evaluation of tcf7l2 and ppar-γ gene polymorphisms and insulin

Acta Medica Mediterranea, 2014, 30: 1203
EVALUATION OF TCF7L2 AND PPAR-Γ GENE POLYMORPHISMS AND INSULIN SECRETION
ABILITIES IN PATIENTS WITH TYPE II DIABETES AND THEIR UNAFFECTED SIBLINGS
KEMAL TÜRKER1, MEHMET NUMAN TAMER2, OĞUZHAN AKSU2, BANU KALE KÖROĞLU2, ISMAIL HAKKI ERSOY2, RAMAZAN
YILMAZ3, EMEL BARAN4, RECEP SÜTCÜ5, SITKI ÖZTAS6
1
Faculty of Medicine, Suleyman Demirel University, Department of Internal Medicine
Isparta - 2Faculty of Medicine, Suleyman Demirel University, Department of Internal Medicine, Division of Endocrinology and
Metabolism, Isparta - 3Faculty of Medicine, Mevlana University, Medical Biology, Konya - 4Faculty of Medicine, Suleyman Demirel
University, Division of Endocrinology and Metabolism, Diabetes nurse, Isparta - 5Faculty of Medicine, Katip Çelebi University,
Department of Biochemistry, Izmir - 6Faculty of Medicine, Suleyman Demirel University, Department of Genetics, Isparta, Turkey
ABSTRACT
Peroxisome proliferator activated receptor-γ (PPAR-γ) and transcription factor 7-like 2 (TCF7L2) gene polymorphisms are
some of the genetic variants that are commonly held responsible for the genetic pathogenesis of type 2 Diabetes Mellitus (T2DM ).In
this study the genetic polymorphisms of TCF7L2 and PPAR-γ genes in affected siblings of families with rich diabetic history and
their unaffected siblings who are thought to be resistant to diabetes development were evaluated. 28 T2DM patient group (Group 1),
27 siblings who still have not developed T2DM 10 years after the earliest T2DM diagnosis age in the family (Group 2) and 20
healthy individuals who do not have T2DM and no history in the first degree relatives (Group 3). PPAR-γ Pro12Ala and TCF7L2
gene polymorphisms (rs12255372 and rs7903146) were searched for in these groups. Moreover, oral glucose tests and insulin secreting abilities of individuals in group 2 were investigated. PPAR-γ Pro12Ala gene polymorphism was not detected in any of the
patients in our study group. There were no significant differences between group 1 and 2 regarding TCF7L2 gene polymorphism
genotyping. TT genotype and T allele ratio was less in group 3 when compared to other groups but this was not statistically significant.As a result, it was observed that PPAR-γ Pro12Ala polymorphisms do not have an affect on T2DM development. However,
TCF7L2 gene polymorphisms, although not a strong determinant, may play a role in diabetes along with other genetic and environmental factors.
Key words:Type 2 Diabetes Mellitus, PPAR-γ, TCF7L2, polymorphism
Received February 18, 2014; Accepted June 19, 2014
Introduction
Diabetes mellitus (DM) is a metabolic disease
that results from insulin resistance and / or insulin
deficiency that is characterized by hyperglycemia(1).
Insulin resistance in peripheric tissues (muscle,
liver, adipose) and disorder of insulin secretion
from pancreatic beta cells play a role in the pathogenesis of T2DM(2). These disorders have a role in
DM pathogenesis and DM development occurs
with the interaction of genetic and environmetal
factors(3) (fig. 1).
Obesity, hypertension, dyslipidemia, insulin
resistance, sedantary life style and family history
increase the risk of DM development(3). In individuals who are genetically vulnerable to insulin secretion disorder and insulin resistance, DM slowly
develops in existence of environmental factors(4).
Apart from rare monogenic DM forms, T2DM is
mostly polygenic(4). The mutations and polymorphisms in the genes encoding the proteins that play
a role in events such as insulin resistance, insulin
secretion and glucose transport that are crucial in
the pathogenesis of DM pathogenesis are the genet-
1204
ic basis of DM(4). The functional candidate genes in
T2DM are genes that are contributing to insulin’s
effects(insulin receptor, insulin receptor substrates
(IRS), PI-3 Kinase, glucose transport and metabolism), genes that are participating to insulin secretion (GLUT2, glucokinase, ion channels, preproinsülin, β cell transcription factors), and obesity
genes (2-4). Proline-alanine substitution in the 12th
codon of PPAR-γ gene (Pro12Ala) is one of the
first genetic polymorphisms shown to be linked to
T2DM(5-7). In this study, the aim was to investigate
the TCF7L2 and PPAR-γ gene polymorphisms of
T2DM patients with rich DM family history, their
unaffected siblings and healthy control group individuals and also to evaluate the relation between
insulin resistance, insulin secretion, pancreatic
capacity and these polymorphisms.
