An intronic polymorphism in the corticotropin-releasing hormone receptor 2 gene increases susceptibility to HBV-related hepatocellular carcinoma in Chinese population

Corticotropin-releasing hormone receptor 2 (CRHR2) plays a role in both the central nervous system (CNS) and the peripheral nervous system. CRHR2 together with its ligands, urocortins (Ucns) and corticotropin-releasing hormone (CRH), functions as a mediator of inflammatory response and inhibitor of angiogenesis. Recently, it has been reported to be expressed in many human cancers. An association between rs2267716 polymorphism in the CRHR2 gene and susceptibility to hepatocellular carcinoma (HCC) was found in patients with chronic hepatitis C virus (HCV) infection. In the present study we analyzed, using a polymerase chain reaction–ligation detection reaction (PCR–LDR), the rs2267716 polymorphism in 364 hepatitis B virus (HBV)-related HCC patients, 196 non-HCC patients with HBV infection, and 404 healthy controls. The aim was to detect the possible association of this single-nucleotide polymorphism (SNP) with susceptibility to HBV-related HCC. Significant differences of rs2267716 allele were detected between HBV-related HCC patients and healthy controls (OR = 1.55, 95% CI 1.13–2.15, P = 0.007) or non-HCC patients with HBV infection (OR = 1.61, 95% CI 1.13–2.31, P = 0.009). These results suggest that the rs2267716 polymorphism in the CRHR2 gene might influence the risk of developing HCC in patients with HBV infection in Chinese population.     

Leukocyte Telomere Length-Related rs621559 and rs398652 Genetic Variants Influence Risk of HBV-Related Hepatocellular Carcinoma

Recent genome-wide association studies (GWAS) have identified eleven leukocyte telomere length (LTL)-related single nucleotide polymorphisms (SNPs). Since LTL has been associated with risk of many malignancies, LTL-related SNPs may contribute to cancer susceptibility. To test this hypothesis in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), we genotyped these eleven LTL-related SNPs in a case-control set including 1186 HBV-related HCC cases, 508 chronic HBV carriers and 1308 healthy controls at the discovery stage. The associations of HCC risk with these SNPs were further confirmed in an independent case-control set. We found that 1p34.2 rs621559 and 14q21 rs398652 were significantly associated with HBV-related HCC risk (both P<0.005 after Bonferroni corrections). There was no significant difference of either rs621559 or rs398652 genotypes between chronic HBV carriers and healthy controls, demonstrating that the association was not due to predisposition to HBV infection. In the pooled analyses (1806 HBV-related HCC cases and 1954 controls), we observed a decreased HCC risk, 0.72-times, associated with the 1p34.2 rs621559 AA genotype compared to the GG genotype (P = 1.6×10⁻⁶). Additionally, there was an increased HCC risk, 1.27-fold, associated with the rs398652 GG genotype (P = 3.3×10⁻⁶). A statistical joint effect between the rs621559 GG and rs398652 GG genotypes may exist in elevating risk of HBV-related HCC. We show, for the first time, that rs398652 and rs621559 might be marker genetic variants for risk of HBV-related HCC in the Chinese population.     

CDC25B is associated with the risk of hepatocellular carcinoma,but not related to persistent infection of hepatitis B virus in a Chinese population

