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Canada Communicable Disease Report

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Volume: 27S3 • September 2001

Viral Hepatitis and Emerging Bloodborne Pathogens in Canada

Hepatitis B Viral Mutants and Their Relevance to the Health Care System

Gerald Y. Minuk, Antonio Giulivi


Over the past 10 years an increasing number of mutations in the HBV genome have been described. Although the majority of these mutations appear to be "silent" or not clinically relevant, some have been described in association with evasion of host immunologic surveillance mechanisms (S escape mutants), increased severity of disease (pre-core, core promoter, and core mutations), resistance to antiviral agents (DNA polymerase mutations), and hepatocellular carcinogenesis (X mutants). This review describes both the molecular events and their clinical consequences.

S Mutants

The target of the host's humoral response to HBV is the hydrophilic region of the HBsAg between amino acid residues 100 and 160. Thus, mutation(s) in this region would afford HBV variants a distinct survival advantage. One such mutation was first described in an Italian child who developed HBV despite vaccination and HBIg given at birth(1). The mutation resulted in a glycine (G) to arginine (A) switch at amino acid 145. Other common mutations that have been described in this region include Asp-144-Ala, Met-133-Leu, Gln -129 - His and ILe/Thr - 126 - Ala(2). HBsAg or S mutations have now been documented in many areas of the world but are most common in Asian infants (2% to 3% of vaccine recipients)(2). High maternal viral loads and mutations elsewhere in the mother's HBV S gene appear to increase the risk of S mutations occurring in the offspring(3). The same mutations also occur in liver transplant recipients receiving HBIg(2). Less frequently, they develop spontaneously during the course of a chronic HBV infection(4,5).

Some of the S mutations have been described in association with other clinical events. For example, the Thr-126-Ala mutation has been identified in vaccinated infants who subsequently developed fulminant hepatic failure(6). Of more diagnostic concern are nucleotide insertions in the area of amino acids 121-124, which can result in false-negative HBsAg testing and thereby represent a risk to the health of the undiagnosed patient and the safety of the blood transfusion system(4-7).

Currently, the clinical concerns associated with S mutants lie in four principal areas:

  • Although uncommon, their failure to be detected by commercially available tests raises the possibility that carriers could enter the blood donor population(8). In this regard, it is important to note that in greater than 95% of cases, antibody to hepatitis B core antigen (anti-HBc) is strongly positive(9). Other diagnostic tests that can also serve to document HBV infection in this setting are HBV-DNA and hepatitis B e antigen (HbeAg)(9).
  • Despite encouraging results in vaccinated chimpanzees, HBIg and HBV vaccination do not protect humans from S mutant infections(10).
  • Like other forms of HBV, S mutants have the capacity to induce both acute and chronic liver disease as well as HCC(11,12).
  • Transmission can occur by both horizontal and vertical routes(9).

Pre-core and core promoter mutants

Because a large region of the pre-core/core ORF is not overlapped by another ORF, more mutations of the viral genome are tolerated in this region. The most common and extensively studied of these is the G to A mutation at nucleotide 1896 of the pre-core region. This mutation transforms codon 23 from TGG to a TAG stop codon, which terminates transcription at this site and thereby abrogates the synthesis of HBeAg(13,14). The same mutation also affords the viral genome increased stability, as the 1896 nucleotide site that is now occupied by A binds more avidly to the corresponding T nucleotide at position 1858, resulting in enhanced stability, pregenomic encapsulation and initiation of DNA synthesis(15). Presumably, this feature has contributed to pre-core mutant infections being very, if not the most, common form of HBV in the Mediterranean, Africa, Southern Europe and Asia (50% to 60% of HBV carriers)(16,17). Although common in these areas, precore mutants are less prevalent in North America and Northern Europe (10% to 50%), where the HBV genotype (type A) has a C rather than a T nucleotide at position 1858 required to stabilize the A base at the corresponding 1896 site(15,16).

Although initially described in association with histologically active liver disease, including fulminant hepatic failure, the pre-core mutant has more recently been identified in chronic HBV carriers without biochemical or histologic evidence of liver disease(16). Indeed, in a recent population-based study of Canadian Inuit, none of approximately 35 pre-core mutant carriers had clinical or biochemical evidence of liver disease(17). Moreover, there are data indicating that reactivation of disease is not associated with the emergence of pre-core mutants, and wild type virus is associated more with inflammation and fibrosis than pre-core mutants(18). Finally, HBV-DNA levels are not increased in patients with pre-core mutant infections when compared with wild type infection(19,20). Thus, in itself the mutation is not uniformly pathogenic. These findings have led to searches for co-mutations that might explain the more virulent forms of pre-core infections originally described.

The status of the 1858 nucleotide site may be clinically important in its own right, as studies have demonstrated that patients without 1896 mutations but a T rather than C nucleotide at 1858 have greater histologic activity/inflammation on liver biopsy(16,21). On the other hand, those with 1896 mutations and T rather than C nucleotides at 1858 have more benign histologic findings(16). G to A mutations at nucleotides 1898 and 1899 have also been reported to be associated with the severity of liver disease(13,22).