Kemal Türker,Mehmet Numan Tamer et Al
the vertical line that goes to iliac crista from the lateral side of the costal arch, was measured during
expiration(10).
DNA Isolation from Peripheral Blood
QIAGEN DNA isolation kit was used for isolating DNA. TCF7L2 (rs12255372 and rs7903146)
ve PPAR-γ (Pro12Ala) genes were amplified by
PCR by Bioer ProGen device using the obtained
genomic DNA. DNA segments amplified by PCR
were compared with restriction fragment lenghts
and mutations were identified (for example figure
1). Following PCR, 295 bp fragments belonging to
PPAR-γ and 119 and 113 bp fragments belonging to
TCF7L2 were identified.
Methods and methods
Selection of Patient and Control Groups
Surveys were done on 295 T2DM patients and
their family and diabetes histories were recorded. 3
study groups were constructed.
Group 1: Diabetic index patients with three or
more first degree relative with T2DM (diabetes rich
family) and the first degree relatives of these
patients that could be summoned.
Group 2: The siblings of the index patients
who have not developed T2DM although they are
10 years older than the age in which the individual
who got the earliest T2DM diagnosis was.
Group 3: Healthy individuals who do not have
diabetes and no history in the first degree relatives.
DM in individuals in group 3 were excluded by oral
glucose tolerance test (OGTT). 0, 30 and 120
minute blood sugar(8) and insulin levels (for measurement of beta cell function and insulin resistance) were recorded during OGTT. In those
thought to be resistant to diabetes development,
HOMA-IR, HOMA-B cell and insulionogenic
index (30th minute) levels were calculated(9).
The diagnosis of diabetic patients were ascertained by fasting blood sugar (FBS) and HbA1c. All
relatives, regardless of T2DM, were checked for
serum lipid profiles. Control group individuals who
did not have DM history in the family as well were
analyzed by FBS and DM was excluded.
Anthropometric measurements and BMI parameters
of DM and non DM siblings in the study were calculated by body mass kg/height² formula. Waist circumference, the diameter that crosses the middle of
Fig. 1: ?????????
Quantitatıve measurements
Insulin releasing abilities of individuals in
group 2 were assessed according to HOMA B-cell
(HOMA-B-cell: 20 x fasting plasma insulin level
(µU/ml) / (fasting plasma glucose level (mmol/L)3,5)) and insulinogenic index (30th minute: (Insulin
30-Insulin 0) / (glucose 30-glucose 0)) measurements
and HOMA-IR (HOMA–IR=[fasting insulin(lU/mL)
x fasting glucose (mmol/L)] / 22.5) measurements
were used to assess insulin resistanc(11).
The statistical analysis of the study was done
using SPSS version 15.0. Constant variables were
expressed in arithmetrical average ± standart deviation, while categorical variables were expressed as
%’s. Mann Whitney U test was used in group comparsion of constant variables. Chi square test was
used in the group comparison of categorical comparison. In the presence of more than two groups
Kruskal - Wallis test was used.
Results
Demographic Characteristics of Patients
28 patients from group 1, 27 from group 2 and
20 from group 3 were included in this study. There
were no statistically significant differences between
Evaluation of TCF7L2 and PPAR-γ Gene Polymorphisms and insulin secretion abilities in patients with type II...
the first and second groups regarding age and gender (p>0,05). TCF7L2 gene’s rs12255372 polymorphism genotype distribution between the groups
were shown in Table 1. When all three groups were
compared in regard to their TCF7L2 gene
rs12255372 genotype distributions, it was observed
that there were no significant differences between
the groups regarding GG, GT and TT genotypes (p
> 0,05). Moreover, when the groups were compared
two by two there were no significant differences as
well (p>0,05) (Table 2).
Group 1
Group 2
Group 3
n
%
N
%
N
%
C allele
36
64,2
36
66,6
29
T allele
20
35,7
18
33,3
11
1205
P1
P2
P3
P4
72,5
0,729
0,309
0,501
0,594
27,5
0,729
0,309
0,501
0,594
Table 4: TCF7L2 gene’s rs7903146 polymorphism allele
frequencies.
