core and their clinical implications

Journal of Clinical Virology 39 (2007) 87–93

Hepatitis B virus (HBV) genotypes/subgenotypes in China: Mutations in core promoter and precore/core and their clinical implications Jing Yuan a,b , Boping Zhou a,b , Yasuhito Tanaka c , Fuat Kurbanov c , Etsuro Orito d , Zuojiong Gong b , Liumei Xu a , Jian Lu a , Xiaoling Jiang a , Weizhen Lai a , Masashi Mizokami c,∗ a Shenzhen East Lake Hospital, 518020 Shenzhen, China Department of Infectious Diseases, Renmin Hospital of Wuhan University, Hubei, China c Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Kawasumi, Mizuho, Nagoya 467-8601, Japan Department of Internal Medicine and Molecular Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan b

d

Received 28 July 2006; received in revised form 8 March 2007; accepted 12 March 2007

Abstract Background: The association of hepatitis B virus (HBV) genotypes with clinical course of infection is increasingly recognized. Objectives: In order to investigate the genetic diversity of HBV and its clinical implications, 241 HBV-infected patients including 34 with hepatocellular carcinoma (HCC) were enrolled in this study. Methods: HBV genotyping was performed with an ELISA assay. HBV subgenotypes were determined by PCR-RFLP. HBV core promoter/precore/core mutations were analyzed by direct sequencing. Results: The overall prevalence of HBV/B and C was 65% and 33%, respectively. Among HBV/C, 42% were Cs/C1 and 58% were Ce/C2. The HBV/C1 was only found in the patients originating from Southern China (p = 0.0001). Among HCC patients, HBV/C2 was only found in the elder age group (≥51 years; p < 0.05) and HBV/Ba was associated with young HCC patients (<35 years). Mutations associated with HCC were V1753 and T1762/A1764 (p < 0.01). The prevalence of the V1753 was higher in HBV/C1 strains (p < 0.04), A1898 was only found among HBV/C1 (p = 0.056). T1762/A1764 was frequently demonstrated in both subgenotypes. The T1858 (90%) and A1896 (40%) mutations were most frequent in HBV/C2 (p < 0.008). Conclusions: HBV/C1 and HBV/C2 have distinct geographic distributions in China. V1753 in addition to T1762/A1764 double mutation in the basal core promoter region seems to be associated with HCC development, especially in the patients with HBV/C1. © 2007 Elsevier B.V. All rights reserved. Keywords: HBV; Genotype; Subgenotype; BCP mutation; CP mutation; PC mutation

1. Introduction Based on the divergence of nucleotide sequences exceeding 8% in the entire genome or 4% in the S gene, HBV has been classified into eight genotypes designated by capital letters, A through H (Okamoto et al., 1986). HBV genotypes ∗

Corresponding author. Tel.: +81 52 8538292; fax: +81 52 8420021. E-mail address: [email protected] (M. Mizokami).

1386-6532/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2007.03.005

have distinct geographical distributions and correlate with the severity of liver disease (Kidd-Ljunggren et al., 2002; Miyakawa and Mizokami, 2003). Genotypes B and C are prevalent in Asia and genotype C is associated with a more severe course of liver disease and a higher risk of HCC development than genotype B (Chan et al., 2004; Orito et al., 2001). Furthermore, two subgenotypes of genotype B circulatie in distinct geographical regions; Ba in Asian countries (except Japan) and Bj (found only in Japan) (Sugauchi et

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al., 2002). Clinical differences between the patients infected with the two different subgenotypes of HBV/Ba or HBV/Bj have been demonstrated, indicating a comparatively better prognosis of HBV/Bj-infection in comparison to HBV/Ba (Orito et al., 2005; Sugauchi et al., 2003). Recently, HBV/C has been classified into four subgenotypes (HBV/C1–C4) (Huy et al., 2004; Norder et al., 2004). However, the designation of the two most widely spread subgenotypes is controversial, in our present study we implemented a classification which is consistent with consecutive reports devoted to the clinical characterization of these subgenotypes HBV/C1, i.e. Cs and C2, i.e. Ce (Chan et al., 2005; Tanaka et al., 2005, 2006). Moreover, little is known about the relationship between the HBV/C subgenotypes and clinical outcomes in China. Mutations in the basic core promoter (BCP) of the HBV (T1762/A1764) and precore region (A1896) were previously reported in association with HBe antigen seroconversion (SC) and viral replication. They were often found in the patients with advanced liver disease and hepatocellular carcinoma (HCC) (Baptista et al., 1999; Blackberg and Kidd-Ljunggren, 2003; Kao et al., 2003). In this study, the epidemiological prevalence of HBV/C subgenotypes, enhancer II/core promoter/precore/core gene mutations was investigated. The association between HBV genotypes and clinical outcomes in HBV infected patients was also analyzed.

