gender-specific association of nfkbia promoter polymorphisms with the risk of sporadic colorectal...

ORIGINAL PAPER

Gender-specific association of NFKBIA promoter polymorphismswith the risk of sporadic colorectal cancer

Shing Cheng Tan • Mohd Shafi’i Mohd Suzairi • Abdul Aziz Ahmad Aizat •

Mustapha Mohd Aminudin • Mohd Shahpudin Siti Nurfatimah •

Venkata Murali Krishna Bhavaraju • Biswa Mohan Biswal •

Ravindran Ankathil

Received: 20 July 2013 / Accepted: 9 August 2013 / Published online: 31 August 2013

� Springer Science+Business Media New York 2013

Abstract The inhibitory protein IjBa, encoded by the

NFKBIA gene, plays an important role in regulating the

activity of nuclear factor-kappa B, a transcription factor which

has been implicated in the initiation and progression of can-

cers. This study aimed to evaluate the association of NFKBIA

-826C[T (rs2233406) and -881A[G (rs3138053) poly-

morphisms with the risk of sporadic colorectal cancer (CRC)

in Malaysian population. A case–control study comprising

474 subjects (237 CRC patients and 237 cancer-free controls)

was carried out. The polymorphisms were genotyped from the

genomic DNA of the study subjects employing PCR–RFLP,

followed by DNA sequencing. The association between the

polymorphic genotypes and CRC risk was evaluated by

deriving odds ratios (ORs) and 95 % confidence intervals

(CIs) using unconditional logistic regression analysis. The two

polymorphisms were in complete and perfect linkage dis-

equilibrium (D0 = 1.0, r2 = 1.0). Overall, no statistically

significant CRC risk association was found for the polymor-

phisms (P [ 0.05). A similar lack of association was observed

when the data were stratified according to ethnicity

(P [ 0.05). However, stratification by gender revealed a

significant inverse association between the heterozygous

genotype of the polymorphisms and the risk of CRC among

females (OR 0.53, 95 % CI 0.29–0.97, P = 0.04), but not

among males (P [ 0.05). In conclusion, the heterozygous

genotype of the polymorphisms could contribute to a signifi-

cantly decreased CRC risk among females, but not males, in

the Malaysian population.

Keywords Colorectal cancer � Genetic association �NFKBIA � Sporadic � Susceptibility � Variation

Introduction

Colorectal cancer (CRC) accounts for a significant burden

of cancer-related morbidity and mortality. Worldwide, it is

the third most common cancer in men and the second most

common cancer in women [1]. The incidence of colorectal

cancer has been increasing over the past decade, especially

in developing countries, and in Malaysia, it has emerged as

the most common type of cancer among men [2]. The

multifactorial nature of the disease suggests that its

occurrence is not only due to environmental factors (such

as lifestyle and dietary habits), but also a result of genetic

factors as well as the interaction between the two [3, 4].

Among the genetic factors, mutations in high penetrance

genes, such as APC, represent a strong determinant for the

pathogenesis of the disease. However, such mutations

accounts for only a small proportion of all CRC cases [5],

and attempts to identify similar high penetrance genes have

been unsuccessful. Therefore, researchers have shifted their

attention to the identification of a panel of low penetrance

genes, which could modestly modulate the risk of CRC

individually, but contribute to a significant risk modifica-

tion effect when acting together [6–8]. Such low pene-

trance genetic variations are relatively common in the

S. C. Tan (&) � M. S. M. Suzairi � A. A. A. Aizat �M. M. Aminudin � M. S. S. Nurfatimah � R. Ankathil (&)

Human Genome Centre, School of Medical Sciences, Health

Campus, Universiti Sains Malaysia, 16150 Kubang Kerian,

Kelantan, Malaysia

e-mail: [email protected]

R. Ankathil

e-mail: [email protected]

V. M. K. Bhavaraju � B. M. Biswal

Department of Nuclear Medicine, Radiotherapy and Oncology,

School of Medical Sciences, Health Campus, Universiti Sains

Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

123

Med Oncol (2013) 30:693

DOI 10.1007/s12032-013-0693-6

general population. Therefore, they contribute to a higher

attributable risk of cancer compared to high penetrance

genes [9].

