AIM: To characterize high mobility group box chromosomal protein 1 (HMGB1) polymorphisms in patients infected with hepatitis B virus (HBV) and determine the different patterns in patient subgroups.

METHODS: A total of 1495 unrelated Han Chinese HBV carriers were recruited in this hospital-based case-control study. The HMGB1 1176 G/C polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism assay.

RESULTS: A significant association was observed between HMGB1 1176 G/C polymorphism and outcome of HBV infection. The subjects bearing 1176G/G genotype had an increased risk of susceptibility to chronic hepatitis B, liver cirrhosis and severe hepatitis B when compared with those bearing at least one 1176C allele.

CONCLUSION: Patients with 1176G/G genotype of HMGB1 gene are more likely to have a progressive status in HBV infection.

Hepatitis B virus (HBV) infection is associated with a variety of diseases, including asymptomatic carrier (AsC), fulminant hepatitis, chronic hepatitis (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Persistent HBV infection has been considered as a multifactorial and polygenic disorder with viral, environmental and genetic components. HBV genomic variability and a number of conventional risk factors, including age, gender, concurrent infection with hepatitis C virus, hepatitis D virus and human immune deficiency virus, are clearly the important factors contributing to the incidence of persistent HBV infection[1-4]. However, segregation analysis and twin studies strongly support the role of host genetic components in determining the chronicity of HBV infection[5,6]. A known and unknown number of identified or unidentified genes are likely to modify the susceptibility to persistent HBV infection[7-10]. Single nucleotide polymorphism (SNP) is currently believed to be a powerful tool for identifying genetic susceptibilities to common complex diseases[11,12].

The intranuclear architectural protein termed high mobility group box chromosomal protein 1 (HMGB1) has recently been identified as a potent proinflammatory mediator when passively released to extracellular by necrotic cells, as opposed to apoptotic cells that will induce inflammation[13,14]. Furthermore, HMGB1 can also be actively secreted by stimulated macrophages or monocytes[15-17]. Active secretion from living inflammatory cells and passive release from necrotic cells implicate that HMGB1 may play a central role in proinflammatory reactions. It is well known that HBV infection is closely related with cytokines. Polymorphisms of cytokine gene, such as human leukocyte antigen, estrogen receptor alpha (ESR1), have been reported to be associated with HBV infection[18-21]. However, so far there has been no report on the association between HMGB1 gene and HBV infection. We conducted a hospital-based case-control study including more than one thousand subjects with HBV infection to characterize the relationship between HMGB1 gene polymorphism and HBV infection.

HMGB1 is a nuclear DNA-binding protein, which also functions as a pleiotropic cytokine, implicated in the pathology of several different immune-mediated diseases. The human HMGB1 gene is located on chromosome 13. Kornblit et al[23] firstly elaborated six polymorphisms and four mutations identified in the HMGB1 gene, located in -1615A/G, 982C/T, 3814C/G, 1779T/G, -196C/A, 1808C/G, 4519_4521delGAT, -1377delA,1747delT, 1888insT, respectively. In other studies, several associations have been observed, revealing the importance of the genetic variation in the HMGB1 gene. In their report, the -1377delA^A/- genotype or the -1377delA^-/- genotype showed a significant association with delayed mortality, independent of age and number of the systemic inflammatory response syndrome (SIRS) criteria[24]. Subsequent estimation revealed that several polymorphisms have a potential regulatory impact on HMGB1 transcription. Genetically determined risk factors associated with early and late mortality and death due to infection have been identified, explaining some of the inherited risks in this heterogeneous patient population. Associations between genetic variation and disease severity parameters are also established. Studies of association between HMGB1 polymorphisms and disease have been also reported with allogeneic hematopoietic cell transplantation (HCT)[25]. Patient homozygosity or heterozygosity for the-1377delA minor allele is associated with increased risk of relapse and increased relapse-related mortality. Furthermore, patient homozygosity for the 3814C/G minor allele is associated with increased overall survival and progression-free survival. Patient carriage of the 2351insT minor allele can reduce the risk from grade II to IV acute graft-versus-host disease whereas donor homozygosity is associated with chronic acute graft-versus-host disease. These findings suggest that the inherited variation in HMGB1 is associated with outcome after allogeneic HCT following myeloablative conditioning regimens. Zeng et al[26] found that three SNPs act as tag SNPs for the entire HMGB1 gene in multiple organ dysfunction syndromes. The rs2249825 and the haplotype TCG can be used as relevant risk estimate for the development of sepsis in patients with major trauma.

