Polymorfizmus –2548 G/A v genu pro leptin u pacientek s endometriálním karcinomem
Východiska: Polymorfizmus –2548 G/A v promotoru genu pro leptin (LEP) byl v předchozích studiích asociován s rizikem nadváhy a obezity; faktorů signifikantně asociovaných se zvýšeným rizikem endometriálního karcinomu. Leptin hraje významnou úlohu v signální transdukci buněk endometriálního karcinomu, přičemž výsledky provedených studií naznačují, že leptin podporuje růst buněk endometriálního karcinomu i jejich invazivitu a předpokládá se, že kritickými mediátory působení leptinu jsou dráhy JAK/STAT a AKT. Cílem naší studie bylo zkoumat možné asociace polymorfizmu LEP –2548 G/A s výskytem endometriálního karcinomu a s ním souvisejících znaků. Design: Do studie bylo zahrnuto 67 pacientek s endometriálním karcinomem (průměrný věk 64,3 ± 10,3 let) a 67 kontrolních žen párovaných s pacientkami podle věku, BMI a etnického původu (62,1 ± 9,8 let); pro zkoumání obecných populačních frekvencí genotypů a alel byla navíc zařazena skupina 543 zdravých žen. LEP –2548 A/G byl zkoumán pomocí PCR s následnou analýzou délky restrikčních fragmentů. Výsledky: Předkládaná studie neodhalila významné rozdíly ve frekvenci genotypů nebo alel zkoumaného polymorfizmu u endometriálního karcinomu nebo s ním souvisejících znaků (věk menarché, menopauzy, počet spontánních abortů v anamnéze nebo čas do doby propuknutí onemocnění) mezi jednotlivými skupinami, což naznačuje, že genetické varianty polymorfizmu LEP –2548 G/A nejsou relevantním markerem rizika endometriálního karcinomu v české populaci. Závěry: Nezdá se, že by polymorfizmus LEP –2548 G/A reprezentoval důležitý genetický marker rizika endometriálního karcinomu u zkoumané české populace, byl nicméně asociován s obezitou, což je v souladu s dřívějšími pracemi.
J. Chovanec1; J. A. Bienertová-Vašků2; Z. Dostálová1
Authors place of work:
Department of Gynecology and Obstetric, The Faculty Hospital Brno 2Institute of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno1
Published in the journal:
Klin Onkol 2009; 22(5): 223-227
Background: Previously, the polymorphism –2548 G/A within the promoter of the leptin (LEP) gene was reported to be associated with overweight and obesity, the factors significantly associated to increased endometrial cancer risk. Leptin has been described to play an important role in signal transduction in endometrial cancer cells indicating that leptin promotes endometrial cancer growth and invasiveness and implicating the JAK/STAT and AKT pathways as critical mediators of leptin action. The aim of the study was to investigate the possible associations of LEP –2548 G/A polymorphism with endometrial cancer and its related traits. Design: Using PCR with following restriction analysis, we studied 67 endometrial cancer cases (mean age 64.3 ± 10.3 years) that were enrolled in the study along with 67 controls matched for age, BMI and ethnic origin (mean age 62.1 ± 9.8 years); an additional cohort of 543 healthy individual was recruited to investigate the general population frequencies. Results: The present study revealed no significant differences between the genotypes or alleles of investigated polymorphism for endometrial cancer risk or its related traits (age of menarche, menopause, number of spontaneous abortions in personal history or waiting time till the onset of the disease) among the groups, thus indicating that the genetic variants of LEP –2548 G/A is not a relevant marker of endometrial cancer risk in this Czech population. Conclusions: To conclude, the polymorphism LEP –2548 G/A doesn’t seem to represent a major genetic marker for endometrial cancer in the studied Czech population; however, it was associated with obesity, which finding is in accordance with previous reports.
Key words: leptin – polymorphism – endometrial cancer – PCR
cancer represents one of the most common gynecological malignancies
in developed countries whereas the incidences of endometrial cancer
in Western are up to 10-times
higher than in Asia of Africa rural regions [1–2].
Obesity represents a significant risk factor for endometrial
cancer and the impact of obesity on endometrial cancer development is
a very interesting intersection of these major health problems.
Although the progress in understanding the genetics of obesity has
moved rapidly in the past few years, the role of white adipose tissue
(WAT) as a crucial endocrine organ still remains to be
elucidated. White adipose tissue is tightly integrated into overall
metabolic control, adipocytes being actively involved in extensive
with other cell types [3–5]
and controlling the energy metabolism within the body by secreting
biologically active peptides named adipokines produced
exclusively or substantially by white adipose tissue pre adipocytes
and mature adipocytes and act by endocrine, paracrine and autocrine
The importance of the
hypothalamus in control of energy homeostasis has been recognized
quite long and it was expected that the brain must receive afferent
inputs in proportion to current body fat level. Coleman in 1973 
demonstrated the existence of a circulating factor playing
a great role in the regulation of body weight. By using
parabiosis methodology, he observed that obesity in the ob/ob
mice was due to lack of this factor, whose gene has been consequently
cloned and this adiposity factor was named leptin .
