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Romanian Biotechnological Letters
Copyright © 2013 University of Bucharest
Vol. 18, No.4, 2013
Printed in Romania. All rights reserved
ORIGINAL PAPER
Insulin-like growth factor genetic variation, colorectal cancer and diabetes
Received for publication, January 5, 2013
Accepted, March 17, 2013
MIHAI TOMA1*; MONICA STAVARACHI1; EMIL POPA2;
CRISTIAN SERAFINCEANU3; SPANDOLE SONIA1; DĂNUŢ CIMPONERIU1;
IRINA RADU1; LAVINIA MARIANA BERCA4; DANIELA ADRIANA ION5
1
Department of Genetics, University of Bucharest, Bucharest, Romania.
2
Surgical Departament, Coltea Hospital, Bucharest, Romania.
3
Diabetes Department, Institute of Diabetes, Nutrition and Metabolic Diseases "Nicolae
Paulescu", Bucharest, Romania.
4
Molecular Biology Laboratory, National Institute of Research&Development for Food
Bioresources – IBA Bucharest, Bucharest, Romania.
5
2nd Department of Physiopathology, “Carol Davila” University of Medicine and Pharmacy,
Bucharest, Romania.
*
Corresponding author:
MD, PhD. Mihai Toma
Postal address: Department of Genetics, University of Bucharest, Intrarea Portocalelor, No. 13, 6th District, Bucharest, 060101, Romania
Telephone: 0040213181576; Fax: 0040213181565; E-mail: [email protected]
Abstract
Insulin-like growth factor 2 (IGF2) has been shown to be involved in proliferation and
apoptosis, playing a significant role in some diseases, like cancer and diabetes. The goal of this study
was to analyze the association between IGF2 ApaI polymorphism (rs680) and susceptibility to
colorectal cancer (CRC) and type 2 diabetes mellitus (T2DM) in Romanian patients.
Blood samples were obtained, after written consent, from 400 subjects (patients and sex- and
age- matched controls). Genomic DNA was extracted from peripheral blood leucocytes using
commercial kits and the IGF2 ApaI polymorphism was genotyped by PCR-RFLP. Statistical analysis
was performed using the χ2 test.
The GG genotype had a higher frequency in the T2DM control group compared with CRC
control group (38% vs. 29%), but the differences are not statistically significant (χ2GGvsAA=0.03;
p=0.86). We didn't find any statistically association relationship between IGF2 ApaI polymorphism
(genotypes or alleles) and CRC (ORGG=0.81, p=0.52, respectively ORG=0.81, p=0.31) or T2DM
(ORGG=0.54, p=0.07, respectively ORG=0.66, p=0.054).
These findings suggest that the IGF2 ApaI polymorphism do not affect the susceptibility to
CRC and T2DM in Romanian patients.
Keywords: IGF2 ApaI polymorphism, loss of imprinting, risk disease
Introduction
The environmental risk factors [1], diet and associated factors, like physical activity
[2] and body size [3] have an important role in colorectal cancer (CRC) etiology. There are
evidences that increased risk of CRC results from the influences of these factors on
hyperinsulinemia [4], hyperglycemia [5] and regulation of IGF1 and IGF2 levels that can
modulate tumorigenesis [6].
Also, recent studies show that CRC [7] and colorectal adenoma [8] are positively
associated with type 2 diabetes mellitus (T2DM), hyperinsulinemia representing the link
between T2DM and CRC [5, 9].
IGF2 gene located 11p15.5 encodes for insulin-like growth factor 2 that is a mitogenic
peptide with important autocrine and paracrine signalling actions [10]. It plays an important
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MIHAI TOMA; MONICA STAVARACHI; EMIL POPA; CRISTIAN SERAFINCEANU; SPANDOLE SONIA;
DĂNUŢ CIMPONERIU; IRINA RADU; LAVINIA MARIANA BERCA; DANIELA ADRIANA ION
role in fetal and adult development processes (like cellular proliferation, differentiation) as
well in apoptosis and tumor growth [11, 12]. IGF2 gene is part of the IGF2 - H19 locus, a
cluster of imprinted genes. In normal tissues, IGF2 is expressed only from the paternally
inherited copy [13]. Loss of imprinting (LOI) of IGF2 gene was associated with diabetes [14,
15] as well with many types of tumors: breast [16], esophagus [17], hepatic [18], gastric [19],
Wilms [20], prostate [21] and colorectal [22 - 24] cancers.
IGF2 expression is modulated by some polymorphisms in other genes or in its self
sequence. Thus, it has been showed that the shorter alleles (class I) of variable number of
tandem repeats (VNTR) of insuline (INS) gene are associated with increased steady-state IGF2
mRNA levels in human placenta [25]. Also, the ApaI restriction fragment length
polymorphism (RFLP) in the 3′-untranslated region (3′-UTR) of IGF2 was associated with an
increased mRNA level for IGF2 gene [26].