2. Material and methods 2.1. Patients A total of 241 HBsAg-positive patients were randomly selected in this study (Table 1). All the 241 patients were living in Shenzhen which is a city with over 10 million inhabitants who have immigrated from different geographical parts of China. The prevalence of HBV geno- and subgenotypes in our patient cohort should reflect the geographical distribution and diversity of HBV in China. All the patients were subdivided into four clinical groups. Namely, inactive carriers (IC, n = 66): persistently normal serum ALT level during at least 6 months of follow up, chronic hepatitis B (CHB, n = 73): elevated serum ALT level, liver cirrhosis (LC, n = 68): clinical evidence for cirrhosis revealed by ultrasonography and clinical and laboratory examinations (such as a platelet count of less than 100,000 platelets/mm3 , ascites, esophageal varices, etc.) and hepatocellular carcinoma (HCC, n = 34) diagnosed by abdominal ultrasonography, angiography, computed tomography or magnetic resonance imaging and elevated serum AFP levels (≥400 ng/mL). The patients who were co-infected with hepatitis A, C, D and E viruses or human immunodeficiency virus (HIV) were excluded from this study. All participants provided written informed consent for participation in the study.

2.2. HBV genotyping/subgenotyping Genotypes of HBV in the 241 serum samples were determined by a commercial ELISA kit using epitopes on the pre-S2 region products (Usuda et al., 1999, 2000) HBV genotype EIA, Institute of Immunology, Tokyo, Japan in accordance with the manufacturer’s instructions. Nucleic acids were extracted from 100 ␮L serum using QIAamp DNA Blood Mini Kits (Qiagen Inc., Hilden, Germany). A novel method for the determination of the HBV/C genotype based on a two-round of PCR with hemi-nested primers followed by an enzyme digestion of the amplified PCR products in specific restriction sites for HBV/C1/Cs or C2/Ce was used (Tanaka et al., 2005). Subgenotypes of HBV/B were also determined by a PCR-RFLP method (Sugauchi et al., 2003). 2.3. Amplification and sequencing of the enhancer II/core promoter and precore/core region In order to examine the association between subgenotypes and gene mutations, HBV DNA sequences of HBV/C strains beaming the enhancer II/core promoter/precore and core regions were amplified by PCR with hemi-nested primers, as reported previously (Sugauchi et al., 2001) with slight modifications. The first-round of PCR was performed with a sense primer (IS2-2, 5 -CAT GGA GAC CAC CGT GAA CGC-3 [nt 1607–1627]) and an anti-sense primer (HC24R: 5 -CCT GAG TGC TGT ATG GTG AGG-3 [nt 2072–2052]). The second-round PCR was performed with a sense primer (IS22) and an anti-sense primer (HBV 1917R: 5 -CTC CAC AGT AGC TCC AAA TTC TTT A-3 [nt 1917–1893]). Thereafter, PCR products were directly sequenced with Prism Big Dye (Applied Biosy-stems, Foster City, CA) in the ABI 3100 DNA automated sequencer. Nucleotide sequences of HBV were aligned using CENETYX-MAC and online tools available at the Hepatitis database (http://s2as02.genes.nig.ac.jp). 2.4. Statistical analysis Collected data were analyzed using the χ2 -test, Fisher’s exact test and student’s test, where appropriate. The differences between compared groups with p values ≤0.05 were considered as statistically significant.

