Interleukin-10 promoter polymorphism predicts initial response of chronic hepatitis B to interferon alfa

We retrospectively enrolled 52 Chinese Han patients with chronic hepatitis B from our outpatients clinics at Fuzhou general hospital, between February 2007 and December 2008. There were 28 non-responders (NR) to IFN treatment with a mean age of 32 years and 24 sustained responders (SR) with a mean age of 35 years. Males outnumbered females (M:F/40:16). All patients' blood samples were hepatitis B virus surface antigen (HBsAg) positive and HBeAg positive and with an elevated ALT of at least 2-fold higher than the upper limits of normal for 6 months. ALTs of SR group and NR group were 180.3 ± 54.5 U/L and 197.2 ± 75.5 U/L respectively. ALT was no significant difference between the SR and NR (P = 0.354). Log₁₀HBV DNAs of the two groups were 6.06 ± 8.3 copies/ml and 6.3 ± 8.2 copies/ml respectively. It was no significant difference between the two groups (P = 0.284). Patients were excluded from receiving IFN-αtherapy if they had any of the following criteria: neutrophil count < 1,500 cells/mm³, Hgb < 110 g/L in women or 120 g/L in men, or platelet count <90 cells/L, history of poorly controlled thyroid disease, and serum creatinine level >1.5 times the upper limit of normal at screening. Eligible patients received IFN-α(2b) at a dosage of 5 million units (MU) 3 times per week for 6 months and were subsequently followed for treatment response via clinical, biochemical, and serologic markers for more than 1 year. The definition of sustained SR to IFN-αtreatment for chronic hepatitis B disease included patients with HBeAg(+) to HBeAg(-) conversion and HBVDNA level <1000 copies/ml after treatment for at least 1 year after follow-up. NR were those with persistent or relapsed HBeAg(+) and HBVDNA level >1000 copies/ml during the follow-up period. Patients coinfected with hepatitis C or D were excluded from the study. In addition, 48 healthy volunteers (31 men and 17 women, a mean age of 33 years), were enrolled as a control group. Informed consent was obtained from each patient, and the study protocol was approved by the Fuzhou general Hospital Ethics Committee.

Genomic DNA was extracted from a 5 ml sample of whole blood collected into EDTA. Extraction was performed using a commercial kit (Omega, USA) according to the manufacturer's instructions.

The 3 biallelic IL-10 promoter polymorphisms were detected by PCR using primers that amplified a short fragment of DNA containing the polymorphism (Table 1).

Amplification of the -592 fragment was performed in a volume of 25 μL containing 250 ng of template DNA, 10 mmol/L Tris-HCL (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂ , 0.8 mmol/L deoxyribonucleotides, 0.5 μmol/L of each primer, and 0.6 U AmpliTaq DNA Polymerase (Takara, DaLian, China). The parameters for amplification of the -819 fragment were the same except that a final concentration of 2 mmol/L MgCl₂ was used. Amplification of the -1082 polymorphism was performed using the TakaraTaq kit (Takara, DaLian, China), and Q-Solution was included in the PCR reaction mix. The parameters for thermocycling were as follows: denaturation at 94°C for 2 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds; annealing at 56°C for 30 seconds; and extension at 72°C for 4 minutes 30 seconds. This was followed by final extension at 72°C for 5 minutes. Identification of the 2 alleles at each polymorphic site was performed by incubating PCR product with a restriction enzyme chosen to cut 1 of the 2 alleles (Table1), followed by electrophoresis on agarose gels (3%) (Figure 1 and Figure 2), All samples were amplified and digested in parallel with 2 samples of known genotype and water. IL-10 -1082 polymorphism performed using direct sequencing because fragment of alleles was not identified clearly after restriction enzyme digestion(Figure 3).

Three biallelic polymorphisms and genotype/haplotype frequencies in the IL-10 gene promoter were analyzed (Table 2). The majority of HBV carriers as well as healthy volunteers had A allele at position -1082 and T allele at position -819 in the IL-10 gene promoter. In addition, there was no significant difference in the frequencies of alleles or genotype/haplotype in the IL-10 gene promoter between HBV carriers and healthy volunteers.

Fifty-two patients received treatment with IFN-α (5 million units, 3 times weekly) for 6 month. Twenty-eight patients were classified as "nonresponders" as a result of persistent or relapsed HBeAg(+) and HBVDNA level >1000 copies/ml during the follow-up. HBeAg(+) to HBeAg(-) conversion and HBVDNA level <1000 copies/ml after treatment for at least 1 year after follow-up was seen in Twenty-four patients ("responders").

Differences between SR and NR in several IL-10 allele, genotype, and haplotype distributions were observed. The -592A and -819T alleles, along with the exclusively linked -592A/A and -819T/T genotypes (Table 3), were more frequent in SRs than in NRs. These two sites are dimorphic, and reciprocal effects (nonresponse) were also seen with the -592C and -819C alleles. Homozygosity for genotypes -592A/A and -819T/T was more strongly associated with sustained response than heterozygosity. Similarly, inheritance of the haplotype ATA was associated with "responder" status to IFN-α therapy.