P1 stands for the relevance values between group 1 and 2 . P2 is
for group 1 and 3. P3 is for group 2 and 3. P4 is for all groups.
pared to others, there were no statistically significant differences in G and T allele frequency among
all three groups (p>0,05). TCF7L2 gene’s
Group 1
Group 2
Group 3
P1
P2
P3
P4
rs7903146 polymorphism genotype distribution
n
%
N
%
N
%
assesment between the three groups were shown
Rs
in table 4. When all three groups were compared
12255372
12
42,9
10
37
9
45
0,660 0,883 0,582 0,842
GG genotype
according to their TCF7L2 gene rs7903146 genoRs
type distributions, there were no significant differ12255372
12
42,9
13
48,1
10
50
0,694 0,624 0,900 0,871
GT genotype
ences observed in their CC, CT and TT genotype
Rs
frequencies (p>0,05).
12255372
4
14,3
4
14,8
1
5
0,956 0,299 0,281 0,530
TT genotype
Moreover, there were no significant differences
observed when all three groups were comTable 1: TCF7L2 gene’s rs12255372 polymorphism genotype
pared
two
by two (p>0,05), (Table 3).
frequencies.
When TCF7L2 gene’s rs7903146 polymorP1 stands for the relevance values between group 1 and 2. P2 is for
group 1 and 3. P3 is for group 2 and 3. P4 is for all groups.
phism was evaluated for alleles, there was a intergroup allele frequency distribution was obtained
Group 1
Group 2
Group 3
which is shown in table 4. Although T allele freP1
P2
P3
P4
quency was less in group 3, there were no signifin
%
N
%
N
%
cant differences between the three groups in
G allele
36
64,2
33
61,1
28
70
0,713 0,644 0,404 0,715
regard to C and T allele frequency (p>0,05) (Table
T allele
20
35,7
21
38,8
12
30
0,713 0,644 0,404 0,715
4). When TCF7L2 gene’s rs12255372 and
Table 2: TCF7L2 gene’s rs12255372 polymorphism’s allele rs7903146 polymorphisms were evaluated in non
diabetic siblings for insulin secretion abilities,
frequencies.
P1 stands for the relevance values between group 1 and 2. P2 is for there were no significant differences between the
group 1 and 3. P3 is for group 2 and 3. P4 is for all groups.
two groups in parameters (age, gender, BMI,
HOMA - β cell, insulin index).
Group 1
Group 2
Group 3
P1
P2
P3
P4
45
0,703
0,364
0,582
0,662
55
0,701
0,517
0,770
0,807
N
%
N
%
N
%
rs7903146CC
genotype
9
32,1
10
37
9
rs7903146CT
genotype
18
64,3
16
59,3
11
Discussion
T2DM is a metabolic disease that develops due
to insulin resistance and / or insulin deficiency by
interaction of genetic and environmental factors. It
rs7903146TT
1
3,6
1
3,7
0
0
0,979 0,393 0,384 0,688
genotype
was reported that parameters such as BMI, HOMATable 3: TCF7L2 rs7903146 polymorphism’s genotype fre- IR, LDL, triglyceride are inclined tro be higher in
quencies.
individuals with T2DM family history compared to
P1 stands for the relevance values between group 1 and 2. P2 is for those who do not have any family history (12) .
group 1 and 3. P3 is for group 2 and 3. P4 is for all groups.
Although there were no significant differences in
BMI of individuals with diabetes and a rich family
When TCF7L2 gene’s rs12255372 polymorhistory and their unaffected siblings, total cholestrol
phism was assessed in regard to its alleles, an allele
and LDL levels of non diabetic siblings have been
frequency distribution was reached which is shown
found to be significantly higher when compared to
in table 2. Although G allele was more frequent and
the diabetic group. The fact that same family memT allele less frequent in the third group when combers carry the same genetic information may be a
1206
reason for this finding. The fact that lipid levels in
the diabetic group was lower was attributed to the
fact that this group was under control and using
hyperlipidemic drugs (56%). Moreover, lipid profile
is a parameter that can be affected by smoking, diet,
exercise and hyperlipidemia drugs. These parameters
were not taken into account in our study, and this is a
weak spot for lipid profile asssesment. Unaffected
siblings with family history had average HOMA-IR
levels of 2.5 as expected. HOMA-IR levels of
healthy individuals with no family history of T2DM
were not checked for comparison and this may seem
as incomplete, however since the aim of the study
was to ascertain any relationship between genetic
polymorphism and IR, this kind of a road map was
chosen.