Other mutations that might explain the increased pathogenicity in patients with (or without) pre-core mutations include point mutations and short deletions or insertions in the core promoter region (nucleotides 1634-1782), which not only limit transcription of pre-core mRNA and thereby HBeAg synthesis but also enhance transcription of core-mRNA and, with it, HBcAg and polymerase enzyme synthesis(23). The most frequently described mutations in this region are A to T nucleotide substitutions at 1762 and G to A at 1764. The frequency of mutations at these sites correlates with disease activity, rates of progression and perhaps hepatocarcinogenesis(24-27). This increased pathogenicity is likely related to decreased synthesis of the immunotolerogen HBeAg and increased core protein synthesis and viral replication. However, not all studies have identified an association between 1762/1764 mutations and severity of liver disease(28). Whether pre-core and/or core promoter mutations predispose to a rapidly progressive fibrosing, cholestatic form of liver disease in the post liver transplant period remains unclear(29-31).

In terms of therapeutic implications, initial reports suggested that if greater than 20% of the viral population within a patient consists of pre-core mutants, then a poor response to interferon therapy can be expected(32). On the other hand, more recent data suggest that a high prevalence of pre-core mutants results in earlier responses to interferon therapy(33). What is clear is that relapses are more common in patients with pre-core mutations when interferon (or nucleoside analogue) therapy is withdrawn, but once again this may be related to associated mutations in the core promoter or core rather than pre-core region(34,35). Indeed, the importance of the core promoter in predicting the response to interferon therapy has recently been emphasized(36).

HBcAg mutants

The most immunodominant epitopes of HBcAg are between amino acids 50-69 and 61-85(37). Mutations in this region have been described in HBV patients with chronic active hepatitis but not during the immune tolerant phase of the infection(35,38). Deletions in the core region have also been described and shown to result in decreased cytotoxic T cell responses and viral replication(39). Similar deletions may play a role in converting an immune tolerant to an immune intolerant state and in progression of acute to chronic HBV infection(40). If the mutations, which likely occur as a result of B and T cell pressure on core antigen, are present prior to interferon therapy, response to treatment is less likely(36,38).

DNA polymerase mutants

Mutations in the catalytic domain of DNA-polymerase (DNA-P), the enzyme responsible for viral replication, tend not to occur naturally but have been described in association with nucleoside analogue therapy(41). In the case of lamivudine, the first nucleoside analogue licensed for the treatment of HBV, a Met-552-ILE or Met - 552 - Val mutation was described in the conserved Tyr-Met-Asp-Asp' (YMDD) motif that is part of the active site (domain C) of the reverse transcriptase(42). Famciclovir, another nucleoside analogue with anti-HBV properties, can induce Val-521-Leu and Leu- 528 - Met mutations within the B domain of DNA-P(43). These mutations decrease but do not eliminate the affinity of nucleoside analogues for the DNA-P enzyme. As a result, viral replication increases but not to levels documented before treatment was initiated(44-46). Because the mutant virus is replicatively compromised in the vital region of DNA-P activity, wild type virus returns on cessation of drug therapy(47,48).

Of interest is that the 'a' determinant of HBsAg is located in the variable linker region between the A (410-426) and B (498-528) domains of DNA-P. Thus, although YMDD mutations could result in HBsAg changes, they are unlikely to do so in the critical antigenic site of HBsAg. Nonetheless, patients with both YMDD and S escape mutations have been described(49).

HBX mutations

Data are only just emerging on mutations in the X ORF. Preliminary findings describe a novel class of HBX mutants in Asian patients with HCC(50). These mutations were found to negate X- induced inhibition of clonal outgrowth and increased apoptosis. Thus, X - mutants increased clonal outgrowth and decreased apoptosis, suggesting a possible carcinogenic role. Although this is intriguing, it should be noted that the HCC tissue sources for the HBX mutations detected also contained HBsAg mutations, which may have been relevant to HCC development(51).

Summary

Mutations have been described in all four ORFs of the hepatitis B virus. From a clinical perspective, the S escape mutant is the most worrisome, because in the absence of surveillance systems and/or a high index of suspicion the diagnosis can be difficult to establish. Undiagnosed cases can progress to liver failure and HCC. Transmission to others, including transmission via the blood transfusion route, might also occur. Fortunately, the prevalence of this mutation appears to be low and is not increasing, despite the widespread application of universal vaccination.

Pre-core mutant infections are less of a clinical concern than originally anticipated. Although they are associated with fulminant and active chronic hepatitis, that association is not universal and appears to be linked to coexisting mutations in the basic core promoter or core regions of the genome. Thus, isolated pre-core mutations more likely reflect the natural evolution of HBV infection from an active to relatively inactive replicative state.

Mutations in the basic core promoter region are worrisome, but it is hoped that their location in terms of overlap with other ORFs will render them relatively infrequent. If confirmed, preliminary findings that mutations to the X gene render the X protein more carcinogenic are disconcerting. Nonetheless, some consolation can be derived from the possibility of exploiting this finding for diagnostic and prognostic purposes in chronic HBV carriers.

References

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