The cell division cycle 25 (CDC25) gene members, including CDC25A, CDC25B and CDC25C, are reported to be associated with several human cancers. Here, we aim to investigate the association of functional polymorphisms of CDC25 gene family with the risk of hepatocellular carcinoma (HCC) and persistent infection of Hepatitis B virus (HBV) in a Chinese HBV-related population. First, we used bioinformatics tools to systematically screen functional polymorphisms within CDC25 gene family. Second, we evaluated the effects of candidate polymorphisms by recruiting 790 HCC cases, 709 persistent HBV carriers (PHC), and 741 subjects with HBV natural clearance (SHNC). MassARRAY platform was used for genotyping. At last, we conducted functional prediction and assay to further explore the pathogenic mechanism of the identified polymorphism. Our results demonstrated that CDC25B rs2295348 played a protective role in HCC risk in a HBV-related Chinese population (adjusted odds ratio [OR]?=?0.77, 95% confidence interval [CI] 0.65–0.93, P?=?0.006). It showed a more significantly reduced HCC risk in the SHNC population (adjusted OR?=?0.73, 95% CI 0.59–0.89, P?=?0.002). However, we did not observe the association between CDC25B rs2295348 and the risk of persistent HBV infection. Further functional prediction and assay demonstrated that the mutant A allele of CDC25B rs2295348 might significantly decrease gene expression to modify the HCC risk. Our results suggest that CDC25B rs2295348 may confer a protective effect on HCC risk in a HBV-related Chinese population, but do not influence the susceptibility to persistent HBV infection.

     

−509C>T polymorphism in the TGF-β1 gene promoter,impact on the hepatocellular carcinoma risk in Chinese patients with chronic hepatitis B virus infection

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The risk for developing HCC increases with severity of inflammation and fibrosis. Transforming growth factor-β1 (TGF-β1) is most frequently upregulated in tumor cells. The most studied −509C>T polymorphism of TGF-β1 gene has been associated with colorectal, gynecologic, and lung cancers. To assess whether this polymorphism in TGF-β1 gene is associated with susceptibility to and/or clinicopathologic characteristics of HBV-related HCC, a total of 575 patients with chronic HBV infection and 299 healthy volunteers with no evidence of recent or remote HBV infection were prospectively enrolled. The patients were divided into two groups: those without (n = 196) and those with HCC (n = 379). These 379 HCC patients with chronic HBV infection were designated as cases, the remaining 196 patients without HCC and 299 healthy volunteers served as disease and healthy controls, respectively. −509C>T polymorphism in the TGF-β1 gene promoter was studied using restriction fragment-length polymorphism. In addition, tumor tissues of liver (n = 60) were obtained from the studied HCC patients for measurement of TGF-β1 mRNA expression levels. We also assessed the plasma TGF-β1 levels of HBV patients without (n = 94) or with HCC (n = 136) and healthy subjects (n = 120). In our study group, the risk of HCC in Chinese patients with HBV infection was significantly lower with the TT genotypes than in those with the CC genotypes at position −509 of TGF-β1 gene (P = 0.01). In addition, in the case group, patients with the CC genotype had a statistically significant higher median plasma TGF-β1 or liver tumor tissue TGF-β1 mRNA level compared with the individuals with the TT genotype. However, in a subsequent analysis of the association between this polymorphism and clinicopathological characteristics including tumor number, size, grade, stage, and invasiveness, there was no significant difference in both the distribution of genotype or allelic frequency within HCC patients, indicating that −509C>T exchange in TGF-β1 gene may play an important role in the occurrence, not the progression of HBV-related HCC through influencing plasma concentrations of TGF-β1 or TGF-β1 mRNA expression of liver tumor tissue.     

Association of Single Nucleotide Polymorphisms in VDR and DBP Genes with HBV-Related Hepatocellular Carcinoma Risk in a Chinese Population

Background

Polymorphisms of genes encoding components of the vitamin D pathway including vitamin D receptor (VDR) and vitamin D binding protein (DBP) have been widely investigated because of the complex role played by vitamin D in cancer tumorogenesis. In this study, we investigated the association between VDR and DBP gene polymorphisms and HBV-related HCC risk in a Chinese population.

Methods

Study subjects were divided into three groups: 184 HBV patients with HCC, 296 HBV patients without HCC, and 180 healthy controls. The VDR rs2228570, and rs3782905 and the DBP rs7041 polymorphisms were genotyped using PCR-RFLP and the VDR rs11568820 polymorphism was genotyped by PCR-SSP, respectively. DNA sequencing was performed to validate the genotype results.

