Author Affiliations: Department of General Surgery, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai 200080, China (Wang PS, Li H, Wang CG, Shi BJ and Zhong L); Department of Hepatobiliary Surgery, Weifang People’s Hospital, Weifang 261000, China (Kuai J)

Corresponding Author: Lin Zhong, MD, PhD, Department of General Surgery, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai 200080, China (Tel: +86-21- 63240090ext3133; Fax: +86-21-63242903; Email: zhongl_3284@hotmail.com)

 

© 2016, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(16)60050-4

Published online January 4, 2016.

Contributors: ZL proposed the study. WPS and KJ performed the research and wrote the first draft. LH, WCG and SBJ collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. WPS and KJ contributed equally to this work. ZL is the guarantor.

Funding: This study was supported in part by grants from the National Natural Science Foundation of China (81170447), the Natural Science Foundation of Shanghai (13JC1404600), and the Shanghai Committee for Science & Technology Project (094119524).

Ethical approval: This study was approved by the Institutional Review Board Liver Transplantation Surgery, Shanghai First People’s Hospital, Shanghai, China.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

BACKGROUND: Mannose-binding lectin 2 (MBL2) plays a key role in the host immune response, but whether it is associated with hepatocellular carcinoma (HCC) is not clear. The present study aimed to identify the association between MBL2 gene polymorphisms and HCC in patients with hepatitis B virus (HBV)-related cirrhosis in the Chinese population.

 

METHODS: A single-nucleotide polymorphism of MBL2, rs11003123, was genotyped and analyzed in a case-control study of HBV-related cirrhotic patients with HCC (n=77) and without HCC (n=40).

 

RESULTS: We found that Child-Pugh profiles, model for end-stage liver disease score, and the incidence of encephalopathy were all higher in the non-HCC group (P<0.05). A significant association between allele mutants and HCC occurrence was demonstrated by allele comparison (A vs G) (OR=0.34; 95% CI: 0.15-0.76; P=0.006). Heterozygous comparison (GA vs GG) revealed that the individuals with GA mutants had a reduced risk of HCC occurrence compared with those with GG wild type (adjusted OR=0.28; 95% CI: 0.10-0.80; P=0.004). In a dominant model (GA+AA vs GG), a decreased risk of HCC occurrence was observed in individuals with variant genotypes (GA and AA) compared with those with the wild type (adjusted OR=0.30; 95% CI: 0.11-0.85; P=0.004). However, no statistically significant associations were observed between rs11003123 and prognosis of patients with HCC after liver transplantation in both recurrence-free survival and overall survival (P=0.449 and P=0.384, respectively).

 

CONCLUSION: MBL2 rs11003123 polymorphism may be a marker for the risk of HCC occurrence in patients with HBV-related cirrhosis in the Chinese population.

 

(Hepatobiliary Pancreat Dis Int 2016;15:282-288)

 

KEY WORDS: mannose-binding lectin 2; polymorphism; hepatitis B; cirrhosis; hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy and ranks the third among causes of cancer mortality in the world; it is estimated that there are 782?500 HCC cases and 745?500 HCC-related deaths throughout the world in 2012.^[1] China has the highest incidence of HCC, and 55% of new cases worldwide occurred in China.^[2] Approximately 70%-90% HCC cases are due to the underlying chronic liver diseases such as cirrhosis and chronic hepatitis. Hepatitis B remains the most common cause of cirrhosis in China.^[3] Sequential development from hepatitis B to cirrhosis and HCC is a typical phenomenon in most Chinese patients. Actually, hepatocarcinogenesis is a multifactorial and complicated process, which is related to various etiological causes including hepatitis B, hepatitis C, aflatoxin B1, high alcohol intake, and metabolic syndrome.^[4] Since the patients with the same genotype of hepatitis B infection have different outcomes and only a minority of these patients eventually develop HCC, the pathogenesis of HCC may be closely associated with genetic mutations, and a high individual risk of HCC may be caused by several unlinked single-gene defects.^[5] It is well established that single-nucleotide polymorphisms (SNPs) are one of the most important forms of genetic mutation, and they have been widely studied.^{[6, 7]} A large amount of SNPs are functional and may lead to pathogenesis of HCC by altering gene expression or protein function.^{[7, 8]}