Leptin is a multifunctional
peptide hormone with numerous biological activities including
appetite regulation, bone formation, reproductive functions and
In the study by Sharma et al , leptin has been described to play
an important role in signal transduction in endometrial cancer cells
indicating that leptin promotes endometrial cancer growth and
invasiveness and implicating the JAK/STAT
and AKT pathways as critical mediators of leptin action. The
promotion of endometrial cancer cell proliferation by leptin involves
activation of STAT3 and ERK2 signaling pathways. Moreover,
leptin induced phosphorylation of ERK2 and AKT was
dependent on JAK/STAT
In an association study by
Petridou et al , leptin plasma levels were found to be strongly
correlated with endometrial cancer risk. However, it remains unclear
whether this association is directly causative or whether increased
leptin plasma levels and endometrial cancer are both secondary
consequences of obesity. In 2000, Mammes et al  reported an
association of LEP –2548 G/A
polymorphism within the promoter of the leptin gene with overweight.
So far, no study focused on the possible relationship of this
polymorphism with the increased risk of endometrial cancer has been
published. Considering the important role of LEP-2548 G/A
(dbSNP ID rs7799039) in obesity development, we hypothesize that it
could also be associated with the endometrial cancer risk and the
disease related traits in the Czech Caucasian population.
total of 67 endometrial cancer cases (mean age
64.3 ± 10.3 years) were enrolled in the study along
with 67 controls matched for age, BMI and ethnic origin (mean
age 62.1 ± 9.8 years). The diagnosis of endometrial
cancer was confirmed by standard histopathological examination
according to the FIGO classification. In controls, the presence of
any malignancy or severe chronic diseases was excluded by
standardized complex physical examination and personal history
evaluation by a specialist. Moreover, additional population of
543 healthy individuals was enrolled in the study to compare the
baseline allelic and genotype frequencies within the general Czech
population (215 men and 328 women, mean age
48.5 ± 13.5 years). All investigated individuals were
This study was approved by the
Committee for Ethics of Medical Experiments on Human Subjects,
Faculty of Medicine, Masaryk University, Brno, and was performed in
adherence to the Declaration of Helsinki Guidelines. Each participant
gave her written informed consent which has been archived.
cell fraction from the peripheral venous blood sample (5ml)
was used to extract DNA according to the standard procedure using
proteinase K. SNP LEP –2548G/A
(dbSNP ID rs7799039), 2 548 bp upstream to the beginning of
the exon 1 was detected as described previously . Briefly,
each of the 12 µl reaction contained 10 ng genomic DNA,
1.5 µl 10X PCR Buffer 1.5 µl MgCl2, 200 µM dNTP,
2 pmol of each primer and 0.4 U of Taq DNA polymerase
(Fermentas). The reactions were performed using XP Cycler (BIOER).
PCR amplification conditions were as follows: LEP –2548G/A
polymorphism: 95˚C for 5 min, 94 ˚C for 30 s, 50 ˚C
for 45 s, 72 ˚C for 50 s for 35 cycles and
72 ˚C for 10 min. Genotyping reliability was assessed by
double genotyping of approx. 20%
samples in independent assays where no differences were found.
Negative controls were included in each reaction batch to exclude
The genotypization success rate was approx. 99%.
in genotype distributions and the consistency with Hardy-Weinberg
equilibrium (HWE) were tested using the chi2 test and the
differences in allele frequencies were tested by the two tailed
Fisher’s exact test. Differences in parameters studied between the
two groups were tested using the Mann Whitney test.
post hoc correction was used to correct for multiple comparisons
where appropriate. Logistic regression analysis was used to analyze
the independence of the association between quantitative variables
and endometrial cancer. Cumulative hazard function plots were
estimated by the Kaplan-Meier
method with the log rank test in order to compare groups. The
Statistica v. 8.0 (Statsoft, Tulsa, OK, USA) program
package was emploeyed in all analyses.
significant differences in distribution of LEP –2548 G/A
genotypes were observed between the endometrial cancer patients and
the matched controls (p = 0.704). When comparing the
genotype distributions of the polymorphism of the cases with the
healthy Czech Caucasian population, no differences were observed as
well (p = 0.248, Tab. 1). No significant differences
in the age of menarche, menopause, parity and number of spontaneous
abortions were observed between the cases and their precisely matched
controls. Of these variables, only the number of spontaneous
pregnancy losses was significantly associated with the increased risk
of endometrial cancer (p = 0.003).
