Molecular and Cellular Endocrinology 325 (2010) 128134

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Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Functional polymorphism of hOGG1 gene is associated with type 2 diabetes

mellitus in Chinese population
Caixia Sun a,b , Xiufang Liu a,b , Huan Zhang a,b , Wenwen Guo a,b , Zhenming Cai a,b , Huimei Chen a,b ,
Kui Zhang c , Dalong Zhu d , Yaping Wang a,b,
a
Department of Medical Genetics, Nanjing University School of Medicine, Nanjing, China
b
Jiangsu Key Laboratory of Molecular Medicine, Nanjing, China
c
Laboratory of Clinic Biochemistry, Medical School Afliated Drum Tower Hospital, Nanjing University, Nanjing, China
d
Department of Endocrinology, Medical School Afliated Drum Tower Hospital, Nanjing University, Nanjing, China

a r t i c l e i n f o a b s t r a c t

Article history: hOGG1 protein excises the 8-hydroxy-2 -deoxyguanine (8-OHdG) which is associated with type 2 dia-
Received 19 March 2010 betes mellitus (T2DM). Our aim of this work is to explore whether the polymorphisms of hOGG1
Received in revised form 7 May 2010 gene are associated with T2DM. We screened the polymorphisms in the 5 -UTR (c.18G>T, c.23A>G,
Accepted 11 May 2010
c.45G>A and c.53G>C) and c.977C>G (Ser326Cys) in exon 7 of hOGG1 gene. A casecontrol study
indicated that c.23A/G heterozygote was markedly associated with diabetes (P = 0.004, OR = 2.648,
Keywords:
95%CI = 1.3555.176) and with an increased level of C-peptide (705.00 versus 545.91 pmol/L, P = 0.044).
Type 2 diabetes mellitus
Furthermore, a signicantly increased risk of T2DM was observed in the subjects carrying heterozygous
hOGG1 gene
Polymorphism
variant of c.23A>G and homozygous mutation of Ser326Cys (OR = 3.684, 95%CI = 1.4009.697). The pro-
8-OHdG moter activity of the variant allele c.23G decreased 3040% in Hela and HEK293 cell lines. In conclusion,
Promoter activity the variant c.23A>G of hOGG1 gene could decrease the gene promoter activity and was a risk factor for
T2DM.
2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
When exposure to the radiation and chemicals, as well as dur-
ing normal cellular metabolism, reactive oxygen species (ROS)
are generated and cause base modications in DNA, including
the oxidation of guanine residues to 8-hydroxy-2 -deoxyguanine
(8-OHdG). During DNA replication, 8-OHdG pairs with adenine
(A) more actively than with cytosine (C), leading to the G:C to
A:T mutation and creating a scenario for a mutator phenotype
(Nakabeppu, 2001). This ROS-induced mutagenesis might alter
function of various genes and play an important role in the etiol-
ogy of several diseases, such as degenerative diseases, cancers and
diabetes (Helbock et al., 1999; Hoeijmakers, 2001; Niedernhofer et
al., 2006). In human, base excision repair (BER) system specically
removes 8-OHdG to repair the oxidative DNA damage, and hOGG1
(human 8-oxoguanine glycosylase 1) is one of the key glycosylases
involved in BER system (Frosina, 2004). hOGG1 protein can excise
the oxidized base from 8-OHdG:A base pairs during DNA replica-
tion, and initiate the BER system to repair 8-OHdG damage (Arai
Corresponding author at: Department of Medical Genetics, Nanjing University
School of Medicine, Nanjing 210093, China. Tel.: +86 25 83686495;
fax: +86 25 83686559.
E-mail address: wangyap@nju.edu.cn (Y. Wang).
et al., 1997; Radicella et al., 1997). If BER system cannot eliminate
8-OHdG in genomic DNA efciently, the G:C to A:T mutation would
occur. Increasing evidences illustrate that the deciency of the BER
pathway is involved in several diseases. Genetic variation in BER
genes may alter repair function and contribute to diseases such as
cancer and some age-related diseases (Goode et al., 2002; Garcia-
Closas et al., 2006; Zhang et al., 2006). Our laboratory reported that
AluYb8MYH polymorphism was associated with increased oxida-
tive damage and age-related diseases (Sun et al., 2010). Paz-Elizur
et al. (2003) demonstrated that reduced hOGG1 activity is a major
risk factor in the formation of sporadic lung cancer. So far, the
variant of c.977C>G (Ser326Cys) in hOGG1 gene has been inves-
tigated as a candidate polymorphism susceptible for many types
of diseases (Weiss et al., 2005). However, hOGG1 gene is highly
polymorphic and most of the other variants have not been well
studied.
Type 2 diabetes mellitus (T2DM), is an insulin-resistant state
characterized by hyperglycemia. It has been shown that hyper-
glycemia could augment free-radical production (Singh et al.,
2001), and the occurrence of T2DM is associated with an elevated
level of oxidative damage to DNA (Seghrouchni et al., 2002). Hence,
oxidative stress is the common pathogenic factor contributing to
T2DM (Evans et al., 2002). In detail, the 8-OHdG is the most often
reported DNA damage in diabetes (Dandona et al., 1996). The asso-
ciation between the BER gene which specically excises 8-OHdG