Results

We found that there were significant differences in the genotype and allele frequencies of the VDR rs2228570 and DBP rs7041 polymorphisms between HBV patients with HCC and healthy controls. The rs2228570 T allele was associated with a significant increased HBV-related HCC risk as compared with the C allele. The rs2228570 TT and TT/TC genotypes were correlated with a significant increased HBV-related HCC risk when compared with the wild-type CC homozygote. Similarly, the rs7041 G allele was associated with a significant increased HBV-related HCC risk as compared with the T allele. The rs7041 GG and GG/TG genotypes were correlated with a significant increased HBV-related HCC risk when compared with the wild-type TT homozygote. However, we did not observe any significant effect of VDR rs11568820, and rs3782905 polymorphisms on HBV-related HCC risk in this population. In haplotype analysis, we also did not find any significant differences in haplotype frequencies of the VDR gene between HBV patients with HCC and the healthy controls.

Conclusions

We conclude that the VDR rs2228570 and DBP rs7041 polymorphisms may contribute to increased susceptibility to HBV-related HCC in the Chinese population. Due to the marginal significance, further large and well-designed studies in diverse ethnic populations are needed to confirm our results.     

Variants Identified by Hepatocellular Carcinoma and Chronic Hepatitis B Virus Infection Susceptibility GWAS Associated with Survival in HBV-Related Hepatocellular Carcinoma

Recent genome-wide association studies (GWAS) have identified several common susceptibility loci associated with the risk of hepatocellular carcinoma (HCC) or chronic hepatitis B infection (CHB). However, the relationship between these genetic variants and survival of patients with hepatitis B virus (HBV)-related HCC is still unknown. In this study, 22 single nucleotide polymorphisms (SNPs) were genotyped among 330 HBV-related HCC patients using the MassARRAY system from Sequenom. Cox proportional hazards regression was used to examine the effects of genotype on survival time under an additive model with age, sex, smoking status and clinical stage as covariates. We identified four SNPs on 6p21 (rs1419881 T>C, rs7453920 G>A,rs3997872 G>A and rs7768538 T>C), and two SNPs on 8p12 (rs2275959 C>T and rs7821974 C>T) significantly associated with survival time of HBV-related HCC patients. Our results suggest that HCC or CHB susceptibility loci might also affect the prognosis of patients with HBV-related HCC.     

Genetic variation in IL28B is associated with the development of hepatitis B-related hepatocellular carcinoma

To evaluate the role of host IL28B (interleukin 28B; interferon lambda 3) single nucleotide polymorphisms (SNPs) in predicting hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) susceptibility, three SNPs in the IL28B gene (rs12979860C/T, rs8099917G/T and rs12980275G/A) were examined in 330 subjects (including 154 HBV-related HCC patients, 86 non-HCC patients with chronic hepatitis B (CHB), 43 HBV self-limited infections and 47 healthy controls). Notably, the frequency of CC homozygosity was 91.5% in healthy controls and 72.9% in CHB, the difference being statistically significant (χ(2) = 6.40, P = 0.01). The statistically difference was seen between healthy controls (91.5%) and HCC (74.7%) (χ(2) = 6.05, P = 0.01). However, this significant finding was not seen between HBV self-limited and healthy controls. Carriers of the minor T allele in rs12979860 had a higher risk of HCC compared with non-carriers (χ(2) = 4.44, P = 0.04). Haplotype analyses revealed significant association between haplotype C-T-A and healthy controls, but not with the HCC group (96.6 vs. 82.0%, χ(2) = 6.08, P = 0.01). Analyses of genotype combination and gene-gene interaction showed that there was a positive interaction between rs12979860 and rs12980275, with an OR rate of 11.79 (likelihood test, P = 0.04). Our results suggest that the IL28B rs12979860 C/T polymorphism might affect susceptibility to the chronic HBV infection and progression of HCC. Of note, the T allele and non-CC genotypes have strong predictive effect of increasing susceptibility of chronic HBV infection and HCC.     