 

The human mannose-binding lectin 2 (MBL2) gene, located on chromosome 10q21, consists of five exons, four of which encode protein.^[9] The serum concentration of MBL is largely genetically determined.^[10] It is verified that various SNPs are associated with MBL concentration.^[11] MBL is a vital component of innate immunity and a central recognition molecule of the lectin pathway of complement, which is likely to represent the most ancient complement activation pathway.^[12] Serum MBL concentration plays a crucial role in regulating the monocytes to produce various inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in response to pathogen infection, and therefore influences disease progression or severity.^[13] Frequent gene polymorphisms found in the MBL2 gene affect the concentration and function of the protein and enhance susceptibility to hepatitis B virus (HBV)^{[14, 15]} and hepatitis C virus (HCV) infection.^[16] The rs11003123 SNP, an upstream variant, is in complete linkage disequilibrium (LD) with some other MBL2 SNPs such as rs4647964, rs7095891, rs7084554, and so on (Han Chinese in Beijing, China SNP genotype data, HapMap Data Rel 24/phase Nov08, on NCBI B36 assembly, dbSNP b126); additionally, rs11003123 SNP was verified as a functional SNP and showed a strong association with MBL concentration.^{[11, 17, 18]}

 

To verify whether MBL2 rs11003123 polymorphism was associated with the development of HCC in the Chinese population following HBV-related cirrhosis, we used patients with HBV-related cirrhosis who had undergone liver transplantation as controls. We also investigated whether rs11003123 was associated with HCC clinical characteristics and survival.

 

 

A total of 117 liver recipients between January 2009 and August 2012 at the department of Liver Transplantation Surgery, Shanghai First People’s Hospital, Shanghai, China, were enrolled. Among them, 77 had HCC based on HBV-related cirrhosis and 40 had HBV-related cirrhosis only. All recruited patients were Chinese. The diagnoses of both HCC and cirrhosis were based on histological examinations. Inclusion criteria were patients with HBV-related cirrhosis; exclusion criteria were (1) patients with HCV infection; (2) patients who had a history of other tumor and received radiotherapy or chemotherapy; (3) patients with other liver diseases, such as autoimmune liver diseases or metabolic liver diseases; (4) if there were genetic relationships between each other, only one patient was involved.

 

Informed consent was obtained from each participant. This study was approved by the Institutional Review Board of Liver Transplantation Surgery, Shanghai First People’s Hospital, Shanghai, China, under the guidelines of the Ethics Committee of the hospital and the Declaration of Helsinki.^[19]

 

The following data were recorded: age, gender, alcohol status, smoking status, Child-Pugh profiles,^[20] Model for End-stage Liver Disease (MELD) score,^[21] encephalopathy, tumor node metastasis (TNM) stage (according to the United Network for Organ Sharing),^[22] tumor size, multinodular type, micro-vascular invasion, histologic grade (differentiated group contains well differentiated and moderately differentiated), serum alpha-fetoprotein (AFP) level (stratification according to previous research),^[23] recurrence-free survival (RFS, HCC group), and overall survival (OS, HCC group). The data prospectively collected were age, alcohol status, smoking status, Child-Pugh profiles, MELD score, encephalopathy, and serum AFP level before transplantation. The recipients were divided into two groups: HBV-related cirrhosis patients with HCC as a study group and those without HCC as a control group.

 

Blood samples were collected in ethylenediaminetetraacetic acid-containing tubes. Genomic DNA was extracted from white blood cell fractions using the Qiagen Blood Kit (Qiagen, Valencia, CA, USA). These samples were previously stored at -20 �� before use. Amplifications were performed according to the manufacturer’s protocol.

 

SNP genotyping was detected using a MassArray high-throughput DNA analyzer with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom, San Diego, CA, USA). Eight tag SNPs were selected based on the tagger algorithm (Haploview 3.32, using R²≥0.8 to indicate redundancy). rs11003123, rs10824792 (complete LD with rs7096206, Han Chinese in Beijing, China SNP genotype data, HapMap Data Rel 24/phase Nov08, on NCBI B36 assembly, dbSNP b126), rs7916582, rs10824795, rs920724, rs1838065, rs2083771, and rs10824793 were analyzed in this study. The procedure was repeated twice, and 10% samples were sequenced to validate the consistency.