0303-7207/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mce.2010.05.005
 C. Sun et al. / Molecular and Cellular Endocrinology 325 (2010) 128134
and the pathophysiology of the islet -cell function was deduced
from the study that there was a signicant reduction of free fatty
acid-induced apoptosis by the overexpression of hOGG1 protein
in mitochondria of INS-1 cells (Rachek et al., 2006). Moreover, the
increase in insulin secretion to compensate for insulin resistance
seemed to be impaired in subjects with the at-risk genotypes of
hOGG1 Ser326Cys polymorphism, indicating a functional involve-
ment of the hOGG1 gene in the pathophysiology of T2DM (Daimon
et al., 2009).
Based on the relationship between the hOGG1 gene and T2DM,
we here hypothesized that the functional variation of this gene
could be a candidate susceptible for diabetes; thus, we examined
the association of the hOGG1 gene polymorphisms with diabetes in
a Chinese population.
2. Subjects and methods
2.1. Subjects
The association test was conducted in an independent group of type 2 dia-
betic patients (n = 459, mean age: 58.3 14.8 years old) serially recruited from
the Afliated Drum Tower Hospital, Nanjing University School of Medicine dur-
ing 20062008. T2DM was diagnosed according to the World Health Organization
(WHO) 1999 criteria (Organization, 1999). The non-T2DM controls (n = 686, mean
age: 58.0 14.4 years old) matched for race, age (5 years) and gender were
randomly chosen from the volunteers who undergoing routine medical checkup
in the same hospital. Detailed interview and various laboratory analyses were
made upon every individual, including fasting glucose. The subjects were recruited
when the fasting glucose <6.1 mmol/L, and they were ruled out if they suffer-
ing from certain diseases, such as acute inammation, tuberculosis, autoimmune
diseases and cancer according to past history and the clinical or laboratory
characteristics.
The 459 T2DM patients were further sub-classied into groups of age <60
years old and 60 years old. All patients provided clinical characteristics including
documentation of family history, blood pressure, body mass index (BMI), rou-
tine biochemical determination, fasting plasma insulin concentrations and HbA1c
(hemoglobin A1c). Obesity was dened as a BMI of 28 kg/m2 (Zhou, 2002).
This study was approved by the Ethics Committee and written informed con-
sents were obtained from all participants.
2.2. Variation analysis by HRM (high-resolution melting)
Genomic DNA was extracted from peripheral blood samples using UltraPureTM
blood kit according to the manufacturers protocol (SBS Genetech Co. Ltd., Shanghai,
China).
Primers for variation detection in hOGG1 gene were designed by use
of LightScanner primer design software (Idaho Technology). The DNA frag-
ment including part of 5 -UTR (5 -untranslated region) and entire exon 1, was
amplied with the primers: forward 5 -aggaggtggaggaattaagt-3 and reverse
5 -ggcttctcaggctcagtca-3 , product size 321 bp. DNA sequence of exon 7 con-
taining the polymorphism Ser326Cys was amplied by the primers: forward:
5 -actgtcactagtctcaccag-3 and reverse: 5 -ggaaggtgcttggggaat-3 , and product size
200 bp. Primers were synthesized by SBS Genetech facility (Shanghai, China).
Polymerase chain reaction (PCR) for hOGG1 gene was initially performed in 10 L
volumes. Reactions included 25 ng of genomic DNA, 0.2 pmol of each primer, 0.8 L
2.5 mM dNTPs, 1 L 25 mM MgCl2 , 1 L 10 Taq buffer with (NH4 )2 SO4 , and 0.4 U
Taq DNA polymerase (Fermentas) and 0.4 L dimethyl sulfoxide (DMSO). Then, PCR
was performed in a PTC-200 thermal cycler (Bio-Rad) with an initial denaturation
at 95 C for 5 min, followed by 35 cycles of 95 C for 30 s, 60 C for exon 1 or 55 C
for exon 7 for 10 s, and 72 C for 30 s. After PCR completed, the solution was added
into 96 well plate (Bio-Rad) by addition of 1 L 1 LCGreen PLUS (Idaho Tech-
nology). Mixtures were overlaid with 20 L of mineral oil (Sigma) and the plates
were centrifuged (1500 g for 30 s). To promote heteroduplex formation, the mix-
tures were subsequently denatured by heating to 95 C for 30 s and cooled to 25 C.
The plates were transferred to the LightScanner (Idaho Technology), with uo-
rescence data collection over the temperature range 7095 C, as samples were
melted.
In all instances, HRM can directly discriminate the heterozygotes and homozy-
gotes, but not the major and minor homozygotes of a polymorphism. Then,
genotyping using spike-in control DNA was performed to allow distinction of minor
homozygotes from major homozygotes. In brief, the PCR products that showed in
homozygous were mixed with an equal amount of DNA product from a known major
allele homozygous subject to apply to the LightScanner . This strategy could convert
the minor allele homozygotes into heterozygotes, but not change the major allele
homozygote samples, providing the distinction between the major allele homozy-