An intronic variant in the GRP78, a stress-associated gene, improves prediction for liver cirrhosis in persistent HBV carriers

Background

Our previous study indicated that a common variant (rs430397 G>A) in the intron 5 of glucose-regulated protein 78 (GRP78) gene was associated with risk and prognosis of primary hepatocellular carcinoma (HCC), including HBV- and cirrhosis-related HCC. rs430397 polymorphism may be a contributing factor or biomarker of HBV infection or HBV-related cirrhosis.

Methodology/Principal Findings

539 non-HBV-infected individuals, 205 self-limited infection and 496 persistent HBV infection were recruited between January 2001 and April 2005 from the hospitals in Southern China. Genomic DNA was genotyped for rs430397. The associations between the variation and susceptibility to liver cirrhosis (LC) in persistent HBV infection were examined. We observed that individuals carrying allele rs430397A were more likely to become HBV-related LC. When persistently infected patients were divided into four subgroups, patients with phase IV had an increased allele A and genotype AG compared with phase I and/or phase III. Decreased serum albumin and prolonged plasma prothrombin time (PT) were showed in LC patients carrying genotype AA. Furthermore, rs430397 genotype had an increased susceptibility to LC with dose-dependent manners (P-trend = 0.005), and the genotype did constitute a risk factor for the development of advanced LC (Child–Pugh classification C and B, P-trend = 0.021).

Conclusions/Significance

rs430397 polymorphism may be a contributing factor to LC in persistent HBV carriers.     

Association of T-Cell Immunoglobulin and Mucin Domain-Containing Molecule 3 (Tim-3) Polymorphisms with Susceptibility and Disease Progression of HBV Infection

Purpose

T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) plays an important role in regulating T cells in hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). However, few researches have reported the association of Tim-3 genetic variants with susceptibility and progression of HBV infection. In this study, we focused on the association of Tim-3 polymorphisms with HBV infection, HBsAg seroclearance and hepatocellular carcinoma.

Methods

A total of 800 subjects were involved in this study. Four groups were studied here, including HBV, HBsAg seroclearance, HBV-associated HCC and healthy controls. Three single-nucleotide polymorphisms (SNPs) of Tim-3, rs246871, rs25855 and rs31223 were genotyped to analyze the association of Tim-3 polymorphisms with susceptibility and disease progression of HBV infection.

Results

Our study found that rs31223 and rs246871 were associated with disease progression of HBV infection, while none of the three SNPs was relevant to HBV susceptibility. The minor allele “C” of rs31223 was found to be associated with an increased probability of HBsAg seroclearance (P = 0.033) and genotype “CC” of rs246871 to be associated with an increased probability of HBV-associated HCC (P = 0.007). In accordance, haplotypic analysis of the three polymorphisms also showed that the haplotype block CGC* and TGC* were significantly associated with HBsAg seroclearance (P<0.05) while haplotype block CAT*, CGT*, TAC* and TGT* were significantly associated with HBV-associated HCC (all P<0.05).

Conclusions

Genetic variants of Tim-3 have an important impact on disease progression of HBV infection. With specific Tim-3 polymorphisms, patients infected with HBV could be potential candidates of HCC and HBsAg seroclearance.     

Association of the microRNA-499 variants with susceptibility to hepatocellular carcinoma in a Chinese population

