SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgments
  6. References

Chronic hepatitis C virus (HCV) infection often results in hepatocellular carcinoma (HCC). Previous studies have shown that there might be some characteristic mutations in the core region of HCV related to HCC. Thus, we downloaded and analyzed HCV genotype 1b core gene sequences from HCV databases online to identify them. Based on the information of the sequences, 63 from patients with HCC and 188 from non-HCC were enrolled into our analysis. Then, the nucleotides at each position were compared by χ2-test between the two groups, and 24 polymorphisms were found to be associated with HCC. Further analysis of these 24 polymorphisms by logistic regression indicated that eight were significantly related to the increased HCC risk: A028C, G209A, C219U/A, U264C, A271C/U, C378U/A, G435A/C, and G481A. Moreover, U303C/A was associated with the decreased HCC risk. These mutations could bring about four amino acid substitutions: K10Q, R70Q, M91L, and G161S. In conclusion, eight characteristic mutations in the HCV-1b core gene related to the occurrence of HCC were identified. The structural and functional alterations of core protein due to these mutations and the relationship with the occurrence of HCC need to be further studied. (Cancer Sci 2009; 100: 2465–2468)

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide and a major risk factor for hepatocellular carcinoma (HCC). More than 70% of patients with HCC in Japan are infected with HCV, and HCC has become the major cause of death in patients chronically infected with HCV.(1–3) In Asia, especially in Japan, HCV-1b is the most prevalent genotype and is more likely to develop into HCC than other genotypes.(4,5) Although there are many published data about the correlation between HCV infection and HCC development, details of hepatocarcinogenesis by HCV remain unclear. HCV core protein is implicated in hepatocarcinogenesis for its ability to modulate cellular gene transcription and protein expression, intracellular signal transduction, cell proliferation and apoptosis.(6–9)

Being an RNA virus, the HCV genome exhibits a considerable degree of sequence variation. Studies have proved that certain kinds of mutations may lead to functional changes of the virus, such as the resistance to antiviral treatments and the association with HCC.(10,11) Furthermore, there are more mutations in the core region in patients with HCC than that in those without HCC. Moreover, the rate of nucleotide substitutions in the core gene is significantly greater for isolates from HCC patients compared to those from individuals with chronic hepatitis.(12) It was also found that there was a significantly higher variability within the core region of tumor tissue isolates than that of isolates from non-tumor tissue. Mutant sequence diversity ranged from silent mutations, as well as amino acid substitutions, to appearance of in-frame stop codons and deletions leading to frame-shifts. In contrast, the variability of the NS5 region sequences between isolates from tumor and non-tumor tissue was not significantly different.(13) Recently, a report indicated that amino acid substitutions in the HCV core region are important predictor of hepatocarcinogenesis.(11) Even in patients without HCC, the substitution of amino acid 70 in the hepatitis C virus core region of genotype 1b is an important predictor of elevated alpha-fetoprotein.(14)

Based on the data above, we suppose that there might be some characteristic mutations in the HCV-1b core gene related to the occurrence of HCC. Because we can find thousands of HCV sequences from databases online, we have tried to identify these mutations by analyzing such sequences in this study.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgments
  6. References

Collection of HCV core gene sequences  HCV core gene sequences were downloaded from the following databases: European HCV database (euHCVdb), HCV Databases from Los Alamos National Laboratory, HCV Database (HCVdb) from the Viral Bioinformatics Resource Center (VBRC), and National Center for Biotechnology Information (NCBI).

Inclusion criteria  All the sequences enrolled into our analysis needed to comply with the following inclusion criteria: (i) the diagnosis of patients could be confirmed; (ii) the genotype of HCV could be confirmed and samples were isolated from sera of patients infected with HCV-1b; and (iii) the core gene sequence was of full length.

Exclusion criteria  Sequences were excluded if: (i) the diagnosis of patients or the genotype of HCV was unknown; (ii) samples came from liver, other tissues, ascites, or pooled sera; (iii) patients were co-infected with hepatitis B virus or human immunodeficiency virus; (iv) patients had ever been treated with interferon (IFN); (v) patients had ever undergone orthotopic liver transplantation (OLT); (vi) samples came from cell culture, animal experiments, chimeric DNA, or fusion protein; (vii) sequences came from repeated samples; and (viii) existence of deletion mutations or insertion mutations.

