A recent major breakthrough in hepatology was the finding that variation in the region of interleukin-28B (IL28B), the gene coding for the IL28B protein, also known as interferon (IFN)-λ3, is highly predictive of the response to treatment with pegylated INF alpha (Peg-IFN-α) and ribavirin (RBV) in chronic hepatitis C (CHC).1-3 Subsequent work also confirmed an association between host IL28B genotype and spontaneous viral clearance.4,5 In addition, this genetic polymorphism was shown to be, at least partially, responsible for previously unexplained ethnic differences in response rates to CHC therapy: Ge et al. estimate that the IL28B-related single-nucleotide polymorphism (SNP), rs12979860, accounts for more than 50% of the variability between responses to Peg-IFN/RBV treatment among African Americans and Caucasians.1
This unexpected association quickly became a central theme in hepatitis C research. A PubMed search for “IL28B” and “hepatitis C” yields more than 380 publications since 2009. Consequently, patient stratification according to IL28B genotype has become standard practice for hepatitis C clinical trials. Despite very solid statistical evidence for this association, its biological underpinning is unknown: Current knowledge remains descriptive in the absence of experimental data to support a mechanistic explanation.
Why is this so? For comparison, it is instructive to consider another example of pharmacogenomic discovery, also derived from hepatitis C treatment response. A separate genome-wide association study (GWAS) from the same group found genetic variation on chromosome 20 as being highly predictive of RBV-induced hemolytic anemia during hepatitis C treatment.6 They further describe the causal variant negatively regulating the promoter region of the inosine triphosphatase (ITPA) gene. RBV depletes adenosine triphosphate (ATP) in erythrocytes, thereby potentially causing hemolysis. ITP can replace ATP unless it is itself broken down by ITPA. Hence, lack of ITPA protects against hemolysis during RBV treatment and this neatly closes the circle on the GWAS findings. However, evidence for a similar regulatory mechanism underlying the IL28B-related effects is currently lacking. Despite concerted efforts, the causative SNP has not been pinpointed, let alone the responsible biological events identified. This alludes to a more-complex situation than for the ITPA example.
How to proceed in this puzzle? It is useful to recall well-established properties of type III IFNs: They have been shown to be antiviral against several viruses, including hepatitis C virus and HBV, and closely resemble the properties of type I IFNs, such as IFN-α.7,8 Although the two IFN families bind distinct receptor complexes, they do share an intracellular signaling pathway (through Janus kinase/signal transducer and activator of transcription), ultimately inducing similar sets of genes. This raises the question as to why both ligand-receptor systems have been conserved in evolution and if type III IFNs possess—yet hidden—distinctive features that render them nonredundant. The above-named GWAS results may reflect such unique functions.
Given its antiviral properties, it seems compelling to propose that the genotype would either regulate IL28B expression levels (similarly to the ITPA SNP) or lead to protein variants with different cytokine activity (e.g., differential affinities for their receptor). In fact, rs12979860, approximately 3 kb upstream of IL28B, turns out to be a marker SNP for the IL28B protein variant, Lys70Arg.1 However, no differences in gene induction or antiviral effect were detected in hepatoma cells upon treatment with these two variants.9
Does this genotype play a role in diseases other than hepatitis C? In this issue of HEPATOLOGY, Lampertico et al. demonstrate that it may indeed matter in chronic hepatitis B (CHB), for which IFN-α exhibits clinical activity.10 The investigators show that genotype CC (rs12979860), which predicts sustained virological response (SVR) as well as spontaneous clearance in hepatitis C, is significantly associated with hepatitis B surface antigen (HBsAg) seroclearance after treatment with INF in a cohort of CHB patients who were mainly infected with viral genotype D. All 101 patients were hepatitis B e antigen (HBeAg) negative. HBV genotype D and HBeAg negativity have both been found to negatively affect outcome in IFN therapy of CHB and are prevalent in Mediterranean populations. The study patients had completed a course with IFN-α2a, IFN-α2b, or Peg-IFN-α2a and had had regular follow-up exams for 1-17 years (median, 11). Both end-of-treatment response and SVR were significantly higher in genotype CC, compared to pooled genotype CT/TT patients. Although this is a retrospective study with diverse treatment conditions and follow-up periods, the data support a predictive value of the IL28B-associated genotype for treatment outcome for a subgroup of difficult-to-treat CHB patients. However, confirmation of the result in larger cohorts is warranted before IL28B genotyping for the prediction of individual treatment response can be generalized.