Genetic variations in genes implicated in cancer initia-

tion and progression could modulate the risk of CRC.

Inhibitor of kappa B alpha (IjBa), encoded by the NFKBIA

gene, is an important inhibitor of nuclear factor-kappa B

(NF-jB), a class of pleiotropic transcription factors which

act as central regulators in many cancer-related pathways,

notably inflammation [10]. In most normal cells, IjBabinds to NF-jB under normal conditions, thereby inacti-

vating the latter [11]. However, such inactivation is often

lost in cancer cells, which highlights the importance of

IjBa in circumventing oncogenesis [12–18]. It has been

hypothesized that polymorphisms in the promoter region of

NFKBIA could lead to transcriptional and functional con-

sequences of the protein product. This could result in the

reduced or enhanced effectiveness of IjBa in suppressing

NF-jB activity, which could in turn modulate the risk of

cancers and other diseases. Two polymorphisms in the

promoter region of NFKBIA, namely the -826C[T

(rs2233406) and -881A[G (rs3138053) polymorphisms,

have been described and their association with the risk of

several immune and inflammatory diseases have been

investigated [19–24]. Thus far, however, no study has been

undertaken to evaluate the association between the poly-

morphisms and CRC risk among Malaysians. Hence, we

conducted a case–control study with the aims to investigate

the frequencies of NFKBIA -826C[T and -881A[G

polymorphic genotypes in Malaysian CRC patients and

cancer-free controls, as well as to evaluate the association

between these polymorphisms and CRC risk in Malaysian

population.

Methods

Study subjects

The study was approved by the Research Review Board

and Human Research Ethics Committee of Universiti Sains

Malaysia (No. USMKK/PPP/JEPeM[201.4(1.2)]) and

Medical Review and Ethics Committee (MREC) of Min-

istry of Health, Malaysia (No. KKM/NIHSEC/08/0804/

P09-581). Written informed consent was obtained from all

subjects before sample collection. For this hospital-based

case–control study, a total of 474 study subjects, which

comprised 237 cases and an equal number of controls, were

recruited from (1) Hospital Universiti Sains Malaysia,

Kubang Kerian, Kelantan, (2) Hospital Raja Perempuan

Zainab II, Kota Bharu, Kelantan and (3) Hospital Sultanah

Bahiyah, Alor Setar, Kedah, Malaysia. Cases were hist-

opathologically confirmed CRC patients, whereas controls

were cancer-free healthy volunteers who visited the par-

ticipating hospitals for regular health examination. Con-

trols were frequency matched to cases in terms of gender,

age (±5 years) and ethnicity. Subjects with familial ade-

nomatous polyposis, ulcerative colitis, Crohn’s disease or

any other previous malignancy were excluded from the

study.