Statistical analysis  According to the diagnosis of the cases, sequences enrolled were divided into two groups: group HCC and group non-HCC. The first base of the start codon (AUG) was designated position 1. All the 573 nucleotides of the core region were compared respectively between group HCC and non-HCC. All the 573 nucleotides of the core region were compared respectively between group HCC and non-HCC by Pearson χ2-test to find out significantly different mutations (when there were cells that the expected count was less than 5 or 1, we chose different χ2-test methods – Continuity correction or Fisher's exact test). Then, the significant bases were further analyzed by logistic regression to identify the mutations significantly related to the occurrence of HCC. A P-value of <0.05 was considered statistically significant.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgments
  6. References

Enrollment of sequences  We downloaded sequences of HCV core region from HCV databases online, mainly from euHCVdb. A total of 2841 sequences of HCV-RNA with full-length core region were downloaded. Then, we checked the information provided by the databases and the published papers so that we could get the details about these sequences such as the diagnoses of patients, genotypes of HCV, the origin of samples, and so on. Finally, we picked out the sequences that complied with the inclusion and exclusion criteria and enrolled them into our analysis. As a result, from 2841 sequences, we found 1336 sequences of genotype 1b. Of these, 547 sequences were from identified samples and the others lacked information that identified the types of the samples. Among the 547 sequences with definite origin, 292 were from sera and the others (228 from livers, 20 from cultured cells, seven from plasmids) were ruled out. Of the 292 sequences derived from sera, seven were from animal experiments, 24 were from repeated cases at different time points, and four and six were from cases that had undergone OLT and IFN treatments, respectively. Finally, 251 sequences complied with the inclusion and exclusion criteria and were enrolled into our analysis. Sixty-three were from patients with HCC and 188 were from HCV carriers or patients with acute or chronic hepatitis without HCC. For the 251 sequences, 73.7% (185/251) were directly sequenced and 26.3% (66/251) represented one clone of available clones. Thus, it is possible that about a quarter of our data could contain mutations due to PCR error. However, the sequences were collected from 42 different studies and the statistical analysis may have overcome this problem.

Identification of the wild-type nucleotides  For the reason that there are frequent mutations of the HCV sequence, it is impossible to know the real wild-type nucleotides. It might be reasonable to identify the wild-type nucleotide based on the consensus of a large number of sequences. In our analysis, we defined the consensus of the 1336 HCV-1b sequences as the wild type, except the position of nucleotide (nt) 209. Because 209G was treated as wild type in almost all the published papers and it was also the consensus type of 2841 all genotypes sequences, we still defined “G” as the wild type even if “A” was the consensus.

Significantly different mutations between group HCC and non-HCC  By performing the χ2-test or Fisher’s exact test, we compared all the 573 nucleotides of the core gene between group HCC and non-HCC. A total of 24 nucleotide mutations were found to be associated with occurrence of HCC. Of the 24 nucleotide mutations, 21 increased the risk of HCC while three decreased the risk of HCC (Table 1).

Table 1.   Significant nucleotide mutations found by χ2-tests
Gene positionTypes of NucleotidePercentage of mutant Amino acidP-values
Wild MutantNon-HCC HCCWild type Mutant
  1. *Pearson χ2-tests; †continuity correction; ‡Fisher’s exact test.

