Original Article Relevance between HLA-DP gene rs2281388 polymorphism and hepatocellular carcinoma risk

Original Article

Relevance between

HLA-DP

gene rs2281388

polymorphism and hepatocellular carcinoma risk

Fangfeng Liu1_{, Jianlu Wang}2_{, Hong Chang}1_{, Jun Lu}1_{, Hongguang Li}1

1_{Department of Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, 9677}

Jingshi Road, Jinan 250021, Shandong, China; 2_{Department of Hepatobiliary Surgery, Shandong Provincial}

Hospital Affiiated to Shandong University, 9677 Jingshi Road, Jinan 250021, Shandong, China

Received January 20, 2015; Accepted March 20, 2015; Epub June 1, 2015; Published June 15, 2015

Abstract: Purpose: We carried out this study to find out the relevance between rs2281388 T/C polymorphism of human leukocyte antigen (HLA) gene and hepatocellular carcinoma (HCC) risk in Chinese Han population. Methods: The method of polymerase chain reaction (PCR) was applied to amplify the genomic DNA. Then the PCR products were sequenced to test the HLA-DP gene rs2281388T/C polymorphism of the case and control groups. Odds ratios (ORs) and 95% confidence interval (95% CIs) were utilized to evaluate the potential correlation between rs2281388 variants and HCC risk. Results: We analyzed the rs2281388 polymorphism distribution among the clinical patho-logical features. The results showed that there existed a significant statistic correlation between rs2281388T/C polymorphism of HLA-DP gene and HBsAg feature, and no significant correlation was found between rs2281388 and other clinical features. Further analysis showed that the TT genotype of rs2281388 was significantly correlated with HCC risk, and the same to T allele, but there was no significant difference of CT genotype distribution in case and control groups. Conclusion: TT genotype and T allele of HLA-DP gene rs2281388 polymorphism may increase the risk of HCC.

Keywords:HLA-DP gene, single nucleotide polymorphism, hepatocellular carcinoma

Introduction

Hepatocellular carcinoma (HCC) is the mainly

primary liver cancer (85%-90%) [1, 2]. China is a high-incidence country of HCC [3], and the

incidence is higher than other countries. HCC seriously affects the individuals’ health, thence many researches have investigated the patho-genesis of it. As we all know, the occurrence of HCC is a complicated biological process, and the pathogenesis has not been totally explained by now. Recent studies demonstrated that the occurrence of HCC was affected by combined effects of many factors, such as cirrhosis ,viral hepatitis and other chemical carcinogen,

envi-ronment and inheritance factors [4-8], and the risk of suffering HCC is mainly determined by genetic background.

Human leukocyte antigen (HLA) complex is the human major histocompatibility complex (MHC). HLA gene is located at 6p21. 31, has

the length of 3600kb, occupies about 0.1% of

the human genome bases, and consists by a

series of related gene sites [9]. As a human genetic marker with high polymorphisms [10, 11], HLA is an important system which controls the body immunity and immune regulation, determines the body histocompatibility and

involves in the differentiation of T-cell.

Researches have proved that HLA, one of the most important areas of genetic predisposition, is related to many diseases. At present, a

num-ber of studies have confirmed that a lot of poly -morphism sites of the gene are linked to the occurrence of many kinds of diseases, includ-ing liver cancer, gastric cancer, breast cancer, hypertension, leukemia, tuberculosis and lung

cancer etc [12-20]. Moreover, a lot of studies

focused on the correlation between HLA

poly-morphisms and hepatitis virus infection [6, 21-24]. According to the reports, rs2281388

polymorphism, which located at the down-stream of HLA-DPB1, has a significant associa

-tion with chronic hepatitis B (CHB) [25], while

Table 1. The relevance between rs2281388T/C and clinical features of

liver cancer

Clinical features n Genotype χ2 _{P value}

CC (n=54) CT+TT(n=85)

Clinical stage I~II 67 29 38 1.071 0.301 III~IV 72 25 47

Differentiated degree high 68 24 44 0.708 0.400 poor 71 30 41

Metastasis yes 77 27 50 1.736 0.188 no 62 29 34

HBsAg positive 71 21 50 5.251 0.022

negative 68 33 35

not observed. So we carried out this study to in-

vestigate the relevance between rs2281388T/C

polymorphism and the occurrence of HCC in northern China, which will help for revealing the pathogenesis of HCC.

Materials and methods

Specimen source

139 HCC patients who have been confirmed by

two pathologists in Shandong Provincial

Hospital, 92 in men and 47 in women. The

mean age of these people is 54.9±14.6. 150 healthy individuals came from the health check-up center, 95 in men and 55 in women, average age of 52.9±14.6, and had no tumor history, hepatitis, cirrhosis and other liver diseases. Clinical features of all participants were detailed in Table 1. All of the research objects belong to Chinese Han population with no blood relationship. All participants signed the written informed consent and provided whole blood

after 12 hours fast. The study was approved by

the Review Boards of Shandong Provincial Hospital, China.

DNA extraction and primer synthesis

2 ml anticoagulant was put in peripheral venous

blood which preserved in -20°C. The whole blood genetic DNA extraction kit was used to extract DNA (Shanghai Xin Ran Biological Technology Co. Limited), according to the man

-ufacturer’s protocol. The primers were designed by Primer Premier 5.0. Upstream primer: 5’-TCT CTG CAA TAC CCT CAA TGA CTG-3’; Downstream primer: 5’-CAA TGG TGA GCA GAC TGC AAA TC-3’. The primers were synthesized by Beijing

Sunbiotech Co. Limited.