Genotyping

Genomic DNA was isolated from the peripheral blood of

the study subjects by using QIAamp DNA Blood Mini Kit

(QIAGEN, Germany) according to the manufacturer’s

protocol. Polymerase chain reaction–restriction fragment

length polymorphism (PCR–RFLP) method was then used

to genotype the polymorphisms. Amplification of the

genomic fragment containing both of the polymorphisms

were performed by using Phusion High-Fidelity PCR

Master Mix (Thermo Scientific, Finland) with a single pair

of PCR primers (forward, 50-GGT CCT TAA GGT CCA

ATC G-30; reverse, 50-GTT GTG GAT ACC TTG CAC

TA-30). The PCR conditions consisted of an initial dena-

turation step at 98 �C for 30 s, followed by 35 cycles of

denaturation (98 �C for 5 s), annealing (63 �C for 5 s) and

extension (72 �C for 5 s), and a final extension at 72 �C for

5 min. The 200-bp product generated was purified by using

QIAquick PCR Purification Kit (QIAGEN, Germany),

followed by digestion with BfaI and TspRI restriction

enzymes (New England Biolabs, USA) for genotyping of

the NFKBIA -826C[T and -881A[G polymorphisms,

respectively. For the former, PCR amplicons containing the

homozygous wild-type (CC) genotype was cleaved into

two fragments of 180 bp and 20 bp, while those with the

homozygous variant genotype (TT) was uncleaved and

remained 200 bp (Fig. 1). Heterozygotes showed all the

three bands on agarose gel. For the -881A[G polymor-

phisms, the homozygous wild-type genotype (AA) did not

contain TspRI restriction site; hence, the PCR product of

200 bp remained undigested. On the other hand, the

homozygous variants were cleaved into two fragments of

129 and 71 bp, and heterozygotes showed all three bands

(Fig. 1). The genotypes were confirmed by sequencing of

approximately 10 % of randomly chosen purified PCR

products, and a 100 % concordance rate was achieved.

Statistical analysis

Statistical analysis was carried out by using SPSS version

19. Deviation of the polymorphic genotype distribution

from the Hardy–Weinberg equilibrium (HWE) was tested

using a chi-square goodness-of-fit test. Linkage disequi-

librium was examined as described [25]. One-way analysis

of variance (ANOVA) was used to evaluate the

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123

significance of the differences between the cases and

controls in terms of gender, ethnicities, age, as well as

genotype and allele frequencies of the polymorphisms. The

association of the polymorphic genotypes with CRC risk

was determined by deriving odds ratios (ORs) and the

corresponding 95 % confidence intervals (CIs) from

unconditional binary logistic regression analysis, with the

homozygous wild-type genotype serving as the reference

(OR 1.00). P \ 0.05 was considered significant in the

analyses.

Results

Characteristics of study subjects

The characteristics of the study subjects are shown in

Table 1. A total of 237 cases and 237 controls were

recruited into this study. Among the cases, 129 (54.4 %)

were males and 108 (45.6 %) were females. On the other

hand, among the controls, 123 (52.3 %) were males and

114 (47.7 %) were females. No significant difference in

gender was observed between cases and controls

(P = 0.581). The age range of the cases was 27–94 years

old, with a mean of 61.5 ± 12.8 years and a median of

62 years, while the age range of the controls was

30–84 years old, with a mean of 58.0 ± 11.0 years and a

median of 58 years. Despite the presence of a significant

difference between the ages of the cases and controls

(P \ 0.01), the difference in the mean ages of both groups

of the study subjects were 3.3 years, which was still within

our initial intention of matching the subjects by 5-year age

difference. Among the cases, 182 (76.8 %) were Malays,

46 (19.4 %) were Chinese and 9 (3.8 %) were Indians,

while 200 (84.4 %), 28 (11.8 %), and 9 (3.8 %) of the

controls were of Malay, Chinese and Indian ethnicities,

respectively. No significant difference was observed

between cases and controls in terms of ethnicity

(P = 0.07).

Distribution of genotype and allele frequencies

of NFKBIA polymorphisms

The distribution of genotype and allele frequencies of

NFKBIA -826C[T and -881A[G polymorphisms are

shown in Table 2. The two polymorphisms were in com-

plete (D0 = 1.0) and perfect (r2 = 1.0) linkage disequi-

librium. The frequency of the homozygous wild-type,

heterozygous and homozygous variant genotypes of the

polymorphisms was 169 (71.3 %), 60 (25.3 %) and 8

(3.4 %) in cases and 163 (68.8 %), 69 (29.1 %) and 5

(2.1 %) in controls, respectively. No significant difference

in the distribution of polymorphic genotypes was observed

between the cases and controls (P [ 0.05, Table 2). The

genotypic distribution was in accordance with the Hardy–

Weinberg equilibrium, both overall (v2 = 0.01, P = 0.92)

and within each group (v2cases ¼ 0:85, Pcases = 0.36;

v2controls ¼ 0:55, Pcontrols = 0.46). On the other hand, the

frequency of wild-type and variant alleles was 0.84 and

0.16 in cases, and 0.83 and 0.17 in controls, respectively.