nt 009G[RIGHTWARDS ARROW]A/U20.2[RIGHTWARDS ARROW]68.3 Thr Thr<0.001*
nt 028A[RIGHTWARDS ARROW]C1.1[RIGHTWARDS ARROW]7.9 Lys[RIGHTWARDS ARROW]Gln0.015†
nt 039U[RIGHTWARDS ARROW]G/A0[RIGHTWARDS ARROW]4.8 Arg Arg0.015‡
nt 048C[RIGHTWARDS ARROW]G/U0[RIGHTWARDS ARROW]4.8 Asn[RIGHTWARDS ARROW]Lys/Asn0.015‡
nt 066C[RIGHTWARDS ARROW]U14.4[RIGHTWARDS ARROW]30.2 Val Val0.005*
nt 078C[RIGHTWARDS ARROW]U28.7[RIGHTWARDS ARROW]3.2[DOWNWARDS ARROW]Gly Gly<0.001*
nt 116G[RIGHTWARDS ARROW]C0[RIGHTWARDS ARROW]7.9 Arg[RIGHTWARDS ARROW]Pro0.001†
nt 209G[RIGHTWARDS ARROW]A28.7[RIGHTWARDS ARROW]52.4 Arg[RIGHTWARDS ARROW]Gln0.001*
nt 219C[RIGHTWARDS ARROW]U/A7.4[RIGHTWARDS ARROW]23.8 Gly Gly<0.001*
nt 264U[RIGHTWARDS ARROW]C12.2[RIGHTWARDS ARROW]36.5 Asn Asn<0.001*
nt 270C[RIGHTWARDS ARROW]U8[RIGHTWARDS ARROW]33.3 Gly Gly<0.001*
nt 271A[RIGHTWARDS ARROW]C/U21.8[RIGHTWARDS ARROW]65.1 Met[RIGHTWARDS ARROW]Leu<0.001*
nt 303U[RIGHTWARDS ARROW]C/A64.9[RIGHTWARDS ARROW]47.6[DOWNWARDS ARROW]Arg Arg0.015*
nt 309U[RIGHTWARDS ARROW]C22.3[RIGHTWARDS ARROW]39.7 Ser Ser0.007*
nt 378C[RIGHTWARDS ARROW]U/A16.5[RIGHTWARDS ARROW]34.9 Leu Leu0.002*
nt 417C[RIGHTWARDS ARROW]U5.9[RIGHTWARDS ARROW]20.6 Leu Leu0.001*
nt 435G[RIGHTWARDS ARROW]A/C10.1[RIGHTWARDS ARROW]34.9 Gly Gly<0.001*
nt 446G[RIGHTWARDS ARROW]A0.5[RIGHTWARDS ARROW]7.9 Arg[RIGHTWARDS ARROW]Lys0.004†
nt 456G[RIGHTWARDS ARROW]A24.5[RIGHTWARDS ARROW]47.6 Ala Ala0.001*
nt 462C[RIGHTWARDS ARROW]U25[RIGHTWARDS ARROW]49.2 Gly Gly<0.001*
nt 471U[RIGHTWARDS ARROW]C/G38.3[RIGHTWARDS ARROW]20.6[DOWNWARDS ARROW]Val Val0.01*
nt 472C[RIGHTWARDS ARROW]G/U2.1[RIGHTWARDS ARROW]9.5 Leu[RIGHTWARDS ARROW]Val/Leu0.026†
nt 481G[RIGHTWARDS ARROW]A2.7[RIGHTWARDS ARROW]20.6 Gly[RIGHTWARDS ARROW]Ser<0.001†
nt 549C[RIGHTWARDS ARROW]U14.4[RIGHTWARDS ARROW]30.2 Ser Ser0.005*

Gene polymorphisms and amino acid substitutions associated with HCC  The 24 significant nucleotide mutations found by χ2-tests were further analyzed by logistic regression. Eight polymorphisms were significantly related to the increased HCC risk: A028C, G209A, C219U/A, U264C, A271C/U, C378U/A, G435A/C, and G481A; and U303C/A was significantly associated with decreased HCC risk (Table 2). Furthermore, four polymorphisms brought about amino acid substitutions which might change the structure and functions of core protein and lead to HCC. For the other five synonymous mutations, it is possible that they might lead to amino acid substitutions of ARFP (alternative ribosomal frame shift protein) which has been reported to be able to affect cell proliferation and apoptosis(15) (Table 3). However, HCC is thought to be a consequence of long-term infection of HCV and chronic hepatitis. Although the gene polymorphisms and amino acid substitutions in the HCV core region identified in this analysis were proven to be significantly associated with HCC, whether they are “causes” or “effects” of HCC remains to be determined.

Table 2.   Significant nucleotide mutations identified in logistic regression
Gene positionMutation typesOdds ratio95% CIP-values
LowerUpper
  1. Nagelkerke R2 = 0.572.

nt 028A[RIGHTWARDS ARROW]C14.651.31163.510.029
nt 209G[RIGHTWARDS ARROW]A3.301.407.780.006
nt 219C[RIGHTWARDS ARROW]U/A4.371.4912.770.007
nt 264U[RIGHTWARDS ARROW]C4.131.6210.520.003
nt 271A[RIGHTWARDS ARROW]C/U5.502.2613.37<0.001
nt 303U[RIGHTWARDS ARROW]C/A0.420.180.980.045
nt 378C[RIGHTWARDS ARROW]U/A2.691.086.730.034
nt 435G[RIGHTWARDS ARROW]A/C4.161.6110.720.003
nt 481G[RIGHTWARDS ARROW]A10.4842.7739.620.001
Table 3.   Amino acid substitutions of core protein and ARFP due to nucleotide mutations
CodonGene positionAmino acid positionNucleotideAmino acid
Wild type MutantWild type Mutant
  1. ARFP, alternative ribosomal frame shift protein.

