Investigating paraoxonase-1 gene Q192R and L55M polymorphism in patients with renal cell cancer.

Abstract

Increased oxidative stress can help promote carcinogenesis, including development of renal cell carcinoma. The enzyme protects low-density lipoproteins from oxidation and can be a factor in this process. PON1 Q192R and L55M paraoxonase gene polymorphisms were assessed in 60 renal cell carcinoma patients and 60 healthy controls. Genotypes were examined by PCR; the restriction enzyme AlwI was used to examine the Q192R polymorphism and Hsp92II for the L55M polymorphism. Significant differences in the PON1 Q192R polymorphism were found between patients and controls. The Q allele was more frequent in the patient group than in controls, while the R allele was more frequent in the control group. No significant differences were found in the L55M polymorphism. Additionally, there were no significant differences in L and M allele frequencies. We conclude that the R allele may protect against renal cell carcinoma.

Full text

Page 1

©FUNPEC-RP www.funpecrp.com.br
Genetics and Molecular Research 10 (1): 133-139 (2011)
Investigating paraoxonase-1 gene Q192R
and L55M polymorphism in patients with
renal cell cancer
O.A. Uyar3, M. Kara2, D. Erol2, A. Ardicoglu1 and H. Yuce2
1Department of Urology, School of Medicine, Firat University Elazig, Turkey
2Department of Medical Genetics, School of Medicine, Firat University,
Elazig, Turkey
3Department of Urology, Elazig State Hospital, Elazig, Turkey
Corresponding author: M. Kara
E-mail: drmuratkara@hotmail.com
Genet. Mol. Res. 10 (1): 133-139 (2011)
Received May 20, 2010
Accepted October 1, 2010
Published February 1, 2011
DOI 10.4238/vol10-1gmr927
ABSTRACT. Increased oxidative stress can help promote carcino-
genesis, including development of renal cell carcinoma. The enzyme
protects low-density lipoproteins from oxidation and can be a factor
in this process. PON1 Q192R and L55M paraoxonase gene polymor-
phisms were assessed in 60 renal cell carcinoma patients and 60 healthy
controls. Genotypes were examined by PCR; the restriction enzyme
AlwI was used to examine the Q192R polymorphism and Hsp92II for
the L55M polymorphism. Significant differences in the PON1 Q192R
polymorphism were found between patients and controls. The Q allele
was more frequent in the patient group than in controls, while the R al-
lele was more frequent in the control group. No significant differences
were found in the L55M polymorphism. Additionally, there were no
significant differences in L and M allele frequencies. We conclude that
the R allele may protect against renal cell carcinoma.
Key words: Renal cell carcinoma; Q192R polymorphism;
Paraoxonase; L55M polymorphism

