1. Gender Based Association Between ACE gene(I/D)
Polymorphism and Type 2 Diabetic Mewari Patients
of Udaipur city, Rajasthan
Pulakes Purkait1
, L. K. Gandharve2
, V. K. Purohit2
J. M. Naidu3
and B. N. Sarkar4*
ABTRACT
Angiotensin-I converting enzyme (ACE) gene I/D polymorphism is shown to have
association with diabetes. The present study examined the prevalence and gender
based association of ACE I/D polymorphism among 111 Mewari individuals of both
sexes from Udaipur, Rajasthan. Among them 50 individuals belong to Type 2 diabetic
(Male = 33, female =17) and 61 normal controls (Male = 33, female =28). Isolated
DNA samples from the studied subjects were genotyped using polymerase chain
reaction. The frequency of DD, ID and II genotypes in total male are found to be
26.7%, 53.3% and 20.0% respectively, whereas the same for total female are 26.7%,
50.0% and 23.3% respectively. Though the odds ratio (OR 3.39, 95% CI=0.80-14.31)
suggests an increased risk of diabetes mellitus male having DD genotypes, but
statistically non significant. The frequency of D allele among the diabetic patients is
found to be 0.533 as compared to 0.516 among only diabetic female subjects. Our
study did not identify any association of D allele with any increased risk of T2DM for
gender (OR=1.58) among the Mewari population of Udaipur city.
Keywords: gender, Type 2 diabetes, ACE, I/D polymorphism, Mewari population.
INTRODUCTION
Angiotensin converting enzyme (ACE) polymorphism is a major component of
Renin-Angiotensin-Aldosterone System (RAAS). The RAAS is a regulator of both
blood pressure and is suggested to play an important role in the development of
complication in Type 2 Diabetes Mellitus (Remuzzi et al., 2008; Wang et al., 2012;
Felehgari et al., 2012).
TheACE gene is located on chromosome 17q23 consists of 26 exons, 25 introns
and it spans 21 kb. The polymorphism of ACE gene results from the insertion (I) or
1
Anthropological Survey of India, WRC, Udaipur- 313001, Rajasthan, India
2
Bhupalpura Govt. Dispensary, N Road, Bhopalpura, Udaipur, Rajasthan, India;
3
VNR VignanaJyothi institute of Engineering and Technology, Hyderabad 500 090, India
4*
Corresponding author: Dr. B, N, Sarkar Anthropological Survey of India, 27 J. N. Road, Kolkata
700016, India. E-mail: diabetes.wrc@gmail.com
Jr. Anth. Survey of India, 62(2) : (169-177), 2013

2. 170 Gender based Association between ACE gene(I/D) Polymorphism and Type 2...
deletion (D) of a 287 bpAlu repeat sequence near the 32 end of intron 16 which has
three genotypes II, ID and DD (Regart B et al. 1992). The function of ACE is the
conversion of Angiotensin I to angiotensin II and is thus implicated in the
pathogenesis of diabetes and diabetic complication like nephropathy (Jeffers et
al., 1997; Estacio et al., 1998; Grzeszczak et al., 1998; Bedir et al.,1999;
Viswanathan et al., 2001).
Various studies have shown that there are differences in the I/D polymorphism
in different ethnic groups (Zhang YM et al., 2001, Pasha et al., 2002, Jayapalan
J Jet al., 2008) and Sikderet al., (2013) reported that as different population from
different region of India as pooled Indian population shown same distribution
pattern of ACE gene polymorphism. D allele and DD genotype of ACE gene has
been reported to be associated with Diabetic nephropathy in T2DM (Hsieh MC
et al., 2000, Ng DP et al., 2005, Tripathi G et al., 2006). Female diabetes patients
with D allele of ACE gene are likely to progress diabetic nephropathy (Tien KJ
et al. 2009). Mansoor Q et al., (2010) reported that, gender affect of ACE I/D
polymorphism and I allele has been associated with progression of diabetic
nephropathy in males with Pakistani T2DM patients. An extensive interethnic
variation in the distribution of ACE gene polymorphism appears to be one of the
causes for such inconsistent findings (Barley et al., 1994; Jayapalan JJ et al.,
2008).
