MATERIALS AND METHODS

The study group comprised 481 unrelated participants of which 305 subjects were with T2DM and 176 were normal healthy controls, who were randomly recruited for the study. None of the controls had any first degree and/or second degree relatives affected with type 1 diabetes, T2DM and hypertension. All the control subjects had fasting glucose <100 mg dL^-1 and post lunch blood glucose <140 mg dL^-1 and were aged above 50 years. All the T2DM patients were recruited from Kamineni Hospitals, Hyderabad and were diagnosed in accordance with the American Diabetes Association guidelines (American Diabetes Association, 2009). Informed consent was obtained from all the participants of the study and the study was approved by the institutional ethical committee.

Anthropometric data for calculation of Body Mass Index (BMI), clinical information and pedigree details were recorded in a specified proforma. Two milliliter of whole blood through venipuncture was obtained from all participants and genomic DNA was isolated by the rapid salting-out method (Lahiri et al., 1992).

The tetranucleotide repeat (TTTC)_n polymorphism in the 3' UTR of the human leptin gene (LEP-tet) was detected by Polymerase Chain Reaction (PCR) as reported previously (Shintani et al., 1996). The primer sequences were as follows: Forward primer-- 5' AGT TCA AAT AGA GGT CCA AAT CA 3' and Reverse primer-- 5' TTC TGA GGT TGT GTC ACT GGC A 3'. The PCR mixture contained 0.1 μg genomic DNA, 25 picomoles of each primer, 0.8 mmol L^-1 of each deoxynucleoside triphosphate (Eppendorf, Hamburg, Germany), 1.5 U Taq polymerase (Bangalore Genei, India) and 1X Taq polymerase reaction buffer in a total volume of 25 μL. The PCR cycling reactions were performed in an iCycler machine (BioRad Laboratories, Hercules, California, USA) with an initial denaturation step of 94°C for 5 minutes followed by 35 cycles of 94°C for 30 seconds, 54°C for 30 seconds and 72°C for 1 minute and a final extension of 72°C for 5 minutes. The PCR products were electrophoresed in 2% agarose gels and were visualized by staining with ethidium bromide. The alleles for the LEP-tet polymorphism are classified into two groups: the shorter one (termed class I) with allele size from 150-166 bp and the longer one (termed class II) with allele size from 220-252 bp.

Statistical analyses: Descriptive characteristics of group variables are expressed as Mean±SE. The variation in lipid profiles and other continuous variables between patients and controls was tested for significance using independent sample t-test. Percentage distribution of the LEP-tet genotypes was calculated and the significance was estimated by using the chi-square (χ²) test. The risk estimates for various combinations of genotypes of the LEP-tet polymorphism were obtained by calculating the Odds Ratio (OR) and its 95% CI and p-value. The two tailed p-values for 2x2 contingency tables were calculated using the Fisher’s exact test. All the statistical analyses were conducted using GraphPad Prism version 5.04 for Windows, GraphPad Software, California, USA.

RESULTS

Table 1 summarises the epidemiological and biochemical characteristics of the study subjects. The mean age at sampling of the control subjects was 54.6±1.2 years and was not significantly different (p = 0.19) from that of the patients which was 56.2±0.5 years. The mean age at onset of T2DM in the patient group was 45.8±0.5 years and the mean duration was 10.8±0.4 years. The control subjects had a mean BMI of 27.0±1.2 kg m^-2 which was not significantly different from that of the patients which was 27.2±0.3 kg m^-2. Significant elevation (p<0.0001) of mean plasma levels of fasting and post lunch blood glucose in the T2DM patients compared with that in the controls was observed.

Table 1:	Mean distribution of different epidemiological and biochemical parameters observed in T2DM and controls
§n: 294 in T2DM group and n: 170 in control group, BMI: Body mass index, FBS: Fasting blood sugar, PLBS: Post lunch blood sugar, TC: Total cholesterol, HDL: High density lipoprotein cholesterol, LDL: Low density lipoprotein cholesterol, TG: Triglycerides, SBP: Systolic blood pressure, DBP: Diastolic blood pressure

Fig. 1:

Representative gel photograph for the LEP-tet polymorphism, Lane 7: 100 bp DNA ladder; Lanes 8, 10, 11: Genotype I/I, Lanes 2, 4, 5, 9, 12, 13: Genotype I/II, Lanes 1, 3, 6: Genotype II/II

With respect to the fasting levels of lipids in our study group, we observed that the mean levels of total cholesterol (Controls: 192.4±5.3, T2DM: 165.9±5.1, p = 0.0008) and LDL cholesterol (Controls: 125.4±4.6, T2DM: 96.5±4.2, p = 0.0001) were significantly lower in the T2DM subjects while the mean triglyceride levels (controls: 131.4±8.7, T2DM: 182.9±15.1, p = 0.014) were significantly higher in the T2DM subjects. Mean levels of HDL cholesterol were lower in the diabetic group (37.3±1.1) when compared with that in the controls (40.9±1.4) and this difference was found to be significant statistically (p = 0.014).

Figure 1 depicts the electrophoretic pattern of the PCR amplicons which have been resolved in a 2% agarose gel. The LEP-tet polymorphism comprised two groups-class I and class II based on the number of tandem repeats of the tetranucleotide sequence (TTTC). The results of the genotyping of the LEP-tet polymorphism are presented in Table 2. The frequency of the class I allele was 47.5% in the T2DM group as against the 49.4% in the control subjects. The class II allele showed a frequency of 52.5% in the T2DM group compared with 50.6% in the control group.