gous samples and the minor ones.
129
2.3. DNA sequencing
All of heterozygotes and 10% of homozygotes detected by HRM were subjected
to DNA sequencing. In brief, the PCR products were separated on a 1% agarose gels,
puried with gel extraction kit (Takara Biotechnology Co. Ltd, Dalian, China) and
sequenced with BigDye Terminator cycle sequencing kits in an ABI 3130xl DNA
sequencer (ABI Biosystems, USA).
2.4. Promoter luciferase activity assays
Functional analysis of a variant identied in the 5 -UTR was performed using
a Dual-Luciferase Reporter Assay System (http://www.promega.com). The pro-
moter region of hOGG1 containing sequence spanning the certain variant was PCR
amplied from genomic DNA using primers containing restriction sites for cloning,
including a variant-type promoter from the individual harbouring the variant and
a wild-type promoter from the individual without variant as the control. Primers
were designed to amplify a fragment from position c.579 to c.+62 bp (reference:
NM 002542.5). It was decided to include the entire 5 -UTR sequence in the luciferase
assay to ensure that all sites important for transcription initiation and regula-
tion. Primer sequences used were: forward 5 -GGGGTACCgacgtggctctgaagacgg-3
and reverse 5 -GGAGATCTgcaggagtggaggctagagta-3 , containing the restriction
sites for KpnI and BglII, respectively. PCRs were performed in PTC-200 thermal
cycler (Bio-Rad). The 25 L reactions contained 50 ng template DNA (wild sam-
ple or variant sample), 10 pmol of each primer, 3 L 2.5 mM dNTPs, 2 L 25 mM
MgCl2 , 2 L 10 buffer, and 1 U LA TaqTM DNA Polymerase (Takara). The PCR
cycling parameters entailed an initial denaturation step at 94 C for 3 min, fol-
lowed by 35 cycles of denaturation at 94 C for 30 s, annealing at 60 C for 30 s
and extension at 72 C for 1 min 20 s, and a nal extension step at 72 C for
10 min. The 641 bp sized PCR products (either wild-type sequence or variant-type
sequence) were subsequently cloned to E. coli TOP10. Single clone was miniprepped
and sequenced to get the pure wild-type promoter and variant-type promoter,
respectively. This two promoter sequences, as well as pGL3-Basic vector were
digested with KpnI and BglII (Takara) and puried (Takara Biotechnology Co.
Ltd., Dalian, China). Vector arms were ligated overnight to the digested PCR frag-
ments. Insertion of the wild-type promoter and variant-type promoter sequence
inframe with the rey luciferase gene in the pGL3-Basic vector was veried by
cloned sequencing. Hence, two plasmids were generated: a wild-type-pGL3 and a
variant-type-pGL3.
These two plasmids constructed above and a blank pGL3-Basic vector were tran-
siently transfected into cells using the Lipofect transfection reagent according to
the manufacturers protocol (Tiangen Biotech, Beijing, Co., Ltd.). The transfection
efciency was determined with the Renilla luciferase gene-containing pRL-CMV
plasmid (Promega). 1 105 Hela cells or 2.5 105 HEK293 cells per well in 12-well
plate were transiently co-transfected with reporter plasmid and pRL-CMV. After
24 h treatment, transfected cells were washed with the phosphate-buffered saline
(PBS) and lysed in 250 L lysis buffer (1 PLB, Promega) with gentle shaking at room
temperature for 20 min. Each assay mixture contained 20 L of the cell lysates and
50 L of a rey luciferase measuring buffer (LARII, Promega). The rey luciferase
activity was measured by a luminometer instrument (Promega). After measuring
the rey luciferase activity, Stop & Glo Reagent (Promega) was added, and the
Renilla luciferase activity was then measured. Each transfection was performed in
triplicate, and all were repeated at four times.
2.5. Statistical analysis
All statistical analysis was carried out using the statistical program SPSS ver-
sion 13.0. Data are given as means SD. A casecontrol association between the
genotypes and disease was analyzed by the Chi-square test and odds ratio (OR)
was shown with 95% condence intervals (CI). The Students t-test was used
for continuous variables. The independent association between the variation in
diabetes and clinic characteristics was examined by multiple logistic regression
analysis. In all cases, P value of less than 0.05 was considered statistically signif-
icant.
3. Results
3.1. Variants detected in hOGG1 gene
In this study, the sequence variations in hOGG1 gene
were detected by HRM and subsequently sequencing (Fig. 1).
Four variants were identied in the 5 -UTR by subsequently
sequencing analysis, including c.53G>C (rs56387615), c.45G>A
(rs56378321), c.23A>G (rs1801129) and c.18G>T (rs1801126).
There was no homozygote or combined variations among them.
In other words, although these variants in the 5 -UTR locate very
closely, they seem to not compose a LD block. The missense muta-
tion Ser326Cys was found in exon 7 of hOGG1 gene (Fig. 1). All of
130 C. Sun et al. / Molecular and Cellular Endocrinology 325 (2010) 128134