Therefore, the aim of the present study was to investigate the gender distribution
of ACE gene (I/D) polymorphism and its relationship with Type 2 Diabetes patients
in Mewari population of Udaipur city, Rajasthan.
MATERIAL AND METHODS
Ethics
Prior to the recruitment of subjects the ethical committee clearance was obtained
from the respective medical institutions and accordingly informed consent was
obtained from all the participants.
Participant’s Inclusion Criteria
In this study out of 111 participant’s only 50 participants are type 2diabetes and
who are taking anti-diabetic medication above 5 years and 61 participants are healthy
control with no family history of any metabolic diseases.
Participants
The present study on ACE gene polymorphism was carried out among 100 Mewari
individuals, consisting of 50 (male = 33, female = 17) Type 2 Diabetes (T2DM)
patients and 61 (male = 33, female = 28) healthy controls in and around Udaipur
city of Rajasthan. Registered patients were recruited from Bhupalpura Govt.

3. Pulakes Purkait, L. K. Gandharve, V. K. Purohit, J. M. Naidu and B. N. Sarkar 171
Dispensary, Udaipur and a detailed medical history of each patient was recorded
accordingly. The detection of Type 2 diabetes was based on physician’s
recommendation. The healthy unrelated controls were randomly selected and
recruited from local community centers.10 ml of venous blood (6 ml was drawn
into EDTA tube for genetic analyses and 4 ml was drawn into serum tube for
biochemistry analysis) was collected from the participants by using sterilized
disposable blood collection kits.
Anthropometric Measurements and Biochemical Analysis
Anthropometric measurements and indices were taken in each subject, using
standard methodology. Clinical data included information of duration of diabetes,
presence of any complication, history of other disorders, systolic blood pressure,
and diastolic blood pressure. Anthropometric data, weight (kg), and Height (cm),
were obtained to calculate body mass index (BMI) using the formula, weight (kg)/
[height (m2)]. The blood pressure was measured on the right arm of the subjects
using an automated blood pressure monitor (Omron, Japan) by seated and rested
for 15 minutes. Blood glucose was measure using the Breez 2 glucometer (blood
glucose monitor).
4 ml of venous blood samples were drawn in the morning. Thereafter, the
blood samples were centrifuged, serum were separated, collected and stored at -
86°C until further analysis. All laboratory measurements were conducted at the
DNA Laboratory in the Anthropological Survey of India, Western Regional Centre,
Udaipur. The serum levels of total cholesterol, triglycerides, high-density lipoprotein
cholesterol (HDL), low density lipoprotein cholesterol (LDL), Urea, Uric Acid,
Creatine, Chloride, total Protein and albumin were measured enzymatically on a
Fully auto analyzer EM360 (TRANSASIA with kits supplied by TRANSASIA
BIO MEDICAL LTD).
Genotype Assessment
Approximately 6 ml of venous blood was drawn from each of the subjects in EDTA
vials and genomic DNA was extracted from whole fresh blood using standard salting
out method using phenol-chloroform (Miller S A, et al., 1998). The ACE I and D
alleles were identified by PCR amplification, using 20 pmol of each primer (flanking
primer pair): oligonucleotide sense primer: 52 -CTG GAG ACC ACT CCC ATC
CTT TCT-32 and anti-sense primer : 52 -GAT GTG GCC ATC ACA TTC GTC
AGA T-32 (Regart B et al. 1992) in a final volume of 10ìl containing 50ng of
genomic DNA, 20 pmol of each primer, 10X Taq PCR buffer, 25 mM MgCl2 ,
100
mM of each dNTPs and 1 U/uL of Taq polymerase.
PCR amplification was performed in a S1000TM
Thermo Cycler (BIO-RAD).