microRNAs (miRNAs) are a new class of small non-coding RNAs that function as tumor suppressors or oncogenes. Single nucleotide polymorphisms (SNPs) in miRNA may contribute to cancer development. We hypothesized that genetic variations of the miRNA could be associated with the risk of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). A total of 100 patients with HCC, 100 cases of chronic hepatitis B and 100 health adults were enrolled in this present study. Two common polymorphisms in pre-miRNAs: Homo sapiens miRNA-146a (hsa-mir-146a) (rs291016, guanine to cytosine [G-C]) and hsa-mir-499 (rs3746444; adenine to guanine [C-T]) were genotyped by PCR-Restriction Fragment Length Polymorphism and confirmed by bidirectional DNA sequencing. Significant differences were found in frequency and distribution of the genotypes of miRNA-499 between the HCC and the control group. Compared with miRNA-499 T/T, the odds ratio (OR) of patients with miRNA-499 C/C for developing HCC was 3.630 (95% CI: 1.545–8.532), and OR for developing HBV-related HCC was 3.133 (95% CI: 1.248–7.861). There was no significant association between miRNA-146a polymorphism and the risk of HCC in all subjects. Our results suggested that hsa-mir-499 polymorphism was associated with susceptibility to HBV-related HCC in Chinese population. Further characterization of miRNA SNPs may open new avenue for the study of cancer and therapeutic interventions.     

Relationships between the Osteocalcin Gene Polymorphisms,Serum Osteocalcin Levels,and Hepatitis B Virus-Related Hepatocellular Carcinoma in a Chinese Population

Background

Available evidence has demonstrated that osteocalcin may play a role in pathogenesis of cancer, and mutation of the osteocalcin gene may be involved in the cancer development. The aim of this study is to determine whether osteocalcin gene polymorphisms are associated with hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) among Chinese population.

Methods

A total of 515 subjects were divided into four groups: 129 patients with chronic hepatitis B (CHB), 62 patients with HBV-related liver cirrhosis (LC), 154 patients with HBV-related HCC, and 170 healthy controls. The polymerase chain reaction-restriction fragment length polymorphism strategy was used to detect osteocalcin gene rs1800247 and rs1543297 polymorphisms.

Results

Compared with healthy controls, the rs1800247 HH and Hh genotypes were associated with a significantly increased susceptibility to HCC (HH versus hh: OR = 6.828, 95% CI 2.620–17.795, P <0.001; Hh versus hh: OR = 6.306, 95% CI 3.480–11.423, P <0.001, respectively). Similarly, the subjects bearing the H allele of rs1800247 had more than a 2.4-fold increased risk for development of HCC (OR = 2.484, 95% CI 1.747–3.532, P <0.001) compared with those bearing the h allele. In addition, we found significant decreased serum osteocalcin levels in HBV-related HCC patients (11.73±8.18 ng/mL) compared with healthy controls (15.3±6.06 ng/mL). Furthermore, the serum osteocalcin levels were significantly lower in HCC patients than healthy controls among the individuals with heterozygous Hh genotype (P = 0.003) and CT genotype (P <0.001). In contrast, there were no significant differences in the genotype and allele of rs1543297 polymorphisms between the groups of patients and healthy controls.

Conclusions

These findings for the first time suggest that genetic variant in osteocalcin gene rs1800247 polymorphisms may be a risk factor for HBV-related HCC. We also find an inverse association of serum osteocalcin levels with HCC.     

Genetic variations of NOD2 and MD2 genes in hepatitis B virus infection

Objectives: Nucleotide oligomerization domain 2 (NOD2) and myeloid differentiation protein 2 (MD-2) have crucial roles in the innate immune system. NOD2 is a member of the NOD-like receptor (NLR) family of pattern recognition receptors (PRRs), while MD-2 is a co-receptor for Toll-like receptor 4 (TLR4), which comprises another group of PRRs. Genetic variations in the NOD2 and MD-2 genes may be susceptibility factors to viral pathogens including hepatitis B virus (HBV). We investigated whether polymorphisms at NOD2 (rs2066845 and rs2066844) or at MD-2 (rs6472812 and rs11466004) were associated with susceptibility to HBV infection and advancement to related liver complications in a Saudi Arabian population. Methods: A total of 786 HBV-infected patients and 600 healthy uninfected controls were analyzed in the present study. HBV-infected patients were categorized into three groups based on the clinical stage of the infection: inactive HBV carriers, active HBV carriers, and patients with liver cirrhosis + hepatocellular carcinoma (HCC). Results: All four SNPs were significantly associated with susceptibility to HBV infection although none of the SNPs tested in NOD2 and MD-2 were significantly associated with persistence of HBV infection. We found that HBV-infected patients that were homozygous CC for rs2066845 in the NOD2 gene were at a significantly increased risk of progression to HBV-related liver complications (Odds Ratio = 7.443 and P = 0.044). Furthermore, haplotype analysis found that the rs2066844-rs2066845 C-G and T-G haplotypes at the NOD2 gene and four rs6472812-rs11466004 haplotypes (G-C, G-T, A-C, and A-T) at the MD-2 gene were significantly associated with HBV infection in the affected cohort compared to those found in our control group. Conclusion: We found that the single nucleotide polymorphisms rs2066844 and rs2066845 at NOD2 and rs6472812 and rs11466004 at MD-2 were associated with susceptibility to HBV infection in a Saudi population.     

