The clinical implication of single nucleotide polymorphisms in deoxycytidine kinase in chronic hepatitis B patients treated with lamivudine

Study Population

A total of 127 patients with chronic hepatitis B who visited Severance Hospital between January 2003 and December 2006 were enrolled in this study. They were followed-up for at least 24 months after LAM treatment. Patients were eligible if they met the following entry criteria: they were 18–75 years of age; the presence of serum HBsAg was observed for at least 6 months; they had an elevated serum alanine aminotransferase (ALT) level on two occasions, at least 1 month apart, with an average value of ≥2 times the upper limit of normal (ULN); the presence of serum HBV DNA was documented on two occasions, at least 1 month apart. Additional requirements included a hemoglobin value of ≥10 g/dl, a platelet count of ≥100,000 mm³, a white cell count of ≥4,000 mm³, a polymorphonuclear count of ≥1,500 mm³, and normal renal function with normal serum creatinine levels. Candidates were required to have compensated liver disease with a prothrombin time of less than 4 sec, prolonged over control values, a serum albumin of ≥3.0 g/dl, a total bilirubin of ≤4 mg/dl, and no history of hepatic encephalopathy or bleeding esophageal varices.

Exclusion criteria were as follows: a history of corticosteroid treatment within 6 months of entry; previous therapy with IFN; the presence of antibodies to human immunodeficiency virus (HIV), hepatitis C virus (HCV), or hepatitis D virus (HDV); a history of malignancy or evidence of other forms of liver disease; a history of intravenous drug abuse. Patients with other significant medical or psychiatric problems were also excluded.

Patients were treated with LAM monotherapy for virological response. LAM was given at a dose of 100 mg per day for at least 12 months. Serum HBeAg, anti-HBe, HBV DNA, and ALT were assessed every 3 or 6 months, and whenever necessary during medication and after drug cessation. In addition, peripheral blood samples from 24 healthy volunteers and 24 patients with CHB were used as control samples. To overcome selection bias, healthy and CHB volunteers were matched for age and sex by simple random sampling (SAS 9.13 software). Two groups did not show any differences in the age and sex. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Review Board of Severance Hospital, and written informed consent was obtained from each patient.

Definitions

Patients without a decline in serum HBV DNA by ≥1 log₁₀ IU/ml after the first 6 months of therapy were classified as “primary non-response (NR).” Virological breakthrough (VB) was defined as patients with an increase in serum HBV DNA of >1 log₁₀ IU/ml (10-fold) above nadir after achieving virological response during continued treatment [Lok et al., 2007]. HBeAg seroclearance was defined as loss of detectable levels of HBeAg and HBV DNA in serum.

Patients who achieved virological response with undetectable HBV DNA for at least 48 weeks were classified as good drug responders. In contrast, patients with NR or VB were considered poor drug responders.

Assay Methodology

Commercially available enzyme-linked immunoadsorbent assays (ELISAs) were used for the detection of serum HBsAg, anti-HBs, anti-HBc, HBeAg, and anti-HBe (Abbott Laboratories, North Chicago, IL). Serum HBV DNA concentration was determined by a commercially available quantitative assay (Lightcycler 2.0, Roche Diagnostics, Branchburg, NJ). The sensitivity of the assay is approximately 300 copies/ml.

Sequence Analysis of the dCK Gene

Peripheral blood (10 ml) was collected from each individual. Genomic DNA from peripheral blood stored samples was isolated using a NucleoGen genomic DNA isolation kit (NucleoGen, Seoul, Korea) according to the manufacturer's instructions, and extracted DNA was subsequently stored at 4°C until analysis. Thirteen primer sets were used to amplify all seven DCK exons and the core promoter region for a total sequence length of 2862 bp (Supplemental Table I). Several fragments of the dCK gene were amplified with forward primers and reverse primers in a final volume of 25 μl containing 50–100 ng of genomic DNA, 20 pmol of each primer, 0.2 mM dNTPs, 2 U of Taq polymerase (iNtRON Biotechnology, Seoul, Korea) and manufacturer-supplied polymerase chain reaction (PCR) buffer. Amplification was performed in a GeneAmp PCR system 9700 (Perkin Elmer Corp., Norwalk, CT). PCR conditions were 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 50 sec, with final extension at 72°C for 7 min. PCR products were identified by electrophoresis, then purified with a PCR purification kit (iNtRON biotechnology, Seoul, Korea) and analyzed by direct sequencing (Bionex Co. Ltd, Seoul, Korea). Twenty-four healthy volunteers and 24 patients with CHB were screened for SNPs in the dCK gene.

Statistical Analysis

All data are expressed as means ± standard deviations (SDs). Student's t-test was used to compare mean age, pretreatment ALT level, and HBV DNA level among subject groups. χ² test or Fisher's exact test was used to compare the sex ratio and frequencies of novel and known dCK genotypes. Based on gene frequencies, predicted phenotype frequencies were calculated according to the Hardy–Weinberg equation and compared with the observed frequencies using the χ² test. Multivariate analyses were performed using logistic regression to identify factors associated with good response, and HBeAg seroclearance. Cumulative HBeAg seroclearance rates were estimated using the Kaplan–Meier method and compared by log-rank test. All statistical analyses were performed using the Statistical Package for Social Science (SPSS Inc., Chicago, IL) version 12.0. A P value of less than 0.05 was considered significant.