PCR was carried out with a Gradient standardize PCR condition with an initial
denaturing time at 95°C for 6 minutes. Then the DNA was amplified for 35 cycles

4. 172 Gender based Association between ACE gene(I/D) Polymorphism and Type 2...
with denaturation at 94°C for 1 minute, annealing at 60°C for 1:30 minutes extension
at 72°C for 2 minutes and final extension 72°C for 7 minutes. Electrophoreses of
PCR products were done in 2.5% agarose gel with ethidium bromide staining and
directly visualized in UV light. The amplified product is a 190 bp fragment in the
presence of the deletion (D) allele and a 490 bp fragment in the presence of the
insertion (I) allele. Therefore, there were three genotypes after electrophoresis: a
490 bp band (genotype II), a 190 bp band (genotype DD), or both 490 and 190 bp
band (genotype ID).
Statistical Analysis
Data were analyzed using statistical package for Social Sciences statistical software
(SPSS Version 16, Chicago, Illinois, USA). Data were presented as mean ± SD and
comparison between T2DM and Control and among group were analyzed by the
independent-sample T tests and one way ANOVA analysis respectively.
Genotype and Allele frequencies were calculated for the SNPs and allelic
association with disease and gender using ÷2-test. A level of P <0.05 was assumed
statistical significance.
RESULTS AND DISCUSSION
In this study we aimed to investigate the gender based association between Clinical
characteristics and ACE gene (I/D) polymorphism in Mewari healthy control and
type 2 Diabetes patients.The study group of this report on total 111 Mewari
individual comprises 66 male (T2DM = 33, Control = 33) and 45 Female (T2DM
= 17, Control = 28).
Table 1 showed the basic Clinical characteristicof the overall studied
participants (Male and Female) of Mewari population. The two study groups
were well-matched for age, body mass index (BMI), SBP, DBP, Glucose,
Cholesterol, Triglyceride, LDL, Chloride, Total Protein and Albumin of
both Male and female individuals. Male had significantly higher Height, Weight,
Urea, Uric Acid, Creatinine values. Only HDL value was significantly higher in
female.
Table 2 summarizes all the subjects into four groups: T2DM (Male & Female),
normal controls (Male & Female). The study group T2DM Was well-matched for
age, BMI, SBP, DBP, Glucose, Cholesterol, Triglyceride, LDL, Urea, Chloride,
Total Protein and Albumin value of both Male and female individuals. Male had
significantly higher Height, Weight, Uric Acid, Creatinine values. Female had
significantly higher HDL value. Control group shown same result as T2DM group
along with Urea value, which was significantly higher in male.
Table 3 shows gender wise distribution of the ACE genotype and allele in
Control and T2DM patients. Among the total male, 19 (28.79%) male had the II

5. Pulakes Purkait, L. K. Gandharve, V. K. Purohit, J. M. Naidu and B. N. Sarkar 173
Table 1
Basic Clinical Characteristic of Male and Female in Mewari
Population of Udaipur City
Variables Male (N =66) Female (N =45) Total (N = 111) F P
Mean ± SD Mean ± SD Mean ± SD
Age (Years) 49.86 ± 11.61 48.07 ± 10.98 49.13 ± 11.34 0.67 0.415