Association of interleukin-18 genotypes (−607C > A) and (−137 G > C) with the hepatitis B virus disease progression to hepatocellular carcinoma

Chronic infection with HBV has been reported to be associated with the development of HCC. The inflammation mounted by cytokine-mediated immune system plays an important role in the pathogenesis of HBV-associated HCC. IL-18 is a pro-inflammatory cytokine whose role in the development of HBV-associated chronic to malignant disease state has not been much studied. The present study was conceived to determine the role of genetic polymorphisms in IL-18, serum levels of IL-18, and expression level of its signal transducers in the HBV disease progression. A total of 403 subjects were enrolled for this study including 102 healthy subjects and 301 patients with HBV infection in different diseased categories. Polymorphism was determined using PCR–RFLP. Genotypic distributions between the groups were compared using odd’s ratio and 95% CI were calculated to express the relative risk. Circulating IL-18 levels were determined by ELISA. Expression levels of pSTAT-1 and pNF?B was determined by western blotting. In case of IL-18(??607C?>?A), the heterozygous genotype (CA) was found to be a protective factor while in case of IL-18(??137G?>?C) the heterozygous genotype (GC) acted as a risk factor for disease progression from HBV to HCC. Moreover, serum IL-18 levels were significantly increased during HBV disease progression to HCC as compared to controls. Also the levels of activated signal transducers (pSTAT-1 and pNF-κB) of IL-18 in stimulated PBMCs were significantly increased during HBV to HCC disease progression. These findings suggest that IL-18 has the potential to act as a biomarker of HBV-related disease progression to HCC.

     

IFNL4 rs12979860 polymorphism influences HBV DNA viral loads but not the outcome of HBV infection in Moroccan patients

ObjectivesThe interferon (IFN) is known to bridge innate and adaptive immune responses, and to play a critical role particularly against hepatitis B virus (HBV) infection. Defects in IFN signals may result, therefore, in attenuated responses against HBV. Accordingly, polymorphisms in genes coding for immune response effectors may affect the clinical outcome of HBV infection. We analyzed the putative association between IFNL4 rs12979860 polymorphism and the outcome of HBV infection in Moroccan patients.MethodsIn this study, 237 chronic HBV (CHB) patients and 129 spontaneously resolved HBV (SRB) individuals were enrolled and genotyped using a predesigned Taqman allelic discrimination assay.ResultsOur data show a significant increase of HBV DNA loads in patients with IFNL4 rs12979860 CC genotype compared to patients with CT and TT genotypes (p = 0.0008). However, there was no consistent association between IFNL4 rs12979860 polymorphism and the outcome of HBV infection.ConclusionsAlthough IFNL4 rs12979860 polymorphism seems to modulate circulating HBV DNA levels, it is disconnected from chronic disease progression. This observation suggests that the role of rs12979860 in liver disease is restricted to viral control and inactive in the deleterious immune pathology that affects liver tissue. Taken together, our data suggest that rs12979860 CC genotypes could be useful as a predictor of success or failure of IFN-based therapy in chronic HBV-infected patients.     