3. Results 3.1. Demographical-clinical background Demographic and clinical data of the 241 patients included in the present study are summarized in the Table 1. The IC group was subdivided into two subgroups according to the HBeAg status. Seroconverted (HBeAg-negative) inactive carriers were considered as a population with good prognosis with respect to the clinical outcome (development of LC

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Table 1 Clinical features of the studied 241 patients with inactive carriers (IC), chronic hepatitis B (CHB), Liver cirrhosis (LC) and hepatocellular carcinoma (HCC) IC

HBV/Ba HBV/Ca HBV/C1a HBV/C2a Birth place (North/South) Gender (M/F) Age (years)c HBeAg (positive)a HBVDNA (logcps/mL)c ALT (IU/L)c AST (IU/L)c T.Bil (umol/L)c Albumin (g/L)c Platelet (×109 /L)c AFP (≥200 ng/mL)a Hyaluronic acid (ng/mL)c a b c d e f g

HBeAg− (n = 32)

HBeAg+ (n = 34)

23 (71.9) 6 (18.8) 1 (16.7) 5 (83.3) 4/28 17/15 30.94 ± 9.37 0 (0) 3.3 ± 1.43 27.8 ± 4.4 26.2 ± 3.3 13.6 ± 2.6 37.8 ± 1.6 128.6 ± 10.2 0 50.0 ± 8.7

25 (73.5) 9 (26.5) 4 (44.4) 5 (55.6) 5/29 18/16 24.15 ± 6.87 34 (100) 7.72 ± 0.71 27.4 ± 4.2 26.1 ± 3.5 13.3 ± 2.8 38.2 ± 1.4 132.2 ± 11.6 0 51.2 ± 7.5

CHB (n = 73)

LC (n = 68)

HCC (n = 34)

p value

43 (58.9) 30 (41.1) 11 (36.7) 19 (63.3) 6/67 62/11 34.12 ± 7.7 33 (45.2) 5.46 ± 1.95 83.8 ± 89.3 52.4 ± 44.4 17.5 ± 7.2 38.1 ± 1.2 131.8 ± 12.3 0 119.5 ± 102.5

45 (66.2) 21 (30.9) 8 (38.1) 13 (61.9) 8/60 55/13 46.19 ± 12.27 19 (27.9) 4.70 ± 1.70 142.8 ± 225.4 123.5 ± 151.6 52.6 ± 70.0 34.8 ± 6.3 76.3 ± 39.7 8 (11.8) 433.8 ± 276.9

21 (61.8) 13 (38.2) 9 (69.2) 4 (30.8) 4/30 31/3 49.53 ± 12.86 2 (5.9) 4.55 ± 1.70 132.3 ± 151.6 126.4 ± 86.8 74.4 ± 101.7 36.1 ± 4.9 122.7 ± 62.7 21 (61.8) 488.2 ± 409.7

NS NS NS NS NS <0.05b <0.05d <0.05e <0.05e <0.05b <0.05b <0.05b NS <0.05f <0.001g <0.05d

Data shown as number and percent of total; n (%). IC vs. LC, CHB and HCC. Data shown as mean value ± S.D. LC and HCC vs. IC and CHB. IC (HBeAg+) vs. IC (HBeAg−), CHB, LC, and HCC; CHB vs. LC and HCC. LC vs. IC, CHB and HCC. HCC vs. IC,CHB and LC.

and/or HCC) and were thus taken as an independent control group (Table 1). Sixty-five percent of the patients (157/241) in this study were found to be infected with HBV/B, while 33% (79/241) were HBV/C carriers. The remaining 2% of the cases (5/241) were unclassified. Using previously reported PCR-RFLP method (Sugauchi et al., 2003), all HBV/B strains were further classified into subgenotype Ba. Using another previously described PCR-RFLP method for HBV/C subgenotyping (Tanaka et al., 2005), 42% of HBV/C cases (33/79) were identified as HBV/C1 and 58% of the cases (46/79) as HBV/C2. The distribution of each of the HBV genotype/subgenotypes did not vary significantly among IC, CHB, LC and HCC groups. The origin of the patients (place of birth) also did not show any difference among the groups. The male/female ratio varied significantly from the lowest among IC (1.1) to the highest among HCC (10.3). As expected, the patients in the LC and the HCC groups were significantly elder than those in the IC and CHB groups (p < 0.05). HBeAg positivity was highest in IC (52%) and lowest among HCC (6%) (p < 0.05). Although the mean age (46.2 and 49.5 years, respectively) as well as the mean HBV DNA levels (4.7 and 4.6 log copies/mL, respectively) in LC and HCC groups were nearly similar, the HBeAg positivity rate was significantly lower in the HCC group (6% versus 28%, p = 0.009). The platelet counts were significantly lower in the patients with LC than in those in the IC, CHB or HCC groups (p < 0.05). 3.2. Geographical distribution of HBV/C subgenotypes In order to analyze the geographical distribution of the genetic variants of HBV in China, the patients were subdi-

