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INTRODUCTION
The hepatitis C virus (HCV) belongs to the family Flaviviridae and is a single strand RNA virus. Its total gene length is 9,600 bp, and it is characterized by the high probability of being mutated. The currently known HCV genotypes include 1 – 11, with the subtypes a, b, and c. Currently, genotypes up to HCV 6 can be clinically diagnosed (12).
Globally, the number of hepatitis C patients has reached approximately 180 million, and the prevalence ratio is 2.8 %. Among the genotypes of HCV, genotype 1 is the most prevalent, followed by genotype 2. These genotypes show differences by region; for example, genotype 1a is the most prevalent in Northern Europe and North America, and genotype 1b is highly prevalent in East Asia and Europe. Genotype 2 also appears frequently in Northern Europe, North America, East Asia, and Europe, but in lower ratios than genotype 1. In South Korea, hepatitis C is an infectious disease under sentinel surveillance, in which approximately 4,000 cases are reported every year, and its prevalence ratio in Korea is approximately 0.78 %. Of the diagnosed cases, genotype 1b accounts for the highest ratio, followed by genotype 2a (1234).
HCV genotypes have important meanings for diagnoses, because their sensitivity levels to antiviral agents are different from each other, leading to different treatment decisions. The antiviral agent' treatment performances against genotypes 1 and 4 have been reported to be lower, when compared to genotypes 2, 3, and 6 (5).
The correlations between HCV and the cholesterol level have been previously proven in many studies. Low cholesterol values are shown in HCV patients, and it has been reported that cholesterol pays an important role when HCVs infiltrate liver cells. More detailed studies have reported that low density lipoproteins (LDLs) and apolipoproteins from cholesterol contribute to an HCV infection and proliferation. Apolipoprotein E (apoE) and B are both components of very low density lipoprotein (VLDL) and are thought to play important roles in the HCV life cycle. In particular, ApoE plays an important role in moving the HCV viral particles to the liver cells (6789). Based on these reports, studies to develop HCV treatment agents through anti-cholesterol functions are being actively conducted (10).
ApoE is one of the plasma protein, and it functions in the transport of cholesterol and lipids. apoE is classified as having ε2, ε3, or ε4 alleles, and since these genetic polymorphisms are associated with some diseases, they are used in the diagnoses as well (111213). However, the relationship between apoE gene polymorphisms and HCV genotypes in patients with HCV is less well understood.
Therefore, the purpose of the present study was to classify the genotypes of the HCVs isolated in Korea. Additionally, the genotypes would be compared with those isolated overseas, a quantitative analysis would be conducted on the blood apoB and apoE according to the HCV genotypes, and genetic polymorphisms of apoE would be identified to study the correlations between them.
MATERIALS AND METHODS
Samples
HCV (n=124) samples (serum and whole blood) were collected at Bundang Jaesang Hospital in 2012, and the HCV samples were defined by anti-HCV tests, looks for antibodies to the HCV. The normal control group (n=100) was selected from clinically healthy people between 45 – 60 years old.
The samples were immediately stored at −70℃, and the patient's medical records were collected for the analysis of the clinical characteristics. This study was approved by the Institutional Review Board of Bundang Jaesang Hospital.
HCV RNA isolation and amplification from serum
The HCV RNA was isolated using Accuzol™ RNA extraction solution (Bioneer, Seoul, Korea), and the isolated RNA was resuspended in 50 µl of 0.1% diethylpyrocarbonate (DEPC). To amplify the 5'UTR region of the HCV RNA for genotyping, a nested PCR was conducted. An initial denaturation was performed at 42℃ for 60 min, and at 95℃ for 5 min. Thereafter, reverse-transcriptase PCR (RT-PCR) was performed for 35 cycles, with denaturation at 95℃ for 1 min, annealing at 50℃ for 1 min, and extension at 72℃ for 1 min using a RT-PCR premix (Bioneer, Seoul, Korea). The primer set consisted of forward CTG TGA GGA ACT ACT GTC TT and reverse ACT CGC AAG CAC CCT ATC AG. A second PCR was performed for 25 cycles, with denaturation at 95℃ for 30 sec, annealing at 55℃ for 30 sec, and extension at 72℃ for 30 sec using a PCR premix (Bioneer, Seoul, Korea). The primer set consisted of forward TTC ACG CAG AAA GCG TCT AG and reserve TAT CAG GCA GTA CCA CAA GG.
HCV genotype analysis
The amplicon was purified using a PCRmix purification kit (Cosmogenotech, Seoul, Korea), and the product was transferred for sequencing to Cosmogenetech (Seoul, Korea). The sequence of the HCV isolates and the sequences available in the GenBank database were used for the comparison. The sequences were aligned using MEGA 4.1 software, and their phylogenetic relationship was analyzed. For this, the Kimura-2-parameter distances were calculated using the neighbor-joining (NJ) algorithm. The reliability of the various phylogenetic groupings was evaluated by using the bootstrap test with 1,000 replications.