IGF2 ApaI (rs680) polymorphism was intensively studied in relationship with diabetes
[27, 28], obesity [29], breast [30] and prostate [31] cancers. To our knowledge, there are no
other reports referring to the association between this polymorphism and CRC risk.
The goal of this study was to analyze the relation between IGF2 ApaI polymorphism,
CRC and T2DM in Romanian patients.
Materials and Methods
Subjects
Between January 2010 and December 2012, blood samples from 400 unrelated
individuals were collected at Coltea Hospital (Bucharest) and N. Paulescu Institute
(Bucharest). The subjects were included in four groups: CRC patients (M:F = 59:41; age
63.3±4.5; with normal values of glycemia); T2DM patients (M:F = 59:41; age 55,7±8,1;
duration of T2DM 13±8,4 years); CRC control (M:F = 59:41; fasting glycemia 90±8 mg/dl),
and T2DM control (M:F = 59:41; fasting glycemia 92±6 mg/dl), both control groups without
known family history of malignancies and clinical signs of CRC or diabetes. The each control
group was matched with CRC and respectivelly T2DM group for both age and gender.
Diagnostic of CRC was confirmed after histopathological examination. The colorectal
tumours were localized in colon and sigmoid (64.2% of cases) and in rectum (35.8% of
cases).
The subjects were enrolled in the study after written consent was obtained, according
to the Declaration of Helsinki. This study was approved by Research Ethics Committee of
Biology Faculty, University of Bucharest.
DNA isolation and genotyping
Five ml of blood were collected in tube containing EDTA from all participants. DNA
was extracted from peripheral blood leukocytes using the Genomic DNA Purification Kit
(Fermentas, Lithuania) according to the manufacturer’s protocol.
The IGF2 ApaI gene polymorphism was detected by PCR-RFLP [32]. Briefly, about
60 ng DNA were amplified in a final volume of 10 μL, containing 1×PCR buffer, 1.5 mmol/L
MgCl2, 1 unit Taq DNA polymerase, 100 μmol/L dNTP, and 0.5 μmol/L of each primer (F 5’CTT GGA CTT TGA GTC AAA TTG G -3’ and R 5’- CCT CCT TTG GTC TTA CTG GG 3’) [32]. PCR was performed in a Corbett research thermocycler (Corbett Research Pty Ltd,
Sydney, Australia) and the program consists of: an initial melting step of 1 min at 94°C;
followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C; a final elongation
step of 3 min at 72°C.
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Insulin-like growth factor genetic variation, colorectal cancer and diabetes
The digestion reaction was performed with 5U ApaI restriction enzyme (Fermentas,
Lithuania) at 30°C for 3 hours. The restriction products were electrophoresed (PAGE 8%) and
were visualized using Bio-Imaging System after ethidium bromide staining. The genotypes
were determined by examining gel fragments: the 236 bp fragment reaveled the AA genotype;
three fragments of 236, 175 and 61 bp indicated the AG genotype and two fragments of 175
and 61 bp indicated the GG genotype.
Statistical analysis
Statistical analysis was performed using SISA software [33]. Alleles and genotypes
distribution was tested for Hardy–Weinberg equilibrium condition. Chi-square test (χ 2) was
used to compare the distribution of genotypes and alleles in patients and control groups. Odds
ratios (ORs) and 95% confidence intervals (CIs) were calculated using the relevant 2 × 2
contingency table. A p value < 0.05 was considered statistically significant.
Results and Discussions
We genotyped the IGF2 ApaI polymorphism in 100 CRC patients, 100 T2DM patients
and 200 healthy controls (number, age and gender matched for each lot of patients). The
frequencies of genotypes are shown in Table 1 and Table 2. The IGF2 ApaI genotype
distribution for patients and controls was in accordance to Hardy-Weinberg equilibrium
expectation (verified by χ2 test) in all groups: CRC patients (χ2 = 0.16, p = 0.68), T2DM
patients (χ2 = 1.54, p = 0.21), CRC control (χ2 = 0.92, p = 0.34) and T2DM control (χ2 = 0.52,
p = 0.47).
As it can be observed, the GG genotype had a higher frequency in the T2DM control
group compared with CRC control group (38% vs. 29%), but the differences are not
statistically significant (χ2GG vs. AA = 0.03; p = 0.86). These differences can be explained by
mean age distribution into the control groups or by selection criteria of the investigated
populations.
For CRC, no statistically significant differences in the distribution of GG genotype
(25% vs. 29%) and G allele (51% vs. 56%) between patients and controls have been identified
(OR=0.816, p=0.52 for GG genotype, respectively OR=0.817, p=0.316 for G allele) (Table 1).