vided according to their origin. The Yangtze River flows from its source in Qinghai Province in Western China, eastwards into the East China Sea. The Yangtze River is traditionally considered as a dividing line between Northern and Southern in China. Significant differences in distributions of the HBV genotypes/subgenotypes were thereby observed in the patients from Northern and Southern parts of China (Fig. 1). In North China, 78% (21/27) of the studied HBsAg carriers were infected with subgenotype C2. Whereas, in Southern China, the prevalence of genotype B (Ba) was the highest with 73% (156/214). It was the predominant subgenotype in Southern China (p < 0.001). HBV/C1 was exclusively found in the carriers from Southern China, where this subgenotype had a prevalence which was quite similar to that of C2 (15% and 12%, respectively).

Fig. 1. Geographical distributions of HBV genotypes/subgenotypes in different parts of China. The prevalences of the subgenotypes were indicated in percent of total examined in corresponding geographical parts. Northern group includes the patients immigrated from the provinces of Shandong, Heilongjiang, Shanxi, Henan, Jilin, Shaanxi, Neimonggu, Gansu, Liaoning province. Southern group includes the patients immigrated from the provinces of Guangdong, Guangxi, Hainan, Hubei, Hunan, Fujian, Sichuan, Zhejiang, Jiangxi, Shanghai province.

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3.3. Clinical characteristics of HBV/Ba, C1 and C2 subgenotypes Clinical characteristics of the patients in IC, CHB, LC and HCC groups were compared according to the genotype/subgenotype carriage (Table 2). No significant differences were observed between HBV/C1 and C2 with respect to gender, age, ALT and viraemia in all the clinical groups. Among LC patients; total bilirubin levels were the lowest among Ba infections and the highest in C2 carriers. The hyaluronic acid levels were significantly lower in the Ba infected patients. Interestingly, among HCC patients, those who were infected with C2 had significantly lower albumin levels, platelets counts (p < 0.05) and higher levels of hyaluronic acid and AFP (p = 0.06). The HBeAg-positive rate of HBV/C2 infections was lower than that of HBV/C1 (p < 0.05). Among IC and CHB patients, but not in the LC group, HBV/C2 tended to be associated with a higher rate of

HBeAg positivity (p = 0.06). None of the HCC patients was positive for HBeAg.

3.4. Age-related distribution of HBV/Ba, HBV/C1 and HBV/C2 in patients with HCC In order to investigate the age-related distribution of HBV genotypes and subgenotypes in HCC patients, they were classified into three age groups (≤35, 36–50 and ≥51 years). All patients of the younger age group (n = 5) were infected with HBV/Ba. In the middle-age group (n = 12), the HBV/Ba and HBV/C prevalence was 58% and 42%, respectively and HBV/C1 was identified in all genotype C cases. Among HCC patients aged more than 50 years (n = 17), HBV/Ba was present in 52% of the cases and HBV/C was also found in 48% of the cases. Interestingly, HBV/C2 was only found in the HCC patients over 50 years of age (p < 0.05).