Fig. 1. The variation detection in exons of hOGG1 gene. (A) Exon 1 of the hOGG1 gene was amplied by high-resolution melting analysis (HRM). The homozygotes cluster
and the heterozygotes cluster were different. (B) Exon 7 of the hOGG1 gene was amplied by HRM. The homozygotes cluster and the heterozygotes cluster were different.
(C) Sequencing conrmed that the wild-type cluster and the c.23A>G cluster were different. (D) Sequencing conrmed the genotype of polymorphism Ser326Cys.

the variants were described based on the sequence of the transcript
corresponding to the isoform 1 of hOGG1 gene (NM 002542.5).
The four variants locating in the 5 -UTR were all adjacent
to the initial AUG. However, none of them were supposed to
be splicing sites or inuence the splicing sites according to the
prediction using software ESEnder 3.0 (http://rulai.cshl.edu/cgi-
bin/tools/ESE3/esender.cgi?process=home).
3.2. The distribution of the hOGG1 gene polymorphism in
casecontrol study
We had screened the ve variants showed above in 459
T2DM patients and 686 non-diabetes controls. Four out of
them, c.53G>C, c.23A>G, c.18G>T and Ser326Cys, were sin-
gle nucleotide polymorphisms (SNPs), while c.45G>A was a
rare mutation of hOGG1 gene in Chinese population. The allele
frequencies of four polymorphisms in hOGG1 gene were in
HardyWeinberg equilibrium in control population, respectively.
A casecontrol study was conducted between these T2DM patients
and non-diabetes controls (Table 1). The statistic analysis indi-
cated that, for c.53G>C, c.18G>T or Ser326Cys, there were
no differences in genotype frequencies between case and con-
trol groups, respectively (P = 0.223, P = 0.419 or P = 0.719). The
differences in allele frequencies were also not found (P = 0.226,
P = 0.422 or P = 0.435). Interestingly, the frequency of heterozy-
gous variant c.23A/G was detected much higher in T2DM patients
than in controls (5.2% versus 2.0%). Hence, c.23A/G heterozy-
gote was markedly associated with diabetes (P = 0.004, OR = 2.648,
95%CI = 1.3555.176). This association was particularly stronger in
the patients with elder age (Table 2). The OR for c.23A/G heterozy-
gote was estimated to be 3.094 (P = 0.026, 95%CI = 1.1448.370) in
elder patients (60 years), compared with corresponding controls.
The different distribution of c.23A>G between diabetes patients
and non-diabetes controls implied that this variant could increase
the risk of diabetes, especially in elder population.
To testify the potential joint effect of different variants in
the same hOGG1 gene, we further evaluated the association
between the combined variants and risk of T2DM. A signicantly
increased risk of disease was observed in combined c.23A>G
and Ser326Cys. As shown in Table 3, when the group of subjects
with genotype c.23A/A combining with one or two wild allele
Ser (ser/ser + ser/cys) was as a reference, a signicantly increased
risk of T2DM was observed in subjects carrying the allele of
c.23G combining with homogeneous variant cys/cys (OR = 3.684,
95%CI = 1.4009.697). No signicant association was found with
other combined genotypes (c.53G>C and Ser326Cys, c.18G>T
and Ser326Cys, data not shown).
We also analyzed the relationship between c.23A>G genotype
of hOGG1 and clinical characteristics in T2DM patients (Table 4). No
signicant difference was found in onset age, gender, BMI, obesity,
hypertension, positive family history, fasting glucose, lipids and
insulin levels, and HbA1c between the genotype groups of c.23A/A
 C. Sun et al. / Molecular and Cellular Endocrinology 325 (2010) 128134 131