No significant difference was observed between cases and

controls in terms of allele distribution (P = 0.79).

CRC risk association of NFKBIA polymorphisms

Since NFKBIA -826C[T and -881A[G polymorphisms

were in complete and perfect linkage disequilibrium, they

Fig. 1 PCR–RFLP analysis of NFKBIA polymorphisms. a -826C[T

polymorphism. Lane M, 100-bp DNA ladder; Lane 1, homozygous

variant (TT); Lane 2, homozygous wild type (CC); Lane 3,

heterozygous (CT). The expected 20-bp band could not be visualized

from the gel. b -881A[G polymorphism. Lane M, 100-bp DNA

ladder; Lane 1, homozygous wild type (AA); Lane 2, homozygous

variant (GG); Lane 3, heterozygous (AG)

Table 1 Characteristics of the study subjects

Characteristics Cases (N = 237) Controls (N = 237) P

Gender 0.58

Male 129 (54.4 %) 123 (52.3 %)

Female 108 (45.6 %) 114 (47.7 %)

Age \0.01*

Range 27–94 30–84

Mean ± SD 61.5 ± 12.83 58.2 ± 11.02

Median 62 58

Ethnicity 0.07

Malay 182 (76.8 %) 200 (84.4 %)

Chinese 46 (19.4 %) 28 (11.8 %)

Indian 9 (3.8 %) 9 (3.8 %)

* Statistically significant

Med Oncol (2013) 30:693 of 6

123

showed an identical CRC risk association profile (Table 3).

The heterozygous genotypes of the polymorphisms were

associated with a decreased risk of CRC, although the

association was not statistically significant (OR 0.84, 95 %

CI 0.56–1.26, P = 0.40). Likewise, no statistically signif-

icant observation was found for the positive association of

the homozygous variant genotypes of NFKBIA and CRC

risk (OR 1.54, 95 % CI 0.50–4.82, P = 0.45).

The results obtained were also stratified by ethnicity, and

the CRC risk association is shown in Table 4. As the

numbers of subjects of Chinese and Indian ethnicities were

too small for sufficiently powered analysis to be made, they

were categorized together as non-Malays in the analysis.

The risk association profile of Malays was similar to the

overall risk association profile of all the ethnic groups

combined. The heterozygous genotypes of the two poly-

morphisms were associated with a reduced CRC risk (OR

0.81, 95 % CI 0.52–1.26), while the variant genotypes were

associated with an increased risk of CRC (OR 1.25, 95 % CI

0.37–4.19), although both associations were statistically not

significant (Pheterozygous = 0.35, Pvariant = 0.72). On the

contrary, for the non-Malay group, a positive CRC risk

association was observed for the heterozygous genotypes

(OR 1.35, 95 % CI 0.45–4.06). Nonetheless, the association

was also not statistically significant (P = 0.59). The asso-

ciation between the homozygous variant genotypes of the

polymorphisms and the risk of CRC could not be computed

due to the absence of individuals carrying the genotypes in

the control group.

In addition, when the results were stratified by the gender,

no significant association was observed for the male subjects

(Pheterozygous = 0.37, Pvariant = 0.67), although it was found

that the heterozygous genotypes of the polymorphisms

conferred a slightly increased risk of CRC (OR 1.3, 95 % CI

0.73–2.30) and the homozygous variant genotypes conferred

a protective role against CRC (OR 0.67, 95 % CI–

0.11–4.13). Similarly, no statistically significant risk asso-

ciation was found for the homozygous variant genotypes

among the females (OR 2.73, 95 % CI 0.53–13.97,

P = 0.21). Interestingly, however, the gender-wise stratifi-

cation analysis revealed a significant inverse association

between the heterozygous genotypes of NFKBIA and the

risk of CRC among females (OR 0.53, 95 % CI 0.29–0.97,

P = 0.04) (Table 5).