Table 1. Genotype distribution of IGF2 gene polymorphism for CRC
Groups
Gender
GG
M
14 (23.73%)
CRC patients
F
11 (26.83%)
Total
25
M
15 (25.42%)
CRC control
F
14 (34.15%)
Total
29
CRC, colorectal cancer; M, males; F, females
Genotype
GA
33 (55.93%)
19 (46.34%)
52
35 (59.32%)
19 (46.34%)
54
AA
12 (20.34%)
11 (26.83%)
23
9 (15.26%)
8 (19.51%)
17
For T2DM (Table 2), we found a possible association relationship between the GG
genotype and T2DM (OR=0.54; p=0.048), but the p-value obtained with the Yates correction
(0.068) showed that the impact of GG genotype on T2DM is not statistically significant.
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MIHAI TOMA; MONICA STAVARACHI; EMIL POPA; CRISTIAN SERAFINCEANU; SPANDOLE SONIA;
DĂNUŢ CIMPONERIU; IRINA RADU; LAVINIA MARIANA BERCA; DANIELA ADRIANA ION
Table 2. Genotype distribution of IGF2 gene polymorphism for diabetes
Genotype
Gender
GG
GA
M
15 (25.4%)
33 (56.0%)
T2DM patients
F
10 (24.4%)
23 (56.1%)
Total
25
56
M
20 (33.9%)
32 (54.2%)
T2DM control
F
18 (43.9%)
18 (43.9%)
Total
38
50
T2DM, type 2 diabetes mellitus; M, males; F, females
Groups
AA
11 (18.6%)
8 (19.5%)
19
7 (11.9%)
5 (12.2%)
12
Also, the alleles distribution revealed an association relationship between G allele and
T2DM (OR=0.66; p=0.043), but not statistically significant when Yates correction is applied
(p=0.054). The comparison of genotypes and alleles distribution (patients vs. controls)
according to gender and age (5 years group) showed no statistically significant differences in
either CRC or T2DM group.
IGF2 ApaI is one of the most common polymorphisms found in this gene, analyzed
for its contribution in different pathologies and complications. To our knowledge, this is the
first research focused on the involvement of IGF2 ApaI polymorphism in the CRC pathology.
Also, is the first report regarding the relationship between this genetic variant and CRC and
T2DM risk in Romanian population.
Our study suggests that there is no association relationship between IGF2 ApaI
polymorphism and CRC or T2DM in Romanian population. Even we didn't find an
association between this polymorphism and CRC or T2DM, we need to take into
consideration that IGF2 expression is a complex process that involves also H19 factor and
imprinting silencing phenomenon [12, 34, 35].
Loss of imprinting (LOI) for IGF2 gene was associated with poor prognosis in
patients with stage IV CRC [36] and with proximal localization of colonic tumors [37]. To
note, LOI for IGF2 was detected in DNA extracted from surrounding peritumoral normal
colonic mucosa of about 30% of colorectal cancer (CRC) patients, as well in 10% of healthy
individuals [22]. Also, LOI for IGF2 was identified in a total of 33% of breast tumor
samples, heterozygous for the ApaI IGF2 polymorphism, but in any of the normal breast
samples [38]. Therefore, as LOI for IGF2 gene allows the expression of maternal allele in
tumor tissues, it is obvious that this phenomenon may modify the risk disease.
Also, when we analyze the contribution of an IGF2 gene polymorphism to a risk
disease, we need to take into consideration the parental origin of the alleles. For IGF2, in the
absence of LOI phenomenon, only paternal allele can affect the risk, being the one that is
expressed. A recent study show that paternally transmitted fetal alleles were associated with
maternal glucose concentration in the third trimester of pregnancy and placental IGF2
contents at birth [28].
In this context, our results may be influenced by the dilution of the statistical risk by
the mixture of paternal and maternal origins. Thus, despite the lack of a statistical significant
association as a result of our study, we cannot exclude a potential involvement of IGF2 ApaI
polymorphism in the predisposition for CRC or T2DM risk.
To overcome these difficulties in assessing the role of IGF2 polymorphisms in cancer
or diabetes, familial studies (which allow analysis of the risk allele transmission’s rate from
parents to affected offspring) are required. In this way, the possible bias due to the population
stratification can be avoided.
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Insulin-like growth factor genetic variation, colorectal cancer and diabetes
Conclusions
Our results indicated that IGF2 ApaI polymorphism does not confer a risk for
colorectal cancer and type 2 diabetes mellitus in Romanian patients. However, additional
researches and replicate studies in other populations are required to confirm our results.
Acknowledgements
This paper was supported by CNCSIS–UEFISCSU, project number PNII–IDEI
2150/2008 and by the Sectoral Operational Programme Human Resources Development,
financed from the European Social Fund and by the Romanian Government under the contract
number POSDRU/89/1.5/S/64109. We are grateful to patients for their collaboration and to
the personnel of Coltea Hospital and N. Paulescu Institute for their cooperation.
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