Table 2 Comparison of the clinical features of the CHB, LC and HCC patients with different genotypes/subgenotypes HBV/Ba

HBV/C1

HBV/C2

p value

CHB patients Gender (M/F) Age (years)a ALT (IU/L)a T.Bil (␮mol/L)a Hyalr. acid (ng/mL)a AFP (≥200 ng/mL)b Albumin (g/L)a Platelet (×109 /L)a HBeAg (positive)b HBVDNA (logcps/mL)a

43 38/5 33.7 ± 7.3 80.8 ± 81.3 19.9 ± 12.1 108.1 ± 64.2 0 (0) 38.4 ± 1.3 134.6 ± 12.6 18 (41.9) 5.5 ± 2.0

11 10/1 34.7 ± 7.8 89.8 ± 63.5 16.6 ± 6.5 94.7 ± 32.3 0 (0) 38.2 ± 1.2 132.4 ± 11.7 7 (63.6) 5.9 ± 1.3

19 15/4 33.2 ± 9.1 87.4 ± 121.0 16.0 ± 6.5 120.9 ± 87.9 0 (0) 38.0 ± 1.1 130.2 ± 12.0 6 (31.6) 5.1 ± 2.1

NS NS NS NS NS NS NS NS NS NS

LC patients Gender (M/F) Age (years)a ALT (IU/L)a T.Bil (umol/L)a Hyalr. acid (ng/mL)a AFP (≥200 ng/mL)b Albumin (g/L)a Platelet (×109 /L)a HBeAg (positive)b HBVDNA (logcps/mL)a

45 37/8 45.6 ± 12.8 167.2 ± 263.0 33.4 ± 23.5 377.8 ± 275 0 (0) 36.3 ± 6.1 83.3 ± 37.1 12 (26.7) 4.9 ± 1.6

8 6/2 52.3 ± 12.8 94.0 ± 152.0 103.9 ± 142.6 584.9 ± 205.2 1 (12.5) 32.9 ± 7.4 37.4 ± 64.4 1 (12.5) 3.7 ± 17

13 10/3 42.8 ± 9.8 140.2 ± 153.9 87.6 ± 94.7 573.1 ± 285.4 4 (30.8) 30.5 ± 4.4 62.5 ± 27.4 6 (46.2) 4.5 ± 1.7

NS NS NS <0.0005c <0.05c <0.05d <0.0005d NS NS NS

HCC patients Gender (M/F) Age (years)a ALT (IU/L)a T.Bil (umol/L)a Hyalr. acid (ng/mL)a AFP (≥200 ng/mL)b Albumin (g/L)a Platelet (×109 /L)a HBeAg (positive)b HBVDNA (logcps/mL)a

21 19/2 48.5 ± 14.9 135.3 ± 173.7 96.2 ± 122.5 512.5 ± 483.8 9 (42.9) 35.7 ± 4.7 122.8 ± 51.6 2 (9.5) 5.0 ± 1.8

9 9/0 48.8 ± 9.8 125.2 ± 105.6 31.2 ± 33.6 316.2 ± 251.1 3 (33.3) 39.2 ± 4.2 141.9 ± 81.0 0 3.8 ± 1.4

4 3/1 56.5 ± 3.7 131.5 ± 147.8 57.1 ± 38.7 629.3 ± 197.3 3 (75.0) 31.5 ± 2.8 64.8 ± 43.8 0 3.8 ± 1.3

NS NS NS NS NS NS <0.05e <0.05e NS NS

a b c d e

Data shown as mean value ± S.D. Data shown as number and percent of total; n(%). HBV/Ba vs. HBV/C1 and HBV/C2. HBV/Ba vs. HBV/C2. HBV/C2 vs. HBV/C1.

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Fig. 2. Parts of the alignment in the enhancer II/core promoter/precore/core coding regions containing featured mutations. Previously reported HBV/C2 strain retrieved from DDBJ/GeneBank (accession number AY641558) was used as a reference. All strains were separated into four blocks according to the HBV/C subgenotype and HBeAg status, indicated in the left upper side of the each block. Strains name indicated on the left side of each block along with a clinical stage of the carrier. Featured mutations indicated above the reference strain, nucleotide position numbering indicated according to the reference HBV strain (NC 003977) deposited in DDBJ/GeneBank. Dots (.) in the sequences were used to indicate that the nucleotide in this position is the same as in reference sequence. Dashes (–) indicated deletions.

3.5. Relationship between HBV/C subgenotypes and the gene mutations in the enhancer II/core promoter/precore/core regions

in HBV/C1 (14.8%, 4/27). Similarly, the A1899 substitution tended to be more frequent in HBV/C2 than in HBV/C1 subgenotype (p = 0.1).