Three common functional polymorphisms in microRNA encoding genes in the susceptibility to hepatocellular carcinoma: A systematic review and meta-analysis

Emerging evidences have shown that common genetic polymorphisms in microRNAs may be associated with the development of hepatocellular carcinoma (HCC); but individually published studies and previous meta-analyses revealed inconclusive results. The aims of this review and meta-analysis are to assess whether common single-nucleotide polymorphisms (SNPs) in the genes encoding the microRNAs are associated with susceptibility to HCC development and clinicopathologic characteristics of hepatitis B virus (HBV) related HCC. A computerized search was performed in PubMed, Embase, Web of Science and China BioMedicine (CBM) databases to identify relevant articles published before January 1st 2013. Ten case–control studies were assessed with a total of 3437 cases and 3437 healthy controls. Three common functional SNPs in miRNA-encoding genes were found, including miR-146a G > C (rs2910164), miR-196a-2 C > T (rs11614913) and miR-499 T > C (rs3746444). This meta-analysis revealed that the miR-146a*C variant was associated with a decrease in HCC risk, especially among Asian and male populations; while the miR-196a-2*T variant was associated with susceptibility to HCC among Caucasian populations. However, we failed to find any significant correlations between the miR-499*C polymorphism and HCC risks. When further stratification on HBV status was conducted, a similar trend of association between the three SNPs and the HBV-related HCC risks was observed, but these results were not statistically significant due to small sample sizes. The current meta-analysis demonstrates that SNPs contained in the genes encoding miR-146a and miR-196a-2 may play a major role in genetic susceptibility to HCC.     

Polymorphism attribution of cSNPs in cancer-related genes located in loss regions with a high frequency of HCC between HBV and health groups

Cancer-related genes harbored in the loss regions containing a high frequency of hepatocellular carcinoma (HCC) were selected. Related information was gathered and the coding single nucleotide polymorphism (cSNP) sequences were obtained from the single nucleotide polymorphism (SNP) database. The appropriate primers and oligonucleotide probes were then designed in accordance with the SNP sites, and subsequently, the gene chips for detecting SNPs were constructed. Genomic DNA was extracted from blood samples of healthy controls and from patients with HBV infection. The sequences, including the SNPs, were amplified via polymerase chain reaction (PCR) and labeled using digoxigenin deoxyuridine tri-phosphate (Dig-dUTP). The labeled products were then hybridized with the SNP chips. Results confirmed that the differences in allele frequencies of three SNPs EGFL3 (rs947345), Caspase9 (rs2308950), and E2F2 (rs3218171) were distinct between HBV-infected patients and controls, suggesting that these SNPs ocuring in high frequency in HBV-infected individuals may be associated with susceptibility to HCC. Translated from Acta Scientiarum Naturalium Universitatis Nankaiensis, 2006, 39(3): 1–5 [译自: 南开大学学报(自然科学版)]     

Non-classical MHC-Ι genes in chronic hepatitis B and hepatocellular carcinoma

The non-classical human leukocyte antigens (HLA)-E, HLA-G, and HLA-F have been shown to modulate immune responses. We examined whether non-classical HLA polymorphisms are associated with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). Fifteen Single-nucleotide polymorphisms (SNPs) in these non-classical class I alleles were investigated by ligase detection reaction. A fragment of 650 bp located in the 3' untranslated region of HLA-G was investigated. Four SNPs (rs17875380, rs41557518, rs114465251, and rs115492845) were associated with altered susceptibility to HBV or HCC, and HLA-F*01:04, HLA-G*01:05N, and HLA-E*01:01 were associated with hepatitis B or hepatitis B complicated with HCC. Six of 16 designated HLA-E, -G, and -F haplotypes were associated with risk of hepatitis B or HCC. Our study provides healthy reference and detailed analyses of non-classical HLA class Ι polymorphisms that provide insight into immune mechanisms involved in susceptibility to hepatitis B and HCC.     