Table 2:	Genotype and allele frequencies of LEP-tet polymorphism in T2DM patients and controls with respect to gender and obesity
§n: 294 for patients and n: 170 for controls for obesity, *p-value for genotypic frequencies between T2DM and control subjects, **p-value for allele frequencies between T2DM and control subjects, χ2: Male patients vs female patients: 0.31 (p = 0.86), χ2: Obese patients vs. non obese patients: 2.35 (p = 0.31)

Table 3:	Test for deviation from Hardy Weinberg equilibrium
nI/I(e): Observed number of subjects with Genotype I/I(expected number), nI/II(e): Observed number of subjects with Genotype I/II (expected number), nII/II(e): Observed number of subjects with Genotype II/II(expected number ), p (Pearson): Pearson's goodness-of-fit chi-square (degree of freedom = 1), p (Llr): Log likelihood ratio chi-square (degree of freedom = 1)

Table 4:	Risk estimations for different combinations of LEP-tet I/II genotypes in T2DM patients and controls
*Statistically significant (p = 0.04) OR and its 95%CI as computed by Fisher’s exact test

A perusal of Table 2 reveals that the allele frequencies did not differ significantly (p = 0.62) between the T2DM subjects and control subjects. No significant differences in the allele frequencies between patients and controls were observed even after stratification of the subjects by gender and obesity. As regards the genotype frequencies, the I/II genotype was the most frequent (53.8% in the T2DM group and 44.3% in the control group). The genotype frequencies also did not differ significantly among the total subjects of the two study groups. No significant differences between genotype frequencies were observed even after stratification of the study subjects by obesity and gender. The results of the test for deviation from Hardy-Weinberg Equilibrium are given in Table 3. The genotype frequencies showed no significant deviations from Hardy-Weinberg equilibrium. The risk estimates for type 2 diabetes with different combinations of genotypes (Table 4) reveal that the I/II genotype is showing a modest OR of 1.46 (95% CI: 1.01-2.12, p = 0.04).

DISCUSSION

T2DM is considered to be a complex disorder involving multiple genetic and environmental triggers. Studies across the world are underway to decode the genetic component of type 2 diabetes. Among a vast array of genetic factors responsible for T2DM, those that control adiposity and energy homeostasis play a crucial role in the setting in of insulin resistance and consequent type 2 diabetes. Obesity, which is a result of disturbance of energy homeostasis, namely, imbalance between energy intake and energy expenditure, is characterised by the presence of excess adipose tissue which is a major source of endogenous production of Non Esterified Fatty Acids (NEFAs) through lipolysis. The increased NEFAs in the circulation result in insulin resistance, which can further lead to the development of T2DM (Randle et al., 1963).

The archetypical view of the adipose tissue being an inert storage compartment has changed significantly over the past twenty years. Apart from the secretion of NEFAs, adipocytes are also a source of several metabolically active molecules, termed as adipocytokines or adipokines, such as leptin, adiponectin, resistin, TNF-α etc., most of which play crucial roles in the modulation of energy expenditure and insulin resistance (Cook et al., 1987; Scherer et al., 1995). The recent cloning and characterization of the human homolog of the mouse ob gene, --- the LEP gene --- raised hopes about explaining the possible connection between T2DM and obesity (Zhang et al., 1994). Plasma leptin and adiponectin levels have been reported to influence insulin resistance and progression to T2DM (Daimon et al., 2002; Abd El-Ghaffar and El-Said, 2006; Assal et al., 2007; Mohiti et al., 2009).

Mutations in the mouse ob gene cause severe obesity and T2DM in mice. While mutations in the human LEP gene have not been able to explain the wide prevalence of obesity, common polymorphic variants have been found to be associated with obesity in humans. Common polymorphic variants in the LEP gene have also been reported to influence serum leptin levels, BMI, hypertension and T2DM.

No significant differences in the allele and genotype frequencies between T2DM subjects and healthy controls were observed. A modest risk of 1.46 (p = 0.04) was observed with I/II genotype in the present study which could not be replicated when the study subjects were stratified by obesity and gender. The risk engendered by the LEP-tet polymorphism could be speculated to be the result of alteration of circulating leptin levels by virtue of the polymorphism. Considering that the polymorphism is present in the 3' UTR of the LEP gene, it can be surmised that the expression of the LEP gene and consequent serum leptin levels might be altered due to the polymorphic variant. It is important to remember that in complex traits such as T2DM, the lack of a strong association with a given polymorphic variant does not necessarily preclude the lack of effect of that variant on disease phenotype, because, the susceptibility to disease may be conferred by combinations of variants rather than a single polymorphism.

CONCLUSION

To conclude, individuals heterozygous for the LEP-tet polymorphism possess a statistically significant 1.46 fold risk of T2DM. To the best of our knowledge, this is the first report pertaining to the association of LEP-tet polymorphism with T2DM in a South Indian population. Future studies in different populations involving the LEP-tet polymorphic variant in conjunction with various other polymorphic variants in the LEP gene and the interactions thereof would help in elucidating the role of leptin in the modulation of T2DM.

ACKNOWLEDGMENTS

We thank all the study participants. Kamakshi Chaithri Ponnaluri is a recipient of Senior Research Fellowship from Council of Scientific and Industrial Research, New Delhi, India. We thank University Grants Commission, New Delhi, India for providing Research Fellowship to Parimala Narne.