1.0 µL of Tag DNA polymerase, and 15 µL of

ddH₂O. PCR conditions: 94°C initial

denatura-tion 4 min, 94°C degeneradenatura-tion 30 s, 60°C annealing 30 s, 72°C extension 30 s, 35

circu-lations, and 72°C final elongation 10 min. PCR

products were sequenced by Sangon Biotech (Shanghai, China).

Statistical methods

Chi-square test was utilized to evaluate

Hardy-Weinberg equilibrium (HWE) in genotype

distri-bution of control group. Odds ratios (ORs) and 95% confidence interval (95% CIs) were utilized

to evaluate the potential correlation between rs2281388 variant and HCC risk. All statistical analyses were performed by SPSS 18.0 statis-tical software package (SPSS, Chicago, IL, USA). P-values less than 0.05 were considered

as significant level.

Results

HWE test

The genotype distribution of the HLA-DP poly-morphism site rs2281388 in the healthy

con-trol was accorded with HWE (χ2_=0.533,

P=0.465), which showed that the community genetic inheritance of the control samples was balanced and it can represent the general population.

Relationship between rs2281388T/C polymor-phism and the clinical features

According to the analysis of clinical stages, dif-ferentiated degree, metastasis and HBsAg, we found that HBsAg positive HCC patients were more than negative patients in the distribution

Amplification of rs2281388T/C

Polymerase chain reac-tion (PCR) was used to

amplify the rs2281388T/ C polymorphism. The

reaction system of PCR was 25 µL, including 1.5

µL of genome DNA, 0.25

µL of upstream primer, 0.25 µL of downstream primer, 3.0 µL of 10× reaction buffer, 2.5 µL of

of CT and TT genotypes (Table 1). There was a significant statistic correlation between rs228-1388T/C polymorphism of HLA-DP gene and HBsAg feature (P=0.022). No significant rela

-tionship was found between rs2281388T/C

polymorphism and other clinical stages (P> 0.05).

Correlation between rs2281388T/C polymor-phism and HCC risk

The results indicated that CT genotype exhibit

-ed no significant difference between case and

control groups (P>0.05). In addition, there

existed significant correlation between HCC risk and TT genotype (OR=2.121, 95% CI=

1.084-4.148, P=0.027), and the same to T allele (OR=1.432, 95% CI=1.021-2.007, P=

0.037). Above all, TT genotype and T allele

might increase the risk of HCC (Table 2).

Discussion

HCC is one of the ten malignant tumors

announced by the world health organization (WHO). About 700, 000 new HCC cases occur

and about one million people die of it every

year. More than 50% of the annual newly increased HCC patients appear in China [1, 3].

In recent years, with the development of molec-ular biology, virology and genetics, the occur-rence of HCC is widely regarded as the conse-quence of the long-term effect of various fac-tors. Environment and genetical factors all

involved in the process of HCC [26], such as virus, aflatoxin, alcohol, single nucleotide poly -morphism (SNP), methylation etc. Not everyone who exposed to the carcinogenic factors will suffer the risk of liver cancer, there exists

indi-vidual difference on HCC susceptibility. The

study of HCC susceptibility will contribute to exploring the pathogenesis of HCC.

Amount of studies indicated that the chronic infection of Hepatitis B virus (HBV) is the most dangerous factor that leads to the occurrence

of HCC in China [27], and more than 85% cases

of HBV infection appear to be positive. Chinese population who infected the HBV has a higher risk of HCC. It is concluded that the host’s genetic predisposition plays an important role in the occurrence and development of HCC. As we all know, there is an obvious relationship between HLA complex protein and the immune reaction. Present studies showed that some

HLA polymorphisms might influence the immune reaction of HBV infection [6, 21-24]. So

we speculated that mutations of HLA could down regulate the human immune capacity. As a result, it is easier for HBV to infect the host. In the present study, we provided preliminary

evidence that the HBsAg feature had a signifi

-cant statistic correlation with rs2281388 T/C

polymorphism of HLA-DP gene. But there was

no significant relationship between rs2281388 T/C polymorphism and other clinical stages.

Based on the study, we suggested that the

rs2281388 T/C polymorphism was apparently related to the HCC risk in northern China. The TT genotype and T allele of rs2281388 signifi

-cantly increased the risk of HCC. That is agreed with the research on Graves’ disease [28].

However, Zhang et al. reported that rs2281388

CC genotype significantly decreased HBV per

-sistence, but no significant association with HBV-caused liver cancer [25].

In summary, the TT genotype and T allele of rs2281388 significantly increased the risk of

HCC. But the pathogenic principle of rs2281388 polymorphism in HCC is still unknown.

Therefore, more comprehensive study with large sample size is needed to explore issue.

Well designed research will contribute to sup-Table 2. The comparison between two groups on rs2281388 T/C polymorphism

Group n Genotype Allele

CCn (%) CTn (%) TTn (%) Cn (%) Tn (%) Health control 150 68 (45.33) 63 (42.00) 19 (12.67) 199 (66.33) 101 (33.67) HCC 139 54 (38.85) 53 (38.13) 32 (23.02) 161 (57.91) 117 (42.09) χ2 _{- 1.00 0.049 4.915 1.00 4.354}

plying accurate evidence on the pathogenic principle of rs2281388 polymorphism in HCC. Disclosure of conflict of interest

None.

Address correspondence to: Dr. Hongguang Li, Department of Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, 9677 Jingshi Road, Jinan 250021, Shandong, China. E-mail: [email protected]

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