Table 1
Germline hOGG1 variations and genotype/allele frequencies in the casecontrol study.

hOGG1 Genotype/allele Number (frequency) P value* OR (95%CI)**

T2DM n = 459 Control n = 686

c.53G>C GC 13 (2.8%) 12 (1.7%) 0.223

GG 446 (97.2%) 674 (98.3%)
C 13 (1.4%) 12 (0.9%) 0.226
G 905 (98.6%) 1360 (99.1%)

c.23A>G AG 24 (5.2%) 14 (2.0%) 0.004 2.648 (1.3555.176)

AA 435 (94.8%) 672 (98.0%) 1.000
G 24 (2.6%) 14 (1.0%) 0.004 2.604 (1.3405.061)
A 894 (97.4%) 1358 (99.0%) 1.000

c.18G>T GT 8 (1.7%) 18 (2.6%) 0.419

GG 451 (98.3%) 668 (98.5%)
T 8 (0.9%) 18 (1.3%) 0.422
G 910 (99.1%) 1354 (98.7%)

c.977C>G*** CC 70 (15.4%) 113 (16.5%) 0.719

CG 225 (49.3%) 345 (50.4%)
GG 161 (35.3%) 227 (33.1%)
C 365 (40.0%) 571 (41.2%) 0.435
G 547 (60.0%) 799 (58.8%)

The bold values (P < 0.05) are considered statistically signicant.

*
P values are analyzed for the frequencies of genotypes or alleles between case and control groups.
**
OR (95%CI) are calculated as the wild genotype c.23A/A or wild allele c.23A as reference.
***
c.977C>G polymorphism was detected in 456 type 2 diabetes and 685 controls.