 

All statistical analyses were performed using the SPSS version 19.0 statistical software (SPSS Inc., Chicago, IL, USA). Descriptive variables were expressed as mean±standard deviation or median (range). Categorical variables were compared using Pearson’s product-moment correlation coefficient, the Chi-square test or Fisher’s exact test. Differences in clinical characteristics such as age, Child-Pugh profiles, MELD score, and encephalopathy were analyzed by Student’s t test or Wilcoxon’s signed-rank test. SHEsis Online Verison (http://analysis.bio-x.cn/myAnalysis.php) and Hardy-Weinberg equilibrium test were used to calculate the allele and genotype frequencies of MBL2 polymorphisms.^[24] A logistic regression analysis was performed to calculate the adjusted genotype odds ratio (OR). RFS and OS were assessed by the Kaplan-Meier method and compared by the log-rank test. A P<0.05 was considered statistically significant.

 

 

The demographics and clinical characteristics of the patients are listed in Tables 1 and 2. This study consisted of 117 subjects including 16 females and 101 males, with a median age of 49 years (range 25-67). All the patients were orthotopic LT (OLT) recipients and the diagnoses were based on histological examinations. Alcohol status, smoking status, and some other important clinical data, such as Child-Pugh profiles, MELD score, and encephalopathy are listed in Table 1. Among these patients, there were 77 HCC cases and 40 non-HCC controls. The 16 Child-Pugh A patients in the non-HCC group were treated with OLT because of upper gastrointestinal hemorrhage, massive ascites, or hepatorenal syndrome.

 

Our data showed that Child-Pugh profiles (P=0.001), MELD score (P<0.001), and incidence of encephalopathy (P=0.002) were significantly higher in the non-HCC group compared with those in the HCC patients. The general characteristics in the HCC patients and controls are presented in Table 1.

 

rs11003123 was the only one among 8 tag SNPs analyzed and showed positive results. The genotype frequencies were tested by the Hardy-Weinberg equilibrium and did not show significant deviations. MBL2 rs11003123 alleles and genotype distributions in HCC cases and non-HCC controls are shown in Table 3. Apparently, a significant association between A allele and HCC development was found in allele comparison (A vs G) (OR=0.34; 95% CI: 0.15-0.76; P=0.006). Similarly, the altered distribution of genotypes and alleles was observed in rs11003123 (G>A) between the two groups. In heterozygous comparison (GA vs GG), the individuals with GA had a lower risk of HCC occurrence than those with GG (adjusted OR=0.28; 95% CI: 0.10-0.80; P=0.004). However, no significant difference was observed between the HCC cases and controls in homozygous comparison (AA vs GG). The HCC risk was lower in the dominant model (GA+AA) than in that with the wild type (GG, OR=0.30; 95% CI: 0.11-0.85; P=0.004). Nevertheless, in the recessive model (AA vs GG+GA), AA mutants did not show a statistical association with the development of HCC.

 

The susceptibility of HCC associated with the MBL2 rs11003123 genotypes was further analyzed by stratifying for age, gender, alcohol status, and smoking status. No statistically significant interactions were observed between stratification factors and rs11003123 (data not shown), suggesting that MBL2 rs11003123 genotypes are independent factors of the HCC occurrence underlying HBV-related cirrhosis.

 

As shown in Table 4, there was no statistically significant association between MBL2 rs11003123 genotypes and clinical characteristics (P>0.05), which indicated that MBL2 rs11003123 genotypes were independent factors in HCC based on HBV-related cirrhosis.

 

Among the 77 patients, 66 (85.7%) had genotype GG and 11 (14.3%) had GA+AA; their mean follow-up time was 33.6 months (range 3.3-71.6). Kaplan-Meier survival curves as well as the log-rank test showed that there were no statistically significant differences in both RFS and OS between the two groups (P=0.449 and P=0.384, respectively; Fig.).