Page 2

134
©FUNPEC-RP www.funpecrp.com.br
Genetics and Molecular Research 10 (1): 133-139 (2011)
O.A. Uyar et al.
INTRODUCTION
Renal cell cancer (RCC) constitutes 3% of all adult tumors and is the third most
common urologic cancer. In general, 8.7 new cases are reported in 100,000 individuals.
The male/female ratio is 3/2 (Landis et al., 1999). It usually occurs in elderly people
mostly within the 6th and 7th decades (Chow et al., 1999). Most RCC are unifocal and
unilateral. Bilateral involvement may be simultaneous or at different times. The rate of
bilateral involvement varies from 2 to 4%, and it is more frequent in familial forms. Mul-
ticentricity occurs in 10 to 20% of all patients and it is more frequent in papillary histol-
ogy and familial RCC (Whang et al., 1995; Campbell, 1997). Clear cell or conventional
RCC constitutes 70-80% of all tumors and they are highly vascularized (Storkel et al.,
1997; Clifford et al., 1998).
Paraoxonase-1 (PON1) is a serum esterase, which is synthesized in the liver. In
humans there are three different PON genes (PON1, PON2, and PON3), closely located. It
is reported that PON genes are located in the q21.3 region of the long arm of chromosome
7 (Connolly et al., 2003). PON1 and PON3 are found in the liver and plasma, whereas
PON2 is found especially in the endothelial layer of liver, kidney, heart, brain, and tes-
ticular tissues. It is also shown to be present in aortic smooth cells (Hassett et al., 1991).
The most widely known protective effect of PON1 is the ability to hydrolyze organophos-
phate neurotoxins, aromatic carboxylic acid esters and insecticides. The second biologi-
cal function of PON1 is its antioxidant activity. Serum PON1 is found in plasma together
with HDL and it plays important role in preventing the oxidation of plasma lipoproteins
(Mackness et al., 1991; Bonnefont-Rousselot et al., 1999).
Epidemiologic and molecular studies showed that there are two important genetic
polymorphisms at positions 55 and 192 of the PON1 gene. These two polymorphisms
arise from the substitution of amino acids at positions 55 and 192. Substitution of glu-
tamine (Q genotype) at position 192 of the PON gene by arginine (R genotype) leads
to the first polymorphism (Q192R). Similarly, substitution of leucine (L genotype) at
position 55 by methionine (M genotype) leads to the second polymorphism (L55M). The
most common one is homozygous QQ, the second is heterozygous QR, and the least is
homozygous RR. Paraoxonase hydrolyzing activity of the protein encoded by R is 8-fold
more than that encoded by the Q allele. This polymorphism also affects serum protein
concentration. Homozygous RR individuals have higher enzyme concentrations than do
homozygous QQ individuals (Mackness et al., 1997; Azarsiz and Sonmez, 2000). In addi-Mackness et al., 1997; Azarsiz and Sonmez, 2000). In addi-
tion to polymorphisms of these positions, five polymorphisms were reported at the PON1
promoter region. Due to these genetic polymorphisms, PON1 activity may show differ-
ences of up to 10- to 40-fold among different individuals and populations (Playfer et al.,
1976; Durrington et al., 2001).
In a study conducted in prostate cancer patients, PON192/QR and PON55LM/
MM genotypes were found to be related to increased risk of prostate cancer (Antognelli
et al., 2005). However, in another study, PON1 Q192R and L55M polymorphisms were
evaluated and no differences or relationships were found (Van Der Logt et al., 2005). We
are not aware of any study in the literature that demonstrates a relationship between RCC
and PON. Therefore, in this study, we investigated the PON1 Q192R and L55M gene
polymorphisms in patients with RCC.
). In addi-

