of T2D. The role of genetic susceptibility in predisposing an individual towards T2D development has been well recognised (Permutt et al., 2005). Advancement of several genetic approaches has led to better understanding of genetic heterogeneity, highlighting the role of various genetic pathways involved in T2D. It has been shown experimentally that abnormality in insulin secretary pathway precedes T2D development and disease can also occur in absence of insulin resistance. Thus, genotyping SNPs in candidate genes involved in insulin secretion pathways can be very informative. Moreover, few studies have also found that some diabetogenic genes conferred increased risk of developing T2D among Indians while some protective genes 4

5 in Europeans did not significantly appear to protect Indians because of different ethnic backgrounds (Radha and Mohan, 2007). Genes such as PGC-1α, UCP2 and SIRT1 are known to play role in insulin secretion pathway. PGC-1α is a transcription co-activator which helps in regulation of genes involved in energy homeostasis, nutritional and physiologic challenges (Finck and Kelly, 2006). Moreover, increasing PGC-1α expression also improves metabolic parameters such as insulin sensitivity and insulin signalling (Wenz et al., 2009). The versatility in regulation of various functions is achieved in tissue specific manner (Liu et al., 2011). PGC-1α (Gly482Ser) polymorphism is highly associated with T2D in various global populations. This polymorphism is also associated with hypertension, cardio vascular diseases and increased BMI (Myles et al., 2011). The potent association of PGC-1α (Gly482Ser) polymorphism with various diseases makes it a significant candidate for case control association studies. Along with PGC-1α, UCP2 is also a very important gene associated with T2D through insulin secretion pathway. UCP2 is a mitochondrial uncoupling protein, which facilitates the transfer of anions from the inner to the outer mitochondrial membrane and the return transfer of protons from the outer to the inner mitochondrial membrane separating oxidative phosphorylation from ATP synthesis. During this process the energy is dissipated as heat, which is also referred to as the mitochondrial proton leak. UCP2 has been found to play role in nonshivering thermogenesis, obesity and T2D (Jia et al., 2009). UCP2 is also known to negatively regulate insulin secretion and protect against ROS production and mitochondrial dysfunction. UCP2-866 G>A polymorphism is commonly associated polymorphism with T2D replicated in various populations (Chai et al., 2012). However, the results of various studies have not been consistent. Therefore, to decipher and confirm the status of UCP2-866 G/A polymorphism in some North Indian populations, this polymorphism was also included for the study. 5

6 SIRT1 is a NAD-dependent protein deacetylase which is known to play role in cell cycle regulation, response to DNA damage, metabolism and apoptosis (He et al., 2012). SIRT1 is also known to repress UCP2 and thus enhancing insulin secretion from pancreas. SIRT1 is also known to deacetylate PGC-1α, hence decreasing its expression having implications on metabolic diseases like obesity and T2D (Gerhart-Hines et al., 2007). The SIRT T/C polymorphism is a promoter polymorphism which has not been reported to have association with T2D in any of the populations. Therefore, to fill the existing lacuna this polymorphism was selected for genotyping in population of Punjab. 1.5 Rationale of the study The main rationale of the present study was, replication of the globally reported association of PGC-1α (Gly482Ser), UCP2 (-866 G/A) and to investigate the association of SIRT1 (-1400 T/C) polymorphisms with T2D in North Indian population groups. This study will also help to define the role of anthropometric (WHR and WC) and Physiometric (hypertension) factors along with these polymorphisms responsible for predisposing the studied population groups towards T2D development. This will further help to construct statistical models which could help in developing personalised medicine and predicting the development of T2D in future in these population groups. Inspite of loads of data provided by genome wide association studies (GWAS) (Bonnefond et al., 2010), little information is available for Indian populations, especially the North Indian population groups. The present study intended to screen SNPs within the candidate genes involved in insulin secretion pathway with a potential to lead tot2d in different ethnic endogamous groups of North Indian population of Punjab and revealing the susceptibility or resistance status of some genotypes towards the disease using. 6

7 Later on, after we designed the present study using candidate gene approach, several papers were published stating that only 10% heritability can be explained by GWAS in european populations and the data was all the more scanty for non European populations (Voight et al., 2010). In addition to this, the signifcant threshold for GWAS is P < 5 x 10-8 which may produce false-negative results. The role of low-frequency risk variants that may have relatively large affects on T2D susceptibility are unrecognised. The principle of GWAS is based on the common disease-common variant hypothesis, so, the variants with minor allele frequency less than 1% are eventually missed out (Imamura and Maeda, 2011). Moreover, there have been several quotes challenging the methodology, utility and research findings of GWAS and describing GWAS as a failure. Some of the main lacunae pointed out GWAS were: 1) flawed assumption that genetics played important role in genetics of common diseases, 2) GWAS was unable to explain the more genetic variations in the populations, 3) biological significance and functional implication of the associated variants could not be explained by GWAS and, 4) finally the results obtained by GWAS were also considered as false positives (Visscher et al., 2012). Therefore, in context with all this, after such huge studies with such large sample size failed, now it seems that way we designed the present study using candidate gene approach proved to be a better option. Although, this is a very initial study and step taken to define the genetic markers which are making the population of Punjab susceptible towards T2D. Further studies and functional validation is required to design appropriate molecular markers, for better understanding of the aetiology of the disease and its better management in Indian population where the disease is suggested to gain epidemic proportions. Keeping this in view, the present case-control study was focused at: 1. Screening and investigating the association of UCP2 (-866G>A), PGC 1α (Gly482Ser) and SIRT1 (-1400T>C) polymorphisms with T2D among patient and 7

8 control from population of Punjab belonging to various endogamous groups i.e. Banias, BCs, Brahmins, Jat Sikhs, Khatris, Rajputs and SCs from population of Punjab. 2. Analysing the association of various confounding parameters such as Waist-hip ratio (WHR), Waist circumference (WC) and hypertension with the development of T2D among the pooled population and various endogamous groups of Punjab. 3. Analysing the allelic and genotypic frequency distribution of above mentioned diabetogenic polymorphisms in cases and controls in various endogamous groups and in pooled population of Punjab. 4. Evaluating the association of studied polymorphisms in providing genetic risk or protection with T2D among various endogamous groups and in pooled population. 5. Exploring the SNP-SNP interactions, if any, to understand the role of these polymorphisms in the aetiology of T2D. 8