 

 

MBL encoded by the MBL2 gene is a collagen-like serum protein that activates the complement system and is of importance for host defense.^[10] A large amount of variant alleles situated in the promoter or structural region of the human MBL2 gene affect the stability, structure, and serum concentration of the protein, which are very important in regulating various inflammatory cytokines such as IL-1β, IL-6, and TNF-α and therefore, MBL is involved in various diseases.^{[11, 13, 14]} In addition, MBL may have a direct effect on some diseases through complement activation.^[15]

 

In fact, there are some evidences supporting the role of MBL in inhibiting neoplastic cells;^[25] lower MBL concentration was associated with a series of human neoplasia: low MBL activity enhanced bacterial colonization of gastric mucosa and increased the risk of gastric cancer.^[26] Similar results were reported for HCC induced by hepatitis C^[16] and some other human malignancies.^[25] However, carcinogenesis is a complex process; whether MBL concentration directly affects carcinogenesis remains controversial.

 

Actually, there are some studies on the correlation between MBL2 SNPs and the development of HCC. However, few of them demonstrated the relations. A case-control study conducted by Segat et al^[27] did not show the role of MBL2 polymorphisms (three structural polymorphisms at codon 52, 54, and 57 in the first exon) in HCC development. However, their control group consisted entirely of healthy people, and there were 20 patients with HCC with no viral infection. The comparison between these two groups might not be suitable because HCC formation is based on multifactors. Cirrhosis triggers the HCC occurrence on the basis of genetic abnormality while genetic impact per se may not be enough to cause HCC. Moreover, it might not be accurate to conclude that there was no association between MBL2 polymorphisms and HCC risk by comparing healthy controls with HCC cases caused by HBV infection or HCV infection because again, any single factor might not be enough to trigger the development of HCC. Also, no association was found between HCC occurrence and some MBL2 SNPs, including rs10824792 (complete LD with rs7096206), rs7916582, rs10824795, rs920724, rs1838065, rs2083771, and rs10824793 (data not shown). However, Lin and coworkers’ case-control study with a larger sample size demonstrated that MBL2 promoter variant rs7096206 was indeed associated with HCC development in a hepatitis-free population.^[28] Their results were not confounded by viral infection and therefore, seemed to be more convincing. Eurich et al^[16] also demonstrated that rs7096206 was associated with the development of HCV-induced HCC. However, we did not find the correlation between rs7096206 and HCC in our patients. This might be partly because our cases were all HBV-related HCC. It is well known that a majority of HCC cases had cirrhosis background, and expressions of some genes would change because of cirrhosis.^[3] It is not provable, as the cirrhotic status of HCC patients was unavailable in the studies by both Segat et al^[27] and Eurich et al.^[16] We therefore recruited HBV-related cirrhotic patients without HCC as controls. We assumed that these 40 controls tended to have a lower possibility of hepatocarcinogenesis, as they had serious cirrhosis and reached an end-stage of liver disease. As shown in Table 1, their Child-Pugh profiles, MELD score, and incidence of encephalopathy were all higher than those in the HCC group. In addition, each patient in our study was examined pathologically and diagnosed accurately because they were all liver recipients. Considering all these factors, we believed that our study had a stronger strength than other studies in exploring the associations between MBL2 gene polymorphisms and HCC occurrence underlying HBV-related cirrhosis.

 

The present study demonstrated that rs11003123 GA genotype was correlated with the reduced HCC risk. A significant association was also observed in the dominant genetic model, but not in the recessive one. No significant associations were found between rs11003123 genotypes and recipient prognosis in both RFS and OS. Compared with the previous work, this study used the patients with HBV-related cirrhosis as controls and therefore, the calculation of P value and ORs was not confounded because the underlying disease is the same in both groups.

 

There are two limitations in this research. First, this is a single center study and the data might not fully represent the genotype distributions of the entire population. Second, the relationships among the MBL2 gene polymorphisms, MBL2 gene expression, MBL concentration and the HCC occurrence after HBV-related cirrhosis are a phenomenon and the direct mechanistic study is still needed.

 

In conclusion, the findings of this study demonstrated that genetic alterations of MBL2 rs11003123 are significantly associated with HCC occurrence following HBV-related cirrhosis, but not the prognosis of HCC after OLT, and it has the potential value to serve as a marker in a Chinese population.

 

 

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