The associations between LEP
genotype and endometrial cancer risk by BMI are summarized in Tab. 2,
no significant differences between groups were observed.
In the next step, we tested
whether the LEP G/A –2548 G/A had any effect on the
characteristics related to endometrial cancer independently on other
known risk factors such as hormonal replacement therapy (HRT) or BMI.
In the multivariate regression modeling, the LEP –2548 G/A
served as an independent predictor for increased BMI (β = 1.1,
p = 0.000004). Furthermore, LEP –2548 G/A
was also significantly correlated with the body height
(β = –0.39,
p = 0.00000001). However, the examined polymorphism didn’t
show independent prediction role for the endometrial cancer
In the following analysis, we
tested, whether the LEP –2548 G/A
exerts an independent prediction role on age of onset of the
endometrial cancer; our data (p = 0.04) suggesting that
a remarkable proportion of the gene effect could be dependent
upon aging. Therefore, we consecutively tested the possible relation
between age at time of endometrial cancer diagnosis and LEP
by subdividing the study group according to individual age (i.e.
below or above 64 years, the median value of the entire cases
cohort). However, these two subgroups did not differ significantly
either in LEP –2548 G/A
genotypes nor allele frequency (pg = 0.070, pa = 0.283).
Fig. 1 shows the cumulative
probabilities for endometrial cancer occurrence and waiting time to
onset of disease. The time to onset of disease wasn’t significantly
different between the A or G allele carriers (p = 0.79).
leptin has been associated with cancers of the breast, ovary,
prostate, small lung cancer and with non Hodgkin lymphoma and
also the important role of leptin in general cancerogenesis has been
proposed by observations from the previous studies [16–20].
The endometrium represents
a classical hormone-dependent
tissue and most of the endometrial carcinomas are hormone-dependent
tumors. Leptin treated monkeys had higher mean plasma
luteinizing hormone (LH) and follicle stimulating hormone (FSH)
levels than controls, demonstrating the ability of leptin to enhance
gonadotropin secretion . Sharma et al  suggested that leptin
rapidly stimulates the JAK/STAT
pathway and induces the phosphorylation of ERK and AKT, thus
activating two key signal-transduction
pathways associated with cell growth. In addition to this finding,
Petridou et al  described significant dysregulation in serum
leptin levels among cases with endometrial cancer compared to
controls, however, it cannot be conclusively inferred, whether leptin
levels elevation, developed in the study subjects as a consequence
of obesity, played a crucial role in endometrial carcinogenesis
or whether it was a simple correlate of obesity.
The common polymorphism in the
promoter of the human leptin gene –2548G/A
influences leptin expression, possibly at the transcriptional level,
and therefore also adipose secretion levels of the hormone ;
adipose tissue leptin mRNA levels is 60% higher in AA subjects when
compared to GA/GG
subjects. Taking this into account, we have hypothesized that this
polymorphism could modulate the endometrial cancer susceptibility.
In this case-control
study carried out in a Czech Caucasian population, we
investigated the effects of –2548 G/A
polymorphism in the gene encoding for leptin on endometrial cancer
risk in the Czech endometrial cancer cases, BMI and age-matched
controls and healthy Czech population. The present study revealed no
significant differences in genotypes or alleles distribution of
investigated polymorphism for endometrial cancer risk or its related
traits (age of menarche, menopause, number of spontaneous abortions
in anamnesis) among the groups, thus indicating that the genetic
variants of LEP –2548 G/A
are not a relevant marker of endometrial cancer risk in this
Czech population. In the study by Mammes et al , the G allele of
the polymorphism was associated with overweight, which is well in
accordance with our results as the G allele was in our study an
independent predictor for increased BMI, but not endometrial cancer,
thus suggesting that the LEP –2548 G/A
polymorphism is associated rather with obesity itself than
endometrial cancer susceptibility.
To conclude, the polymorphism LEP
doesn’t seem to represent a major genetic marker for
endometrial cancer in the Czech population; however, it is associated
with obesity, which is well in accordance with previous reports.
authors are grateful to Dana Polášková and Petra Přikrylová for
technical assistance, and to all the study participants for
participation in the project.
was supported by grant of Ministry of Education of the Czech Republic
No. 881/2006 and by project of DANONE/2007 by Danone Institute
focused on genetic variability of adipokines in obese individuals.
authors declare they have no potential conflicts of interest
concerning drugs, pruducts, or services
used in the study. Autoři
deklarují, že v souvislosti s předmětem studie nemají žádné
Editorial Board declares that the manuscript met the ICMJE “uniform
requirements” for biomedical papers. Redakční
rada potvrzuje, že rukopis práce splnil ICMJE kritéria pro
publikace zasílané do biomedicínských
Julie Bienertová-Vašků Ústav
patologické fyziologie lékařské
fakulty MU Kamenice
00 Brno e-mail:
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