As is well known, the susceptibility of HBV infection is closely related to the variation of some important genes. Deng et al[27] have demonstrated that polymorphisms at the ESR1 gene locus are associated with persistent HBV infection. Subsequently, Yan et al[18] have also demonstrated an association between cis-acting regulatory variation of the ESR1 gene and hepatitis B virus-related liver cirrhosis. Some important variations of cytokine gene also influenced the susceptibility to HBV infection. Deng et al[28] have found that the novel regulatory polymorphism G-201A in the promoter of interferon gamma-inducible protein of 10 kilodaltons (IP-10, CXCL10) gene might be a part of the genetic variation underlying the susceptibility of individuals to disease progression of chronic HBV infection. In another study, Yan et al[29] demonstrated that the -592C allele and the -1082A-819C-592C haplotype in the IL-10 gene promoter were associated with an increased susceptibility to acute liver failure in HBV carriers.

Nevertheless, there are few reports about the association between HMGB1 gene and HBV infection, especially reports about the association between HMGB1 polymorphisms and HBV infection. In this study, we used the current recommendations of human genome SNPs described at http://www.ncbi.nlm.gov/SNP under accession number NT024524. The higher allele variation frequency was selected in position 1176 G/C, the intron 4 of HMGB1 gene. There has been no report about this SNP up to date. We genotyped the polymorphisms of 1495 cases, including AsC, CHB, SHB, LC and HCC. The distribution of HMGB1 1176G/C genotypes in studied sample of unrelated men and women from the referral center were in Hardy-Weinberg equilibrium (P > 0.01), so it is important to consider whether our studied sample could be representative. Yan et al[18] and Deng et al[28] had scanned the polymorphisms on the same cohort. Because differences in age or sex existed in the studied subgroups, we detected the association by logistic regression between observed SNP and HBV infected subgroups, and the age and sex factors were considered. As a result, there was statistically significant evidence of association. The fraction calculated by relative risk indicated that HMGB1 1176G/G genotype was more susceptible to CHB, LC and SHB than 1176C/C and 1176G/C genotype. In other words, the patients with 1176G/G genotype of HMGB1 gene are more likely to have a progressive status in HBV infection. The results suggest that allele 1176G is closely related to the ponderance of disease. These findings underscore a potentially important role of HMGB1 in influencing the development of HBV infection.

In another study, we had successfully cloned and analyzed 154 bp nucleotides in intron 4 near the fourth exon-intron boundary, and found that the region contained sequences 1176 G/C polymorphism characteristic of an enhancer using PGL3 reporter gene systems. We demonstrated that the SNP 1176 G/C could affect the function. Furthermore, this activity was enhanced by the SNP: G→C change in position 1176，providing the basis for molecular investigations of the HMGB1 gene in HBV infection. Subsequent reports would focus on this investigation.

In conclusion, our results showed that the HMGB1 1176G/G genotype was related to the outcomes of hepatitis B infection, and patients with 1176G/G genotype of HMGB1 gene are more likely to have a progressive status in HBV infection. The subjects bearing 1176G/G genotype have an increased risk of susceptibility to CHB, LC and SHB compared with those bearing at least one 1176C allele. However, further work is needed to validate our results, and clarify more potential functions of human HMGB1 gene.