Page 3

135
©FUNPEC-RP www.funpecrp.com.br
Genetics and Molecular Research 10 (1): 133-139 (2011)
Renal cell cancer and paraoxonase-1 gene
MATERIAL AND METHODS
Patient selection
After obtaining approval of the Firat University Ethics Committee, 60 patients with
clear cell carcinoma of the kidney (RCC classic type) and 60 healthy control subjects were
included in the study. Written informed consent was obtained from patients and controls after
explanation of study details. After a 20-min rest, 2 mL blood samples were collected from the
antecubital vein into tubes prepared with EDTA. All blood samples were stored at -20°C until
DNA purification.
Methods
Determination of the PON1 192 gene polymorphism
Polymerase chain reaction (PCR) (25 μL) for the PON1 192 gene polymorphism
was performed in 10 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs,
20 pmol of each primer (Biobasic, Ontario, Canada), and 1 U Taq polymerase (Fer-(Biobasic, Ontario, Canada), and 1 U Taq polymerase (Fer-
mentas, Inc.) in a thermocycler under the following conditions: 94°C for 5 min (initial
denaturation) followed by 35 cycles of 95°C for 1 min (denaturation), 60°C for 1 min
(annealing), 72°C for 1 min (extension), and a final extension of 72°C for 10 min. A 99-
bp fragment was amplified from 10 ng genomic DNA. PCR products were restricted us-
ing AlwI restriction endonuclease and separated by 2% agarose gel electrophoresis. PCR
products of 99 bp were digested with 2 U AlwI (MBI Fermentas, Lithuania) at 55°C for
20 h, and resulted in 99-, 69- and 30-bp fragments for the QR genotype, 69- and 30-bp
fragments for the RR genotype and a non-digested 99-bp fragment for the QQ genotype.
Digested products were resolved by gel electrophoresis (2% agarose gel) and visualized
by ethidium bromide staining.
and 1 U Taq polymerase (Fer- Fer-
Determination of the PON1 55 gene polymorphism
In genomic DNA samples of individuals, alleles of the PON1 55 locus were
amplified by PCR. PCR (25 μL) was performed in 10 mM Tris-HCl, pH 8.4, 50 mM
KCl, 1.5 mM MgCl2, 200 μM dNTPs, 20 pmol of each primer (Biobasic), and 1 U Taq
polymerase (Fermentas, Inc.) in a thermocycler under the following conditions: 94°C
for 2 min (initial denaturation) followed by 35 cycles of 94°C for 1 min (denaturation),
60°C for 1 min (annealing), 72°C for 1 min (extension), and a final extension of 72°C
for 10 min. A 170-bp fragment was amplified from 10 ng genomic DNA. A fragment
belonging to the PON1 55 locus, after PCR amplification, was restricted using Hsp92II
(Promega, USA) restriction endonuclease and separated 2% agarose gel electrophoresis.
PCR products (170 bp) were digested with 2 U Hsp92II at 37°C for 20 h, and resulted in
170-, 126- and 44-bp fragments for the ML genotype, 126- and 44-bp fragments for the
MM genotype and a non-digested 170-bp fragment for the LL genotype. Digested prod-
ucts were resolved by gel electrophoresis (2% agarose gel) and visualized by ethidium
bromide staining.

Page 4

136
©FUNPEC-RP www.funpecrp.com.br
Genetics and Molecular Research 10 (1): 133-139 (2011)
O.A. Uyar et al.
Statistical analysis
Concordance of genetic distribution with Hardy-Weinberg equilibrium was tested by
the χ2 goodness-of-fit test. Differences between genotype distributions of patients and con-
trols were evaluated using the chi-square test. Allele distribution differences of controls and
patients were evaluated by the Fisher exact test. P < 0.05 was considered to be statistically
significant. All statistical tests were performed using SPSS® for Windows, version 12.0.
RESULTS
Sixty clear cell RCC patients and 60 controls were included in the study. The ages of
patients varied between 32 and 82 years, with a mean age of 58 years. Only one patient had
bilateral clear cell RCC. Demographic characteristics of patient and control groups are given
in Table 1. All patients evaluated were classified according to the TNM system. Stage I-II pa-
tients were grouped as early stage, whereas stage III and IV patients were grouped as advanced
stage (Table 2).

Age (mean ± SD)
Gender (male/female)
RCC classic type (N = 60)
60.42 ± 12.8
38/22
Control (N = 60)
58.96 ± 11.71
36/24
RCC = renal cell carcinoma.
Table 1. Demographic characteristics of patient and control groups.
Genotype distributions of classic type RCC patients and controls and Q and R allele
distributions are summarized as numbers and percentages in Table 3. At the end of our study,
the PON1 gene Q192R polymorphism genotype distribution was obtained. According to our
results, the QQ genotype was the most common (38; 63.3%) in the patient group, and the QR
genotype was the second most common type (21; 35.5%). The RR genotype was found in only
one subject in the patient group while it was present in 6 subjects in the control group. Statisti-
cal analysis demonstrated a significant difference between genotype distributions (P = 0.045).
In addition, when the RCC group was compared to the control group with respect to Q and R
allele frequencies, there were statistically significant differences between the patient and con-
trol groups (P = 0.013). The Q allele frequency was higher in the patient group than in the con-
trol group, whereas the R allele frequency was higher in the control group. LL, LM, and MM
genotype numbers and percentages obtained for the PON1 gene L55M polymorphism and L
and M allele distributions are summarized in Table 4. In our study of the PON1 gene L55M
Table 2. Classification of patients according to the TNM system.
Early stage Advanced stage
I II III IV
Patients [N (%)] 24 (40.0%) 14 (23.3%) 12 (16.6%) 10 (20,0%)
TNM = primary tumor (T), regional nodes (N), and metastases (M).