The HBV DNA sequences bearing the enhancer II/core promoter/precore/core regions were successfully amplified and determined for 67 HBV/C strains. Following nucleotide substitutions were observed among studied strains; T1653, V1753, T1762/A1764, T1856, T1858, T1862, A1896, A1898 and A1899 (Fig. 2). V1753, T1856 and C1858 mutations were specifically associated with HBV/C1 (p < 0.027). Conversely, T1858 and A1896 were specifically associated with HBV/C2 (p < 0.002) (Table 3). Although differences in the prevalence of A1898 substitutions between subgenotypes did not reach statistical significance (p = 0.056), it was found only

3.6. Association of the mutations occurring in the enhancer II/core promoter/precore/core coding regions with the clinical outcome of HBV infection

Table 3 Comparative prevalences of mutations in the enhancer II/core promoter/precore/core genes between HBV/C subgenotypes

T1653 V1753 T1762/A1764 T1856 T1858 C1858 T1862 A1896 A1898 A1899

HBV/C1 (n = 28)

HBV/C2 (n = 39)

p value

4 (14.3%) 11 (39.3%) 20 (71.4%) 7 (25.0%) 6 (21.4%) 22 (78.6%) 0 2 (7.1%) 4 (14.3%) 1 (3.6%)

7 (17.9%) 6 (15.4%) 19 (48.7%) 1 (2.6%) 36 (92.3%) 3 (7.7%) 1 (2.6%) 16 (41.1%) 0 8 (20.5%)

NS 0.027 0.06 0.016 <0.0001 <0.0001 NS 0.002 0.056 0.10

Using sequence data, the relationship between genetic mutations and clinical outcome was analyzed. The V1753 and T1762/A1764 were strongly associated with HCC (p < 0.01) (Table 4). The prevalence of the A1898 substitution was also significantly higher in HCC than in IC or CHB (p < 0.05), although HCC patients numbers were relatively small, i.e. only 2 (25%) cases were observed. No significant differences were observed in other mutations’ prevalence among the studied strains.

4. Discussion Accumulated data from virological and clinical studies on HBV indicate that the genotype C was associated with more aggressive clinical course as compared to genotype B (Kao et al., 2000; Kidd-Ljunggren et al., 2004). Based on phylogenetic analyses of the complete genome, genotype C is subdivided into four subgenotypes (C1–4) having distinct geographic distribution (Huy et al., 2004; Norder et al., 2004; Sugauchi et al., 2001; Tanaka et al., 2005). Lit-

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Table 4 Associations of the muations in the enhancer II/core promoter/precore/core coding regions with the clinical stage of HBV/C infection

T1653 V1753 T1762/A1764 T1856 T1858 C1858 A1896 A1898 A1899 a b

IC (n = 14)

CHB (n = 28)

LC (n = 17)

HCC (n = 8)

p value

2 (14.3%) 1 (7.1%) 3 (21.4%) 2 (14.3%) 10 (71.4%) 4 (28.6%) 3 (21.4%) 0 3 (21.4%)

3 (10.7%) 8 (28.6%) 17 (60.7%) 1 (3.6%) 18 (64.3%) 10 (35.7%) 9 (32.1%) 0 2 (7.1%)

4 (23.5%) 3 (17.6%) 12 (70.6%) 3 (17.6%) 12 (70.6%) 5 (29.4%) 4 (23.5%) 2 (11.80) 3 (17.6%)

2 (25.0%) 5 (62.5%) 7 (87.5%) 2 (25.0%) 2 (25.0%) 6 (75.0%) 2 (25.0%) 2 (25.0%) 1 (12.5%)

NS 0.011a 0.0062a NS NS NS NS 0.040b NS

HCC vs. IC. HCC vs. IC and CHB.