Table 2
The distribution of hOGG1 c.23A>G variant in the casecontrol study.

hOGG1 c.23A>G Genotype/Allele Number (frequency) P value* OR (95%CI)

T2DM n = 459 Control n = 686

<60 years AG 12 (5.1%) 8 (2.3%) 0.102

AA 223 (94.9%) 344 (97.7%)

60 years AG 12 (5.4%) 6 (1.8%) 0.026 3.094 (1.1448.370)

AA 212 (94.6%) 328 (98.2%) 1.000

The bold value (P < 0.05) is considered statistically signicant.

*
P values are analyzed for the frequencies of genotypes between case and control groups.

Table 3
Possible joint effect of hOGG1 c.23A>G and Ser326Cys variants associated with type 2 diabetes.

hOGG1 c.23A>G hOGG1 Ser326Cys Number OR (95%CI)

T2DM n = 456 Control n = 685

A/A ser/ser+ser/cys 285 450 1.000

A/A cys/cys 147 221 1.050 (0.8131.357)
A/G ser/ser+ser/cys 10 8 1.974 (0.7705.060)
A/G cys/cys 14 6 3.684 (1.4009.697)

The bold value refers to P < 0.05 and is considered statistically signicant.

Table 4
The characteristics of patients with type 2 diabetes by c.23A>G genotypes.

c.23 A/G (n = 24) c.23 A/A (n = 435) P value

Age (years old) 57.80 15.70 58.31 14.82 0.857

Onset age (years old) 50.46 12.80 52.82 13.12 0.391
Male/female* 14/10 217/218 0.276
BMI (kg/m2 ) 24.62 3.53 24.73 11.95 0.965
Obesity (%)* 6 (25.0%) 61 (14.0%) 0.141
Hypertension (%)* 17 (70.8%) 282 (64.8%) 0.663
Family history (%)* 8 (33.3%) 178 (40.9%) 0.527
Fasting glucose (mmol/L) 11.33 4.80 10.94 4.61 0.724
Fasting insulin (mIU/L) 5.97 3.08 7.71 7.12 0.446
Triglyceride (mmol/L) 2.34 1.70 2.38 3.12 0.954
HDL-cholesterol (mmol/L) 0.93 0.30 1.00 0.43 0.451
LDL-cholesterol (mmol/L) 2.61 0.81 2.44 0.86 0.372
Fasting C-peptide (pmol/L) 705.00 454.75 545.91 345.00 0.044
HbA1c(%) 9.36 2.25 9.39 2.71 0.966