The effects of PPAR- γ gene’s Pro12Ala polymorphism on IR, lipid metabolism and BMI are
under the control of other factors as well and pretty
complicated. PPAR- γ gene’s Pro12Ala polymorphism has been found to be associated with T2DM in
many studies(13-15). On the contrary, in a study by
Hasstedt S.J. et al in which relatives of type II diabetes patients took part Pro12Ala polymorphism was
shown to have no effect on diabetes development(16).
In a study conducted by Bendlová B. et al, there
were no differences in glucose, lipid profile parameters and Pro12 Ala polymorphisms between Czech
individuals with and without T2DM family history
when corrected according to their BMIs(17).
In the metaanalysis assessement by Tönjes et al
which consisted of 57 studies, there were no relations between Pro12Ala polymorphism and BMI,
insulin, HOMA-IR and glucose in the nondiabetic
population(18). Pro12Ala polymorphism frequency
differs by population and race. There is no extensive
study that investigates Pro12Ala polymorphism rate
and effects in the Turkish population.
In the study of Tok et al, Pro12Ala polymorphism was found in 19.4% of mothers with gestational diabetes and in 16% of non diabetic pregnancies(19). In Zengi et al’s study in which carotis intima
media thickness, metabolic parameters and PPARγ gene Pro12Ala polymorphism rate was compared
in the non diabetic first degree relatives of type 2
diabetic patients, Pro12Ala polymorphism rate was
found to be 9.9% in the non diabetic group with
negative family history while this rate was 14.6 %
in the non diabetic group with positive family history(20). Also, there were no significant correlations
between Pro12Ala polymorphisms and metabolic
parameters.
Kemal Türker,Mehmet Numan Tamer et Al
The fact that there were no significant correlations between Pro12Ala polymorphisms and metabolic parameters in our study is consistent with the
previous studies. We have not observed any
Pro12Ala polymorphisms in all our groups and this
may be due to our limited study group.
Lately, it is postulated that beta cell secretion
(insulin secretion) defect is an important genetic
pathogenic factor in the T2DM ethiopathogenesis in
addition to ID(21-23). In a study in which genotype
scoring was performed on 2377 individuals, 18
SNPs including rs7903146 was checked.
rs7903146 polymorphism was found to be associated with T2DM development(3).
In another study which comprised 17,284
Swedish and 2770 Finnish individuals that had an
average follow up of 23,5 years, presence of T2DM
was found to be correlated with family history,
increased BMI, smoking, elevated blood pressure
and triglyceride levels.
11 gene variants including TCF7L2 and PPAR
- γ were examined. All 11 genes were found to have
correlation with T2DM development, 8 gene variants, including TCF7L2, were observed to be related to decreased insulin secretion(24).
In all the groups of our study, rs7903146 and
rs12255372polymorphism and genotyping in
TCF7L2 gene, which was found to be influential in
insulin secretion in all previous studies, was performed. There were no significant differences
between groups 1 and 2 regarding genotype. In the
third group, genotype frequencies of TT and CT /
GT were found to be lower than the other groups
but this decrease was not statistically significant.
When the allele distribution was viewed from the
perspective of rs12255372 and rs7903146,no differences were observed in groups 1 and 2, but T allele
rate was found to be lower in the third group compared to other groups however this was not statistically significant.
This may be becaus of the limited number of
individuals in the study group. The fact that T allele
is decreased in non diabetic individuals with negative family histories when compared to diabetic and
non diabetic individuals with positive family history underlines the importance ofTCF7L2 gene polymorphisms in the development of type II diabetes,
and this finding is consistent with previous studies(25-27). However, similar T allele distribution in the
diabetic relatives and the non diabetic individuals
with positive family history shows that TCF7L2
gene polymorphisms are not solely adequate for the
Evaluation of TCF7L2 and PPAR-γ Gene Polymorphisms and insulin secretion abilities in patients with type II...
development of diabetes and that it may evolve
with the interaction of genetic and environmental
factors. As a result, there were no differences in
PPAR-γ gene Pro12Ala and TCF7L2 geners7903146 and rs12255372polymorphisms
between diabetic individuals from rich diabetes
families and their unaffected siblings. The authors
state that more extensive studies should be conducted in this field.
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_________
Corresponding Author
Dr. OĞUZHAN AKSU
Department of Endocrinology and Metabolism, Suleyman
Demirel University, School of Medicine
3200, Isparta (Turkey)