tle is known about the virological features associated with different subgenotypes and the clinical relevance of subgenotyping. In this study, we analyzed geographical, virological and clinical features of both subgenotypes C1 and C2 and Ba in chronic hepatitis B carriers in China, where these genetic viral variants are most widely distributed. Among the 241 serum samples randomly collected from HBV-infected patients with different clinical stages, genotype B was predominant (65%) and genotype C was second in importance (33%). Subgenotype Ba was found in all cases of HBV/Binfection. Among the genotype C, 42% and 58% of the strains belonged to the C1 and C2 subgenotypes, respectively. The geographical distribution of the subgenotypes was distinct, which indicated that HBV/C1 was the major subgenotype in the Southern part of China and C2 was predominant in the Northern part of China. The prevalence rates and the geographical pattern of the genotypes/subgenotypes in China were in agreement with previous reports (Huy et al., 2004; Norder et al., 2004; Sugauchi et al., 2001; Tanaka et al., 2005). In the IC and CHB groups, a higher number of HBV/C1 carriers was HBeAg positive in comparison to patients infected with HBV/C2 (p = 0.06). An opposite trend was observed in the LC group. The highest HBeAg prevalence was present in subgenotype C2 carriers. This might be explained by the relatively younger age of the LC patients infected with HBV/C2 than those who were subgenotype C1 carriers (43 years versus 53 years, respectively, p = 0.06). The association between HBeAg and subgenotypes requires more studies from age-matched groups. The results of hematological and biochemical markers of liver fibrosis (such as AFP, hyaluronic acid, albumin and platelets counts) indicated a possible trend for carriers infected with HBV/C2 to develop HCC after liver cirrhosis opposite to those infected with HBV/Ba and C1. This might also explain an observation concerning age related prevalence of the subgenotypes among HCC patients in this study, revealed HBV/C2 incidence only in elder group of HCC patients. Furthermore, the HBV/Ba was only present in the younger age group (≤35 years), whereas the prevalence of HBV/C increased along with patient age. These findings are in agreement with the results of a previous study on genotypes B and C among HCC patients in Taiwan and in Japan (Kao et al., 2000; Orito

et al., 2005). To our opinion, this is the first report to describe a possible association of subgenotype C1 infection with HCC at a younger age in comparison to C2. However, more studies need be performed in the near future to confirm our findings. The analysis of the nucleotide sequences of the core promoter region indicated a significantly higher prevalence of V(not T)1753 among HBV/C1 strains (p = 0.039) as previously reported by (Tanaka et al., 2006). In our study, this mutation was associated with HCC, independently of the subgenotype (p = 0.011), the prevalence was similar to that observed in Japanese HCC patients (Takahashi et al., 1998; Tanaka et al., 2006). In addition, another mutation T1762/A1764, which affects basal core promoter, has also been found to be associated with a high risk of HCC in Africa (Baptista et al., 1999) and in Asia (Kao et al., 2003; Kuang et al., 2004) In the present study, the association was demonstrated for HBV/C1 strains. Previous reports indicated a significantly higher incidence of HCC in the Southern part of China (Wang et al., 1996; Zhang et al., 1999). Our results also indicate that HBV/C1 was only found in this area. Further studies should be performed to confirm the significance of HBV/C1 together with V1753 and T1762/A1764 mutations in the development of HCC. Analysis of core promoter and precore/core nucleotide sequences revealed subgenotype-related patterns. The T1856 and C1858 mutations were associated with HBV/C1. C1856 and T1858 were frequently found in HBV/C2 in accordance with previous observations (Chan et al., 2005; Tanaka et al., 2005). The T1858 substitution is a provocateur of the A1896, which was is a “stop-codon mutation”. This substitution abolishes synthesis of HBeAg. However, on the other hand, T1858 and A1896 stabilize the structure of the epsilon loop which may result in increasing viral replication (Li et al., 1993; Lok et al., 1994; Tong et al., 1992, 1993; Yuan et al., 1995). No significant association of the A1896 mutation with the clinical stage of the HBV infection was present in our study. Although the number of cases was relatively small in our study, A1898 was frequent demonstrated among HCC patients (p < 0.04). Chan et al. (2006) observed a particular high prevalence of T1856 in C1 strains, which was associated with a more aggressive liver damage in comparison with the wild type strain (Chan et al., 2006). However, clini-

J. Yuan et al. / Journal of Clinical Virology 39 (2007) 87–93

cal implications and mechanisms of associations between the HBV genotypes/subgenotypes and mutations need be further studied.

Acknowledgments Authors thank Dr Bernard Weber (Laboratoires Reunis Junglinster, Luxembourg) and anonymous reviewer for their critical reading of the manuscript and valuable suggestions.

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