Page 5

137
©FUNPEC-RP www.funpecrp.com.br
Genetics and Molecular Research 10 (1): 133-139 (2011)
Renal cell cancer and paraoxonase-1 gene
polymorphism, the LL genotype was found in 29 (48.3%) patients, whereas the LM genotype
was found in 25 (41.7%) patients. The MM genotype was present in 6 (10.0%) patients. In
the control group, LL, LM and MM genotypes were found in 21 (35.0%), 29 (48.3%), and
10 (16.7%) subjects, respectively. There was no statistically significant difference between
patient and control groups in terms of the PON1 gene L55M polymorphism (P = 0.276). In
the patient group, the L allele frequency was 83 (69.2%) and the M allele frequency was 37
(30.8%). On the other hand, in the control group, the L allele frequency was 71 (59.2%) and
the M allele frequency was 49 (40.8%). Allele distributions were different but this difference
did not reach statistical significance (P = 0.069).
DISCUSSION
Although the etiology of renal cell carcinoma is not yet clear, oxidative stress is con-
sidered to be a factor in the development of RCC as in the other cancer types. In a study of
antioxidant enzymes in RCC, catalase and selenium-dependent or selenium-independent glu-
tathione peroxidase enzymes were shown to be low (Pljesa-Ercegovac et al., 2008). Another
study showed that cytoplasmic superoxide dismutase and glutathione peroxidase activities
were lower in RCC tissues than in normal tissues (Lusini et al., 2001). Furthermore, there are
several reports suggesting a relationship between RCC and lipid peroxidation (Gago-Domin-
guez et al., 2002; Gago-Dominguez and Castelao, 2006).
Synthesized in human liver, PON1 is a serum esterase, which has the ability to hydro-
lyze paraoxone, the active metabolite of parathione. Human serum PON1 is bound physically
to HDL (Mackness et al., 1996; Primo-Parmo et al., 1996). The physiological role of PON1
is not fully understood, but it protects LDL from oxidation via hydrolyzing lipid peroxides.
In addition, it has some protective effects against cellular damage resulting from toxic agents
such as organophosphates (Mackness et al., 1996).
As paraoxonase has antioxidant effects, such as in preventing LDL oxidation, it is con-
sidered that it may have role in the pathogenesis of many disorders related to oxidative stress.
Nevertheless, the relationship between serum PON1 levels and cancer is not fully known.
Gago-Domin-
Table 3. PON1 Q192R genotype distribution of classic type renal cell carcinoma (RCC) patients and the
control group.
QQ QR RR Q allele distribution R allele distribution
RCC patients (N = 60)
Controls (N = 60)
38 (63.3%)
27 (45.0%)
21 (35.5%)
27 (45.0%)
1 (1.7%)
6 (10.0%)
97 (80.8%)
81 (67.5%)
23 (19.2%)
39 (32.5%)
Data are reported as number with percent in parentheses.
Table 4. PON1 L55M genotype distribution of classic type renal cell carcinoma (RCC) patients and the
control group.
LL LM MM L allele distribution M allele distribution
RCC patients (N = 60)
Controls (N = 60)
Data are reported as number with percent in parentheses.
29 (48.3%)
21 (35.0%)
25 (41.7%)
29 (48.3%)
6 (10.0%)
10 (16.7%)
83 (69.2%)
71 (59.2%)
37 (30.8%)
49 (40.8%)