Discussion

The inhibitor of kappa B (IjB) family of inhibitory pro-

teins plays an central role in regulating the nuclear factor-

kappa B (NF-jB) signaling pathway, which has been

implicated in various carcinogenic processes, including

immune response, cell proliferation, apoptosis, angiogen-

esis and perhaps most notably, inflammation [10]. With the

exceptions of proliferating T cells, B cells, thymocytes,

Table 2 Distribution of NFKBIA polymorphisms

Cases (N = 237) Controls

(N = 237)

P

-826C[T polymorphism

Genotype

Wild type (CC) 169 (71.3 %) 163 (68.8 %) 0.55

Heterozygous (CT) 60 (25.3 %) 69 (29.1 %) 0.35

Variant (TT) 8 (3.4 %) 5 (2.1 %) 0.4

Allele

Wild type (C) 0.84 0.83 0.79

Variant (T) 0.16 0.17

-881 A[G polymorphism

Genotype

Wild type (AA) 169 (71.3 %) 163 (68.8 %) 0.55

Heterozygous

(AG)

60 (25.3 %) 69 (29.1 %) 0.35

Variant (GG) 8 (3.4 %) 5 (2.1 %) 0.4

Allele

Wild type (A) 0.84 0.83 0.79

Variant (G) 0.16 0.17

Table 3 Association of NFKBIA polymorphisms with colorectal

cancer risk

Genotype Cases

(N = 237)

Controls

(N = 237)

Odds ratio

(95 % CI)

P

Both polymorphisms

Wild type 169 163 1.00

(Reference)

–

Heterozygous 60 69 0.84

(0.56–1.26)

0.40

Variant 8 5 1.54

(0.50–4.82)

0.45

Table 4 Stratification of CRC risk association of NFKBIA poly-

morphisms by ethnicity

Genotype Cases Controls Odds ratio (95 % CI) P

Malays

Total (N) 182 200

Wild type 127 132 1.00 (Reference) –

Heterozygous 49 63 0.81 (0.52–1.26) 0.35

Variant 6 5 1.25 (0.37–4.19) 0.72

Non-Malays

Total (N) 55 37


Heterozygous 11 6 1.35 (0.45–4.06) 0.59

Variant 2 0 NA NA

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123

monocytes and astrocytes, IjBs persistently bind to NF-jB

in normal cells, thereby masking the nuclear localization

signals of the latter and inactivating it by cytoplasmic

trapping [11, 26]. In the presence of pro-inflammatory

stimuli, IjB kinases (IKKs) phosphorylate the serine resi-

dues of IjBs in the canonical pathway, rendering IjBs for

destruction via the ubiquitin-dependent pathway [26, 27].

This results in the activation, nuclear translocation and

aberrant functioning of NF-jB, leading to oncogenesis.

Indeed, such phenomenon has been observed in most

cancer cell lines and tissues, including those of the pan-

creas [12], colon and rectum [13, 14], stomach [15], breast

[16], liver [17] and prostate [18], which highlights the

importance of NF-jB signaling in carcinogenesis, and of

IjB in circumventing tumorigenesis. As such, polymor-

phisms in the genes within the NF-jB and its inhibitory

pathways could play an influencing role in cancer risk.

Indeed, our previous study has established an association

between the variant genotype of NFKB1 and CRC risk

[28].

In mammals, the evolutionary conserved family of IjB

consists of seven members, namely IjBa, IjBb, IjBc,

IjBe, Bcl-3, and the precursor proteins p100 and p105

[29]. Among these, IjBa represents the most abundant

form of the family and serves as an essential requirement

for the normal inhibition of NF-jB [30]. IjBa is encoded

by the NFKBIA gene on chromosome 14q13, which is

located close to the inflammatory bowel disease-4 (IBD4)

locus [31]. We therefore hypothesized that the interindi-

vidual variations within the NFKBIA gene could modulate

the risk of CRC. Hence, promoter polymorphisms of

NFKBIA which could potentially affect the transcription of

the gene were selected for evaluation in the present study.