Values are expressed as means standard deviation (SD). Abbreviations: BMI = body mass index; HDL = high density lipoprotein; LDL = low density lipoprotein;
HbA1c = hemoglobin A1c.
The bold value (P < 0.05) is considered statistically signicant.
*
Data are counts or frequencies and P values are derived by Chi-square test.
132 C. Sun et al. / Molecular and Cellular Endocrinology 325 (2010) 128134
Fig. 2. Effect of c.23A>G variant on hOGG1 promoter activity. HEK293 and Hela
cell lines were transfected with a reporter plasmid containing the hOGG1 promoter
sequences that drive the expression of the luciferase gene. The luciferase activities in
the pGL3-Basic vector were arbitrarily set to 1, and the relative luciferase activities
in the wild-type and variant-type promoter were calculated accordingly. Values are
presented as the mean SD from four separate experiments. *P < 0.05.
and c.23A/G. However, the level of C-peptide was higher in the
patients with c.23A/G than that with c.23A/A (705.00 454.75
versus 545.91 345.00 pmol/L; P = 0.044), suggesting that the vari-
ant of c.23A>G in hOGG1 gene may be associated with insulin
resistance which was a hallmark of T2DM.
3.3. c.23A>G variant and the promoter activity of hOGG1 gene
To explore the mechanism of the association of c.23A>G vari-
ant with T2DM, we detected the inuence of the variant on the
promoter activity of hOGG1 gene using the luciferase promoter-
reporter expression assay. The wild-type and variant-type reporter
plasmid, was respectively constructed by inserting the 641 bp
(c.579 to c.+62) KpnIBglII genomic fragment containing the
hOGG1 promoter c.23A or c.23G allele upstream of the rey
luciferase cDNA in pGL3-Basic vector. Both of reporter plasmids
were transfected to HEK293 and Hela cell lines to identify putative
inuence of c.23A>G on promoter activity. The results indicated
that the luciferase activity was much lower in the variant allele than
in the wild type (P < 0.05, Fig. 2). In HEK293 cell line, the luciferase
activity of the variant allele decreased 40% compared to that of the
wild type. Similarly, the promoter activity declined 30% in Hela cell
line. No matter in either normal or tumor cell lines, c.23A>G vari-
ant caused the decreased promoter activity, suggesting this variant
could affect the gene function due to reducing gene transcription.
4. Discussion
We investigated several variations in hOGG1 gene by HRM in the
casecontrol study of T2DM, found the c.23A>G in 5 -UTR was
associated with diabetes and there was a possible joint effect of
c.23A>G and the missense mutation Ser326Cys. The subsequently
function evaluation of c.23A>G showed this variant reduced the
promoter activity of hOGG1 gene, indicating c.23A>G was a risk
factor for T2DM, possibly by decreasing the hOGG1 gene transcrip-
tion.
A variety of traditional mutation screening methods are
available, including denaturing gradient gel electrophoresis,
temperature gradient capillary electrophoresis, denaturing high
performance liquid chromatography (dHPLC), and so on. These
methods have varying sensitivities and require intensive labor or
sophisticated instruments to perform analysis. However, HRM is
easier to perform and is a better mean for targeted SNP screening.
Compared with dHPLC, HRM had better sensitivity and specicity
and had greater potential for rapid, closed tube mutation screening
(Chou et al., 2005).
Various types of genetic polymorphisms have been identied
in the human hOGG1 gene locus so far, and parts of them have
been reported to be associated with several diseases (Shinmura
and Yokota, 2001). For example, the missense mutation Ser326Cys
in hOGG1 was indicated that the genotype Cys/Cys showed a sig-
nicant association with lung cancer compared to Ser allele carrier
(ser/ser + ser/cys) status (Okasaka et al., 2009). The positive associa-
tion of the Ser326Cys polymorphism of the hOGG1 gene with insulin
sensitivity or diabetes has also been reported (Wang et al., 2006;
Daimon et al., 2009), thus demonstrating the functional involve-
ment of the hOGG1 in the pathophysiology leading to diabetes.
8-OHdG level in tissues has been used as a marker of oxidative
stress-related DNA injury to the nuclei or mitochondria (Toyokuni
et al., 1997). Increasing evidences show that elevated 8-OHdG con-
tributed to the islet pathology in T2DM, including that the rise
of 8-OHdG was correlated with HbA1c values (Leinonen et al.,
1997; Toyokuni, 1999), and 8-OHdG positive cells was increased
in the islets from diabetic subjects (Sakuraba et al., 2002). On the
other hand, the expression of hOGG1, the major repair enzyme
involved in the defense against the accumulation of 8-OHdG, was
also increased in islets from diabetic humans in mRNA and pro-
tein level (Tyrberg et al., 2002). Therefore, it seems that, in diabetes
patients, the increased free radicals could cause DNA damage (such
as 8-OHdG) in islet -cells, and hOGG1 expression would sub-
sequently be up-regulated to attenuate such DNA damage. If the
equilibrium between DNA damage and repair is disturbed, the
accumulation of oxidative damage will cause the islet -cell dys-
function. The results in our works were consistent with it. Our