Height (cm) 167.61 ± 6.92 157.03 ± 6.58 163.32 ± 8.54 65.105 0.000*
Weight (kg) 72.09 ± 11.11 63.89 ± 9.79 68.76 ± 11.30 16.014 0.000*
BMI (kg/ m2
) 25.65 ± 3.63 25.94 ± 3.76 25.77 ± 3.67 0.151 0.698
SBP (mmHg) 132.24 ± 19.55 127.22 ± 23.69 130.21 ± 21.37 1.483 0.226
DBP (mmHg) 87.30 ± 10.26 83.78 ± 9.42 85.87 ± 10.04 3.37 0.069
Glucose (mg/dl) 150.42 ± 56.33 132.38 ± 49.18 143.11 ± 54.05 3.038 0.084
Cholesterol (mg/dl) 166.27 ± 42.63 173.37 ± 45.96 169.15 ± 43.95 0.697 0.406
Triglyceride (mg/dl) 176.09 ± 97.04 158.28 ± 70.48 168.87 ± 87.34 1.113 0.294
HDL (mg/dl) 39.42 ± 8.36 46.96 ± 10.44 42.47 ± 9.94 17.765 0.000*
LDL (mg/dl) 101.34 ± 28.47 105.78 ± 29.84 103.13 ± 28.98 0.627 0.43
Urea (mg/dl) 21.97 ± 11.95 16.87 ± 6.55 19.91 ± 10.38 6.797 0.01*
Uric Acid (mg/dl) 6.31 ± 1.29 5.21 ± 1.41 5.86 ± 1.44 18.046 0.000*
Creatinine (mg/dl) 1.09 ± 0.17 0.95 ± 0.15 1.04 ± 0.17 19.326 0.000*
Chloride (mmol/L) 100.32 ± 3.29 101.55 ± 3.25 100.82 ± 3.32 3.762 0.055
Total Protein (g/dl) 7.17 ± 0.57 7.27 ± 0.54 7.21 ± 0.56 0.838 0.362
Albumin (g/dl) 3.93 ± 0.26 3.90 ± 0.24 3.92 ± 0.25 0.263 0.609
genotype, while the ID genotype was present in 32 (48.48 %), the DD genotype in
15 males (22.73%) and the D phenotype (DD+ID) was 47 (71.21%). In female, the
DD, ID II genotypes and D phenotype (DD+ID) were present in 13.33 %, 55.56 %,
31.11% and 68.89% respectively. In this study, we found the frequency of D allele
slightly higher in total male (46.97%) than in total female (41.11%) but statistically
non significant (X2
= 0.743, P =0.389).
Among the total male group 33 individuals were T2DM patients and 33
individual belongs to control group. In T2DM-Male group, the DD, ID II genotypes
and D phenotype (DD+ID) were present in 21.21 %, 48.48 %, 30.30% and 69.70%
respectively. Among the Control- male, 24.24% male had the DD genotype, while
the ID genotype was present in 48.48 %, the II genotype 27.27% and the D phenotype
(DD+ID) was 72.73%. D allele slightly higher in Control-Male (48.48%) than in
T2DM-male (45.45%) but statistically non significant, X2
= 0.122, P =0.727
(Table 3).
Among the total female group 17 individuals were T2DM patients and 28
subjects belongs to control. Among the T2DM- Female, 11.76% female had the
DD genotype, while the ID genotype was present in 41.18%, the II genotype 47.06%

6. 174 Gender based Association between ACE gene(I/D) Polymorphism and Type 2...
Table2
BasicClinicalCharacteristicsofMaleandFemaleinT2DMandcontrolofMewariPopulation
VariablesT2DM(N=50)Control(N=61)
MaleFemaleMaleFemale
(N=33)(N=17)FP(N=33)(N=28)FP
Mean±SDMean±SDMean±SDMean±SD
Age(Years)56.64±10.0053.88±10.000.8510.36143.09±8.9044.54±10.150.3510.556
Height(cm)165.53±7.75155.93±5.1721.1330.000169.70±5.32157.70±7.3154.7620.000
Weight(kg)73.21±13.1463.71±9.357.030.01170.97±8.7064.00±10.228.2770.006
BMI(kg/m2
)26.69±4.2126.27±4.110.1160.73524.62±2.6325.73±3.591.9320.17
SBP(mmHg)140.48±20.43135.53±23.370.5990.443124.00±14.81122.18±22.830.1410.709
DBP(mmHg)89.58±12.3584.00±7.652.8770.09685.03±7.1283.64±10.490.3750.543
Glucose(mg/dl)180.94±62.35162.47±69.670.9090.345119.91±25.25114.11±12.161.2330.271
Cholesterol(mg/dl)171.94±49.71188.29±45.601.2820.263160.61±33.99164.32±44.570.1360.713