SNPs in the dCK Gene in Korean Subjects

All 5480 bp of the dCK gene were analyzed in a group of 24 healthy volunteers and 24 patients with CHB. Six SNPs were detected in the 5′ regulatory region. They were −2052C/A, −1431A/G, −1359A/G, −1329C/T, −360C/G, and −201C/T with allele frequencies of 0.979:0.021, 1:0, 1:0, 0.979:0.021, 0.74:0.26, and 0.734:0.266, respectively. We also detected 10 SNPs, 12 SNPs, and 1 SNP in introns, exons, and the 3′ UTR, respectively. Among these, 23 SNPs had been previous reported in public SNP databases (NCBI reference number, NT_006216.14). Six novel dCK SNPs were found (−2052C/A, IVS3 − 46G/del, IVS4 + 40G/T, IVS5 + 39T/C, IVS5 − 72A/T, and 966–975T₁₀/T₁₁) (Supplemental Table II). Two promoter SNPs of −360C/G and −201C/T have been reported previously in Caucasian, Japanese, and Chinese subjects. These two SNPs were in full linkage disequilibrium and found at an allele frequency of 26%. The allele frequencies of these two SNPs were more frequent than what has been reported in Caucasian, Japanese, and Chinese subjects (26% vs. 2%, 13.1% and 15.6%). Ten SNPs were found in introns (IVS1 + 37G/C, IVS1 − 174G/A, IVS1 − 110T/G, IVS2 + 114G/A, IVS3 − 46G/del, IVS3 − 45T/del, IVS4 + 40G/T, IVS5 + 39T/C, IVS5 − 72A/T, and IVS7 + 41A/T), of which IVS1 − 174G/A, IVS2 + 114G/A, and IVS7 + 41A/T were found at allele frequencies of 3%. Twelve SNPs were found in exons (A100A, P122S, 948T/C, 966–975T₁₀/T₁₁, 1796A/G, 1797A/G, 1827del/A, 1904del/C, 1952A/C, 2031A/G, 2105A/T, and 2254TT/del). One SNP was nonsynonymous and resulted in the substitution of proline at amino acid position 122 by serine (364C/T in exon 3, P122S). The allele frequencies of 948T/C and 966–975T₁₀/T₁₁ were 2%. The SNP located in the 3′ untranslated region of the gene was a synonymous SNP, and this SNP had an allele frequency of 2%. Linkage analysis showed significant disequilibrium between −360C/G and −201C/T (rho-square 1.0, P value <10⁻⁵).

DCK SNP Genotypes and Lamivudine Responses

Three dCK SNP genotype patterns (−360CC/−201CC, −360CG/−201CT, and −360GG/−201TT) were found among 127 CHB patients. Allele frequency analysis of −201C/T and −360C/G in 127 CHB patients as well as peripheral blood samples from 24 healthy volunteers and 24 patients with CHB revealed that these alleles were in Hardy–Weinberg equilibrium (HW P = 0.561). Sequence analysis exhibited that −360C/−201C and −360G/−201T were in complete linkage disequilibrium.

Patients did not show any differences in the distributions of dCK genotypes according to age (P = 0.551), and sex (P = 0.201). We next analyzed the relationship between dCK SNP genotypes and treatment responses. The 127 CHB patients were divided into two groups according to their responses to LAM monotherapy. Forty-seven patients achieved a good drug response, namely a virological response with undetectable HBV DNA for at least 12 months. Eighty patients showed a poor drug response, including 21 patients who did not have a decline in serum HBV DNA by ≥1 log₁₀ IU/ml after the first 6 months of therapy and 59 patients whose serum HBV DNA increased by >1 log₁₀ IU/ml (10-fold) above nadir after achieving virological response at 12 months. No obvious differences in clinical data such as sex, age, baseline serum ALT level, or HBV DNA level were found (Table I). There was no statistically significant difference between the two groups in terms of the distributions of SNP genotypes or allele frequencies (Table II). Multivariate analysis was performed using one categorized value (dCK SNP genotype) and three continuous values that were previously reported to be predictive of a good drug response. Age and baseline serum HBV DNA level were independent predictive factors of a good drug response (Table III).

DCK SNP Genotypes and HBeAg Seroclearance

Among 127 patients, 13 patients with HBeAg-negative CHB were excluded. Three dCK SNP genotype patterns were found among the 114 HBeAg-positive CHB patients. Allele frequency analysis of −201C/T and −360C/G in the 114 HBeAg-positive CHB patients, as well as in peripheral blood samples from 24 healthy volunteers and 24 patients with CHB, revealed that these alleles were in Hardy–Weinberg equilibrium (P = 0.605).

The 114 HBeAg-positive CHB patients were divided into two groups according to HBeAg seroclearance during LAM monotherapy. Forty-six patients achieved HBeAg seroclearance during the treatment period, whereas another 68 patients did not achieve HBeAg seroclearance. There were no obvious differences in sex, age, baseline serum ALT, or HBV DNA level among the two groups (Supplemental Table III). Interestingly, there was significant difference in the allele frequency of −360G/−201T between HBeAg seroclearance group and HBeAg non-seroclearance group (P = 0.045). (Table IV). However, multivariate analysis revealed that age was the only independent predictive factor for HBeAg seroclearance (Table V). Follow-up data concerning cumulative HBeAg seroclearance rates were available for 114 HBeAg-positive CHB patients. With a median follow-up of 51 (24–103) months, the 3-year cumulative HBeAg seroclearance rate for the −360CG/−201CT (N = 30) and −360GG/−201TT (N = 2) groups was 55.5%, whereas that for the −360CC/−201CC (N = 82) group was 31.2%. Cumulative HBeAg seroclearance rates of the two groups are presented in Figure 1. Although there were slightly more genotypes containing the −360G/−201T haplotype in the seroclearance group than the non-seroclearance group, there was no significant difference between the two groups (P = 0.087).