Association of Ghrelin Gene Polymorphisms and Serum Ghrelin Levels with the Risk of Hepatitis B Virus-Related Liver Diseases in a Chinese Population

Background

The functions of ghrelin (GHRL) include anti-inflammatory effects, reduction of the fibrogenic response, protection of liver tissue, and regulation of cell proliferation. Genetic variations in the GHRL gene may play an important role in the development of chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Therefore, we investigated whether GHRL gene polymorphisms and its serum levels are associated with hepatitis B virus (HBV)-related diseases risk in a Chinese population.

Methods

176 patients with CHB, 106 patients with HBV-related LC, 151 patients with HBV-related HCC, and 167 healthy controls were recruited in the study. Genotyping of GHRL rs26311, rs27647, rs696217, and rs34911341 polymorphisms were determined with the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and DNA sequencing. The serum GHRL concentrations were determined using enzyme-linked immunosorbent assay (ELISA).

Results

Binary logistic regression analyses adjusting for gender and age revealed that a significant increased risk of LC was found in the GHRL rs26311 GC genotype and combined GC+CC genotypes when compared with the GG genotype (GC vs. GG: OR = 1.671, 95% CI = 1.013–2.757, P = 0.044; GC+CC vs. GG: OR = 1.674, 95% CI = 1.040–2.696, P = 0.034). In subgroup analysis by gender, binary logistic regression analyses adjusting for age showed that the GHRL rs26311 C allele and combined GC+CC genotypes were associated with a significantly increased risk to LC in males (C vs. G OR = 1.416, 95% CI = 1.017–1.972, P = 0.040; GC+CC vs. GG: OR = 1.729, 95% CI = 1.019–2.933, P = 0.042). In addition, we found significant decreased serum GHRL levels in LC patients compared with the healthy controls. However, there was no significant association of the GHRL rs26311 polymorphism with serum GHRL levels in LC patients.

Conclusions

These observations suggest that the GHRL rs26311 polymorphism is associated with an increased risk to HBV-related LC, especially in men. We also found an inverse association of serum GHRL levels with LC.     

p53 codon 72 polymorphism is associated with susceptibility to hepatocellular carcinoma in the Turkish population: a case–control study

The tumor suppressor p53 gene plays a crucial role in preventing carcinogenesis through its ability to induce cell cycle arrest and apoptosis following DNA damage and oncogene activation. A guanine (G)/cytosine (C) common single nucleotide polymorphism (SNP) at second position of codon 72 in exon 4 of p53 gene determines a arginine (Arg) to proline (Pro) (Arg72Pro) aminoacidic substitution within the proline-rich domain of p53 protein. Arg72 and Pro72 allele are different from a biochemical and biological point of view and many reports suggest that they can modulate individual cancer susceptibility. To determine the association of the p53 Arg72Pro polymorphism with the risk of hepatocellular carcinoma (HCC) development in a Turkish population, a hospital-based case–control study was designed consisting of 119 subjects with HCC and 119 cancer-free control subjects matched for age, gender, smoking and alcohol status. The genotype frequency of the p53 Arg72Pro polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Our data shows that the Pro/Pro genotype of the p53 Arg72Pro polymorphism is associated with increased risk of HCC development in this Turkish population (OR = 3.20, 95% CI: 1.24–8.22, P = 0.02). Furthermore, according to stratified analysis, a significant association was observed between the homozygote Pro/Pro genotype and HCC risk in the subgroups of male gender (OR = 3.01, 95% CI: 1.14–7.97, P = 0.03) and patients with hepatitis B virus (HBV)-related HCC (OR = 4.04, 95% CI: 1.46–11.15, P = 0.007). Because our results suggest for the first time that the Pro/Pro homozygote of p53 Arg72Pro polymorphism may be a genetic susceptibility factor for HCC (especially in the male gender and HBV-infected patients) in the Turkish population, further independent studies are required to validate our findings in a larger series, as well as in patients of different ethnic origins.