Triglyceride(mg/dl)187.48±116.92172.88±65.410.2270.636164.70±72.04149.43±73.110.6710.416
HDL(mg/dl)40.19±7.7750.78±8.9818.730.00038.64±8.9744.65±10.745.6660.021
LDL(mg/dl)106.03±33.45115.37±31.010.9190.34396.65±21.9699.96±28.070.2670.607
Urea(mg/dl)25.45±15.7519.60±7.712.0710.15718.51±4.1815.22±5.227.4780.008
UricAcid(mg/dl)6.19±1.515.26±1.524.2090.0466.44±1.055.18±1.3716.5220.000
Creatinine(mg/dl)1.15±0.201.01±0.156.7510.0121.04±0.110.93±0.1412.1520.001
Chloride(mmol/L)100.35±3.05101.08±3.410.5850.448100.29±3.58101.84±3.193.1340.082
TotalProtein(g/dl)7.29±0.597.41±0.570.4680.4977.05±0.547.19±0.520.9620.331
Albumin(g/dl)3.90±0.303.98±0.191.0670.3073.95±0.223.85±0.252.7520.102

7. Pulakes Purkait, L. K. Gandharve, V. K. Purohit, J. M. Naidu and B. N. Sarkar 175
Table3
FrequencyDistributionofACE(I/D)GenotypeandAlleleamongMaleandFemaleSubjectinT2DMand
CONgroupofMewariPopulation
ACEGeneMALEFEMALEMALE(N=66)FEMALE(N=45)
Genotypen(%)n(%)T2DMn(%)CONn(%)T2DMn(%)CONn(%)
DD15(22.73)6(13.33)7(21.21)8(24.24)2(11.76)4(14.29)
ID32(48.48)25(55.56)16(48.48)16(48.48)7(41.18)18(64.28)
II19(28.79)14(31.11)10(30.30)9(27.27)8(47.06)6(21.43)
DD+ID47(71.21)31(68.89)23(69.70)24(72.73)9(52.94)22(78.57)
DDVsIDVsIIX2
=1.557X2
=0.119X2
=3.301
P=0.459;DF=2P=0.942;DF=2P=0.192;DF=2
X2
=5.161
P=0.523;DF=6
DDVsIIX2
=1.056X2
=0.119X2
=0.952
P=0.304;DF=1P=0.730;DF=1P=0.329;DF=1
(DD+ID)VsIIX2
=0.069X2
=0.074X2
=3.242
P=0.793;DF=1P=0.786;DF=1P=0.072;DF=1
Allele
D62(46.97)37(41.11)30(45.45)32(48.48)11(32.35)26(46.43)
I70(53.03)53(58.89)36(54.55)34(51.52)23(67.65)30(53.57)
IVsDX2
=0.743X2
=0.122X2
=1.731
P=0.389;DF=1P=0.727;DF=1P=0.188;DF=1
X2
=2.562
P=0.464;DF=3

8. 176 Gender based Association between ACE gene(I/D) Polymorphism and Type 2...
and the D phenotype (DD+ID) was 52.94%. In Control-Female group, the DD, ID
II genotypes were 14.29%, 64.28%, and 21.43% where as D phenotype (DD+ID)
was present 78.57% (Table 3). In female subjects, D and I allele frequencies have
no significant difference in T2DM and controls subjects (X2
= 0.122, P =0.727).
We also investigated the association between the D phenotype (DD + ID) and
II genotype in two groups. The D phenotype frequency in Male group was found
slightly higher than that of the female and that indicated the D phenotype was not
statistically significant between male and female (X2
= 0.069, P = 0.793 ). When
we compared the Male and Female group in Case – control model, but there was
no association between T2DM and Control group (X2
= 0.074, P =0.786; X2
= 3.242,
P =0.072) respectively (Table 3). In conclusion, we do not found any significant
association of ACE I/ D polymorphism with male and female T2DM subjects of
Mewari Population of Udaipur city.
CONFLICT OF INTERESTS
The authors declare no conflict of interest for the present research outcome.
ACKNOWLEDGMENTS
We would like to thank the members of the study populations for their cooperation during data
collection. We wish to express our deep gratitude to the Director, Anthropological Survey of
India, for his kind permission to initiate the work and also for providing financial support.
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