Quantitative analysis of apo
The Apolipoprotein B Human ELISA kit and Apolipoprotein E Human ELISA kit were used for analysis (Abcam, Cambridge, UK). The obtained serum was used for testing, and we followed the protocols thoroughly.
ApoE genotyping
The DNA was isolated using a DNA isolation kit (Cosmogenotech, Seoul, Korea) From whole blood. The isolated DNA was used for apoE genotyping, and an EzWaay™ Direct ApoE Genotyping kit (Komabiotech, Seoul, Korea) was used. This kit is based on the multiplex PCR method, and the genotype was determined by electrophoresis in a 2 % agarose gel(Fig. 1.)
Statistical analyses
SPSS 18.0 statistical software was used for the data analysis, and continuous variables, like the clinical characteristics, were compared with the Student's t-test. Categorical variables, like apoE genotype, were compared with a cross tabulation test. P-values of <0.05 were considered statistically significant.
RESULTS
HCV genotype distribution and gene characteristics
The HCV genotypes in the 124 HCV patient' serum samples were analyzed, and as a result, the HCV genotypes could be identified in a total of 124 samples. The most frequently identified genotype was genotype 1b, which was identified in a total of 80 samples, accounting for 64.5% of all samples. Genotype 2a was identified in 42 samples, accounting for 33.9%, and genotype 1a was identified in 2 samples, accounting for 1.6% (Table 1).
The isolated HCV genotypes were classified as shown in Table 2. Genes that were similar to each other based on the results of the gene analysis were grouped for analyses. Genotype 1b sequences formed five major separated groups (namely KOR-A to E). Group KOR-A (n=33) and KOR-B (n=21) contained the majority of 1b isolates. Five isolates identified genotype 1b, was not contained any other groups. Genotype 2a sequences formed four major separated groups (namely KOR-A to D). Group KOR-A (n=16) and KOR-B (n=19) contained the majority of 2a isolates. Three isolates identified genotype 2a, was not contained any other groups.
Analysis of age and gender according to HCV genotype
The ages and genders were analyzed according to the HCV genotypes, and the results showed that the mean age of the patients with HCV genotype 1a was 51.5 years, which was younger by approximately 10 years when compared to the patients with genotypes 1b or 2a. However, the mean ages of the patients with genotype 1b or 2a were both 60.8 years. The gender distributions according to the genotypes were analyzed and according to the results, all of the patients with HCV genotype 1a, although the ratio of females was slightly higher (Table 4).
Comparison of the clinical laboratory test results of HCV genotypes 1b and 2a
The clinical properties and states of the patient groups with HCV genotypes 1a were excluded from this analysis, since the number of isolated patients was too small, and was judged as not being statistically significant (Table 5).
Both HCV 1b and 2a patients had higher AFP, AST, ALT, ALP, γ-GTP, apoB, and apoE values compared with the normal control group. In particular, apoB and apoE levels were statistically significantly higher in the HCV 2a patients (p<0.05) and apoE levels were significantly higher in the HCV 1b patients (p<0.000). Total cholesterol, HDL, and LDL levels were lower in the both HCV 1b and 2a patients (p<0.05) and TG levels were higher in the HCV 2a patients. However, there were no significant differences in the all clinical biomarkers between the HCV 1b and 2a patients (p>0.05) (Table 5).
The apoE genetic polymorphisms were determined according to the classified HCV genotypes. According to the results of all of the patients with HCV genotype 1a, they were apoE type ε3/ε3. Among the patients with HCV genotype 1b, type ε3/ε3 was the most prevalent at 78.5%, followed by ε2/ε3 and ε3/ε4 in order of precedence. Among the patients with HCV genotype 2a, type ε3/ε3 was the most prevalent at 85.7%, followed by ε3/ε4, ε2/ε3, and ε2/ε4 in order of precedence. Among the patients with HCV genotype 2a, type ε2/ε4 was specifically identified, and the isolation ratio of ε3/ε3 was shown to be higher by at least 7.0% than in genotype 1b. The isolation ratios of the other subtypes were lower. The apoE genotypes according to the HCV genotypes were cross-analyzed, and the correlations were found to be statistically significant (p<0.05) (Table 6).
The apoE genotypes were identified using the apoE alleles according to the classified HCV genotypes, and the results indicated that the ratios of the apoE ε3 and ε4 alleles among the patients with genotype 1b and 2a were similar to each other. However, the ratio of the ε2 allele among the patients with genotype 1b was higher by approximately 2 times that among the patients with genotype 2a (Table 7).