It is important to note that studies investigating the association between the IL28B polymorphism and IFN therapy for CHB have yielded conflicting findings (Table 1). There is considerable heterogeneity with respect to study populations, treatment regimens, and outcomes. Similar to Lampertico et al.'s characterization of HBeAg-negative individuals, Sonneveld et al. recently found a significant association between IL28B genotype and Peg-IFN treatment outcome in 205 HBeAg-positive patients with a range of viral genotypes.11 Both studies found genotype CC as a positive predictor of therapeutic response. In contrast, a study in 512 Chinese patients (HBV genotypes B and C) undergoing Peg-IFN-based treatment suggested the opposite effect: The minor (putatively unfavorable) IL28B-related allele was more frequent in the group with SVR.12 Yet another Taiwanese study enrolling 115 HBeAg-positive patients (HBV genotypes B and C) who underwent Peg-IFN therapy did not find significant differences in outcome between host genotypes.13 Similarly, when 95 HBeAg-positive and -negative hepatitis B patients were treated with a combination of Peg-IFN and adefovir in a Dutch study, IL28B genotype was not predictive of virological response as well as HBsAg and HBeAg seroclearance in HBeAg-positive or -negative patients.14
|Reference||Setting||HBeAg||HBV Genotype||Treatment||Conclusion/Association With IL28B Polymorphism|
|Lampertico et al.10||101 patients (Italy)||Negative||92% D||IFN-α or Peg-IFN-α||Homozygosity for major allele predicts HBsAg clearance in HBeAg-negative patients with HBV genotype D (29% versus 13% in nonhomozygous patients; P = 0.039)|
|Sonneveld et al.11||205 patients (11 hospitals in Asia and Europe)||Positive||A/B/C/D||Peg-IFN-α2a or Peg-IFN-α2b ± lamivudine||Homozygosity for major allele predicts HBeAg seroconversion at end of treatment (P < 0.001) and during long term follow-up (P = 0.018)|
|Wu et al.12||512 patients (Han Chinese, Beijing Youan Hospital)||Positive||B/C||Peg-IFN-α2a ± nucleoside analog||Minor allele more frequent in SVRs than nonresponders (8.3% versus 3.9%; P = 0.003)|
|Tseng et al.13||115 patients (five hospitals, Taiwan)||Positive||B/C||Peg-IFN-α2a (for 6-12 months)||No association between IL28B genotype and HBeAg seroconversion at 6 months post-therapy (P = 0.928)|
|de Niet et al.14||95 patients, two centers (The Netherlands)||Positive (46) and negative (49)||No information||48 weeks Peg-IFN-α2a plus adefovir||No association between IL28B genotype and HBeAg seroconversion or HBsAg clearance|
There are several issues to consider in the interpretation of these five, mainly retrospective, studies. Treatment regimens and durations of therapy as well as follow-up data are quite heterogeneous. The heterogeneity of the populations studied and their diverse genetic backgrounds may additionally hamper generalizability of a given set of findings. Overall, at best, these studies suggest that IL28B effects may be limited to certain subgroups, such as HBeAg-negative patients or persons infected with specific HBV genotypes. Hypothetically, IL28B genotype may contribute to the distinct features of infections with different HBV genotypes and contribute to distinct virus-specific immune responses. Indeed, Lampertico et al. propose an IL28B effect specific to viral genotype D infection with HBeAg negativity; the extendability of this finding awaits comparable robust data from other viral genotypes.10 For instance, Sonneveld et al. did not report a subanalysis addressing the association of IL28B genotype with IFN response in specific viral genotypes, likely because of limited patient numbers.11 Thus, extended study of larger, prospective cohorts will be necessary to establish the validity and strength of the IL28B/HBV association.
Additionally, several studies analyzing spontaneous clearance in hepatitis B and human immunodeficiency virus/HBV coinfection, failed to reveal a link with IL28B related genotype.15-18 A GWAS in East Asians demonstrated that genetic variation in the region of HLA-DP on chromosome 6, but not around IL28B, was associated with CHB.19
How do we reconcile these findings? The effect of IL28B genotype in hepatitis B, if present, is clearly not as profound as it is for hepatitis C. The current literature, as discussed above, provides early evidence that the IL28B-associated genotype may indeed play a role in the response of some forms of CHB to IFN treatment. However, a role in the natural course of the disease comparable to the situation in hepatitis C is not readily apparent from currently available data.
A central question arising from the GWAS data is how genetic variation around IL28B affects the response to IFN-α. Does IL28B signaling overcome resistance in cells that have become refractory to IFN-α signaling, thereby complementing IFN-α's action? Does IFN-α induce type III IFNs or vice versa? What cell types are involved? Is direct action of IL28B on hepatocytes key or is rather an indirect effect mediated by a chain of events, such as involving cells of the innate (i.e., dendritic cells, hepatocytes, monocytes, macrophages, or natural killer cells) or adaptive immune system at work?
A better understanding of the evolutionary forces that drove the striking geographically and ethnically distinct allele distribution, making the T allele rare in East Asia and prevalent in regions of sub-Saharan Africa, may also help. It is tempting to speculate that there is a tradeoff or evolutionary cost that comes with the C allele, which we currently would refer to as the “protective” or “favorable” allele because of its beneficial role in hepatitis C. For instance, there may be another, yet-to-be identified infectious disease (one may speculate this to be endemic in tropical Africa) for which the T allele is protective. Further evidence of the role of the genotype in regulating responses to environmental stimuli comes from recent work demonstrating that IL28B alleles may predispose to allergic disease susceptibility in children.20
Further basic and clinical investigation is clearly necessary to extend our knowledge of the type III IFN system and to permit linkage of this important genotype to phenotype in hepatitis B.