results showed that a variant c.23A>G in 5 -UTR of hOGG1 gene
would decreased the promoter activity and was associated with
T2DM. Furthermore, the accumulation of ROS plays an important
role in the etiology of insulin resistant and diabetes. ROS not only
directly oxidize and damage DNA, proteins, and lipids, but also indi-
rectly induce damage to cells by activating a number of cellular
stress-sensitive pathways such as NH2 -terminal Jun kinases/stress-
activated protein kinases, which can lead to both insulin resistance
and impaired insulin secretion (Evans et al., 2003; Goldstein et
al., 2005). Eventually, the type 2 diabetes patients who are asso-
ciated with insulin resistance could exhibit an elevated C-peptide,
which has been a surrogate marker of insulin release. We found
that the risk allele c.23G was associated with higher serum C-
peptide levels. It suggested this variant, c.23A>G in hOGG1, could
be involved in the pathophysiology of insulin resistance. However,
there is a limitation in our study. The clinical parameters in the
diabetic subjects may be inuenced by many other factors such as
hyperglycemia or drugs to treat diabetes. To better clarify the link
between the genetic disposition and the phenotypes, the differ-
ences in the clinical parameters between the genotype groups in
the non-diabetic subjects should be further examined.
The variants in 5 -UTR of hOGG1 gene we detected here were
rstly described in Japanese (Kohno et al., 1998). The subsequent
study demonstrated that c.18G>T in hOGG1 was signicantly
more prevalent among Japanese patients with lung adenocarci-
noma by a small sample and closely associated with colorectal
multiple adenoma families in Korean, whereas the relationship
between c.23A>G and risk of lung cancer or colorectal adenoma
was not identied (Ishida et al., 1999; Kim et al., 2007). Never-
theless, no investigation has been reported on the disease risk of
c.53G>C yet. Our results in this study indicated that c.23A>G in
hOGG1 was a risk factor for T2DM, whereas c.53G>C and c.18G>T
were not. In addition, we also analyzed the association of the mis-
sense mutation Ser326Cys in hOGG1 with T2DM, but no positive
result was found. Considering potential joint effects among variants
involved in the same hOGG1 gene, we also evaluated the association
 C. Sun et al. / Molecular and Cellular Endocrinology 325 (2010) 128134
between the combined variants and risk of T2DM. Interestingly, a
signicantly increased risk of type 2 diabetes was observed in the
subjects carrying heterozygous variant of c.23A>G and homozy-
gous mutation of Ser326Cys.
We rstly reported the variant c.23A>G in 5 -UTR of hOGG1
gene was associated with T2DM and the luciferase promoter-
reporter expression assay indicated this variant can decreased
the activity of promoter, whereas its mechanism remained to be
addressed. 5 -UTRs in mRNA are known to play crucial roles in
the post-transcriptional regulation of gene expression, including
modulation of the transport of mRNAs out of the nucleus and of
translation efciency, subcellular localization and stability (van der
Velden and Thomas, 1999; Bashirullah et al., 2001; Jansen, 2001).
The previous studies have revealed that regulation by 5 -UTRs is
mediated in several ways, and one of them is involved in transcript
factors which can specically interact with nucleotide patterns or
motifs located in 5 -UTR. Mutations that alter the 5 -UTR can lead
to serious pathology by regulating gene expression (Conne et al.,
2000; Paulin et al., 1998; Chappell et al., 2000). On the other hand,
inactivation of transcript factor Sp1 could suppress hOGG1 tran-
scription, and will subsequently result in an impaired oxidative
damage repair (Youn et al., 2005). While, the induction of the tran-
scription factor, NF-YA, can up-regulated hOGG1 expression and the
increased transcription level of the hOGG1 gene will be correlated
with an increase in enzyme activity providing functional protec-
tion from oxidative injury (Lee et al., 2004). Bioinformatic analysis
(http://jaspar.genereg.net) suggested that the variant c.23A>G in
5 -UTR could change the binding sites of transcript factors. There-
fore, we reasonably proposed that this variant could induce the
gene dysfunction by down-regulating the gene expression, possibly
through changing the transcript factor binding site and subse-
quently impacting the gene transcription.
In summary, we rstly reported that c.23A>G variant in 5 -
UTR of hOGG1 gene was associated with T2DM. This variant could
decrease the promoter activity of gene. Furthermore, the variants
c.23A>G and Ser326Cys in hOGG1 gene have a joint effect on the
risk of T2DM.
Conict of interest
None.
Acknowledgements
This work was partly supported by the National Natural Science
Foundation of China (grant number: 30800546 and 30972535),
the Doctoral Foundation of Education Ministry of China (grant
number: 20070284015 and 200802841008), Natural Science Foun-
dation of Jiangsu Province, China (grant number: BK2009236 and
BK2008269), the Jiangsu Science and Technology Foundation (grant
number: BZ2008055).
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