In the HCV 1b patients, the ε3/ε3 group had higher AFP and ALP levels than the ε2/ε3 apoE genotype group and the ε2/ε3 group had higher AST and ALT levels than the ε3/ε3 apoE genotype group. In the HCV 2a patients, the ε2/ε3 group had higher AFP and ALP levels than the ε3/ε3 apoE genotype group. And the ε2/ε3 group and ε3/ε3 apoE genotype groups had the same levels of AST, and ALT.
The ε2/ε3 group in the HCV 1b, and 2a patients had higher levels of γ-GTP. The ε3/ε3 group in the HCV 1b, and 2a patients had higher levels of TG. The ε2/ε3 group in the HCV 1b, and 2a patients had the lowest LDL levels. HDL levels were generally low when not accounting for apoE genotype or hepatitis type. In the HCV 1b patients, apoB and apoE levels were higher in the ε3/ε3 group. In the HCV 2a patients, apoB and apoE levels were higher in the ε3/ε3 and ε3/ε3 groups, respectively (Table 8).
DISCUSSION
Many previous studies have investigated the distribution of HCV genotypes, in Korea. Whereas genotypes 1b and 2a have been isolated at similar frequencies in Korea, in the present study, genotype 1b was more frequently isolated by approximately two times than genotype 2a, as the ratio was 64.5% (80/124). This bias is considered attributable to the fact that the clinical samples were obtained from only one medical institution located in the Gyeonggi-do Province. In previous study, Shon et al. reported that the prevalence of HCV infection differs by regions as well as towns in the Korea, and is highest in Busan, Jeonnam, and Gyeongnam. Therefore, the genotypes of the isolated HCVs may be different by region (14151617).
We obtained 124 HCV positive samples that were successfully sequenced. To analyze the distribution of HCV genotypes, three phylogenetic trees were generated, each representing one of the three genotypes identified in this study, 1a, 1b, and 2a (Fig.2.). The phylogenetic tree for genotype 1a was showed two clear separate groups. In the genotype 1b and 2a trees, major groups distributed throughout the trees. These showed that the distribution of HCV genotypes of isolates from this study is complex. Our phylogeny analysis need to be verified in new further studies with large random sampled sequences.
HCV infection is associated with a decrease in serum levels of total cholesterol, LDL cholesterol (18). In previous study, we already confirmed that HCV infection are strongly associated with low levels of main cholesterol biomarker (HDL, LDL, and total cholesterol) (12). We reconfirmed it through this study. In addition, apoB and apoE levels were higher in both HCV 1b and 2a patients than in healthy controls. ApoE and B are both components of VLDL and are thought to play important roles in the HCV life cycles. Some studies have reported that both apolipoproteins are required for HCV assembly and secretion (192021). HCV 1b has more of an effect than HCV 2a due to high levels of TG, apoB, and apoE. However, there were no statistically significant differences between HCV genotype 1b and 2a.
Price et al. reported that apolipoprotein ε3 allele is associated with persistent HCV infection. Also, Gomma et al. suggest that apo ε3 allele is considered as a particular risk factor in HCV patients (2223). In the present study, the correlations between the HCV genotypes and apoE genotypes were analyzed. The HCV 1b and 2a both had a high distribution of haplotype ε3/ε3 78.7% (63/80), 85.7% (36/42), respectively. The distribution of ε3/ε3 type is about 75% in normal people (1124). Therefore, ε3/ε3 is more common type in HCV 1b and HCV 2a. Especially, genotype 2a was shown to be higher than the ratio of type ε3/ε3 generally identified in Korea, indicating that genotype 2a is correlated with type ε3/ε3. We anticipate that the apoE ε3 allele is the most common type in HCV genotype 1b (89.2%) and 2a (91.7%). As a result of apoE genotyping, we confirmed an association with HCV infection and the apoE ε3 allele. However, the ratios of the apoE ε3 allele among the patients with genotype 1b and 2a were similar to each other (were not statistically significant). The present study has some limitations; The problem of clinical samples (relatively small sample size and limitation of collection region), and exclusion of the patient's underlying medical conditions. Therefore, we need further studies to verify our findings.
In conclusion, the HCV genotypes were identified among the 124 patients infected with HCV, and the genetic characteristics of the HCV genotype were analyzed. In addition, the results of clinical laboratory test were comparatively analyzed according to the classified genotypes. According to the results the patients with HCV genotype 1b showed higher values of liver damage related indicators and apoB expression than the patients with HCV genotype 2a. The fat related indicators and apoE expression were not different between the genotypes. The apoE ε3 and ε2 allele were more frequently identified in the patients with genotype 2a and 1b, respectively. Analyses with large scale samples collected from diversity of region are needed in order to verify the relationship between HCV genotype and apolipoprotein.
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