Indirect evidences have shown that the variant alleles of

-826C[T and -881A[G polymorphisms were responsi-

ble for the decreased promoter activity of the gene [32].

The -826C[T polymorphism is located within a putative

binding site of GATA-2, and as such, the C/T substitution

could disrupt the binding of the transcription factor [33].

On the other hand, it has been suggested that the

-881A[G polymorphism could alter the binding ability of

retinoic acid-related orphan receptor a (RORa) transcrip-

tion factor, which has been implicated in carcinogenesis

[34]. Based on these, we hypothesized that an association

may exist between the -826C[T and -881A[G poly-

morphisms and CRC risk.

However, our results indicated that there was no sig-

nificant difference between the cases and controls in terms

of genotypic distribution. This observation was in agree-

ment with He et al. [22] and Cheng et al. [23], who studied

the association of these two polymorphisms with hepato-

cellular carcinoma in China and Taiwan, respectively.

However, Lin et al. [24] reported that the homozygous

wild-type genotypes of these two polymorphisms were

significantly overrepresented in Taiwanese oral cancer

patients and the heterozygous genotypes were significantly

overrepresented in cancer-free controls. In addition, the

present study also established that the two polymorphisms

were in complete and perfect linkage disequilibrium. All

the three authors above did not test for linkage disequi-

librium in their analysis, but He et al. [22] reported a

slightly different genotypic distribution between the two

polymorphisms. All these differences highlight the varia-

tions in genotypic distribution of the polymorphisms across

different populations and cancer types.

Our results also showed no evidence of an association

between the polymorphisms and the risk of CRC. The lack

of cancer risk association observed was in concordance

with Cheng et al. [23], but not with the other two authors

mentioned above. He et al. [22] reported an association

between -826TT and -881AG genotypes and the risk of

hepatocellular carcinoma, while Lin et al. [24] reported an

association between the variant genotypes of the two

polymorphisms and oral cancer risk. These inconsistencies

in risk association could be attributed to the different

backgrounds of the study subjects in different studies.

Indeed, our results also indicated that the heterozygous

genotypes of the polymorphisms conferred opposing

effects (risk vs. protective) to Malays and non-Malays

when ethnic-based stratification was performed. Interest-

ingly, we also found a significant inverse association

between female heterozygotes and CRC risk when the

results were stratified by gender. This observation suggests

the important role of gender as a major determinant in

colorectal carcinogenesis.

In conclusion, our results indicated a female-specific

inverse association of the heterozygous genotypes of

NFKBIA -826C[T and -881A[G polymorphisms with

the risk of CRC. To the best of our knowledge, this is the

first study evaluating the association between the

Table 5 Stratification of CRC risk association of NFKBIA poly-

morphisms according to gender

Genotype Cases Controls Odds ratio (95 % CI) P

Males

Total (N) 129 123


Heterozygous 36 28 1.30 (0.73–2.30) 0.37

Variant 2 3 0.67 (0.11–4.13) 0.67

Females

Total (N) 108 114


Heterozygous 24 41 0.53 (0.29–0.97) 0.04*

Variant 6 2 2.73 (0.53–13.97) 0.21

* Statistically significant

Med Oncol (2013) 30:693 of 6

123

polymorphisms and the risk of CRC in Malaysian popu-

lation. To validate our findings, further replicative studies

with a larger sample size are warranted.

Acknowledgments We wish to thank Dr. Zaidi Zakaria (Hospital

Raja Perempuan Zainab II), Dr. Ahmad Shanwani Mohd Sidek

(Hospital Raja Perempuan Zainab II) and Dr. Mohammad Radzi Abu

Hassan (Hospital Sultanah Bahiyah) for their help in recruiting the

study subjects. The study was supported by Universiti Sains Malaysia

Research University Grant (No. 1001/PPSP/812001).

Conflict of interest The authors declare that they have no conflict

of interest.

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