Normalnt 028aa 010AAA[RIGHTWARDS ARROW]CAALys[RIGHTWARDS ARROW]Gln
nt 209aa 070CGG[RIGHTWARDS ARROW]CAGArg[RIGHTWARDS ARROW]Gln
nt 219aa 073GGC[RIGHTWARDS ARROW]GGU/AGly Gly
nt 264aa 088AAU[RIGHTWARDS ARROW]AACAsn Asn
nt 271aa 091AUG[RIGHTWARDS ARROW]C/UUGMet[RIGHTWARDS ARROW]Leu
nt 303aa 101CGU[RIGHTWARDS ARROW]CGC/AArg Arg
nt 378aa 126CUC[RIGHTWARDS ARROW]CUU/ALeu Leu
nt 435aa 145GGG[RIGHTWARDS ARROW]GGA/CGly Gly
nt 481aa 161GGC[RIGHTWARDS ARROW]AGCGly[RIGHTWARDS ARROW]Ser
+1 Codonnt 028aa 010GAA[RIGHTWARDS ARROW]GACGlu[RIGHTWARDS ARROW]Asp
nt 209aa 070GGC[RIGHTWARDS ARROW]AGCGly[RIGHTWARDS ARROW]Ser
nt 219aa 073GCA[RIGHTWARDS ARROW]GU/AAAla[RIGHTWARDS ARROW]Val/Glu
nt 264aa 088AUG[RIGHTWARDS ARROW]ACGMet[RIGHTWARDS ARROW]Thr
nt 271aa 091GCA[RIGHTWARDS ARROW]GCC/UAla Ala
nt 303aa 101GUG[RIGHTWARDS ARROW]GC/AGVal[RIGHTWARDS ARROW]Ala/Glu
nt 378aa 126UCA[RIGHTWARDS ARROW]UU/AASer[RIGHTWARDS ARROW]Leu/Stop
nt 435aa 145GGG[RIGHTWARDS ARROW]GA/CGGly[RIGHTWARDS ARROW]Glu/Ala
nt 481aa 161ACG[RIGHTWARDS ARROW]ACAThr Thr
−1 Codonnt 028aa 010AAA[RIGHTWARDS ARROW]ACALys[RIGHTWARDS ARROW]Thr
nt 209aa 070CCG[RIGHTWARDS ARROW]CCAPro Pro
nt 219aa 073CAG[RIGHTWARDS ARROW]T/AAGGln[RIGHTWARDS ARROW]Stop/Lys
nt 264aa 088UGA[RIGHTWARDS ARROW]CGAStop[RIGHTWARDS ARROW]Arg
nt 271aa 091CAU[RIGHTWARDS ARROW]CC/UUHis[RIGHTWARDS ARROW]Pro/Leu
nt 303aa 101UGG[RIGHTWARDS ARROW]C/AGGTrp[RIGHTWARDS ARROW]Arg
nt 378aa 126CAC[RIGHTWARDS ARROW]U/AACHis[RIGHTWARDS ARROW]Tyr/Asn
nt 435aa 145GGG[RIGHTWARDS ARROW]A/CGGGly[RIGHTWARDS ARROW]Arg
nt 481aa 161CGG[RIGHTWARDS ARROW]CAGArg[RIGHTWARDS ARROW]Gln

Recently, a similar study(16) determined four amino acid substitutions associated with increased HCC risk, and three of them were the same as our results (G209A, A271C/U, and G481A). Moreover, up to now, the G209A polymorphism has been thought to be related with both IFN/Ribavirin (RBV) treatment resistance and HCC.(10,11) The relationship between them is an interesting question. We guess that apoptosis might make an important function in this process. Apoptosis is considered to be a common pathway of virus clearance. HCV core protein could suppress apoptosis and escape this clearance mechanism.(9) On the other hand, inhibition of apoptosis might be able to make the genetically damaged hepatocytes survive and lead to neoplastic transformation. Whether the amino acid substitution of R70Q could enhance these effects is worth studying.

Alteration of the RNA and protein secondary structures of the HCV-1b core gene  Gene mutations of A271C/U, G435A/C, and G481A, were found to be able to cause the changes of the RNA secondary structure of HCV-1b core gene using RNAdraw software(17) (Karolinska Institute, Stockholm, Sweden). Furthermore, Chou–Fasman protein secondary prediction with the gene analysis software Genetyx (Genetyx, Tokyo, Japan) showed that A028C might turn the coil structure of AA8-12 into β-sheet and G209A might turn the coil stucture of AA71 into α-helix (Fig. 1). The results indicated that the point-mutations of the core gene might change the secondary structure of not only RNA but also protein. As a result, the functions of both RNA and protein of the core region, such as interaction with other DNA/RNA or proteins (lymphotoxin B receptor, heterogeneous-nuclear ribonucleoprotein, tumor necrosis factor receptor 1, etc.),(18–20) might change and lead to HCC.

image

Figure 1.  Secondary structure of wild-type (a) and mutant (b) hepatitis C virus (HCV)-1b core protein predicted by Chou–Fasman analysis with Genetyx. The straight, bent, looped, and zigzag lines represent coil, β-sheet, α-helix, and turn structures, respectively. The numbers indicate the amino acid positions. A028C might turn the coil stucture of AA8-12 into β-sheet and G209A might turn the coil stucture of AA71 into α-helix. (The secondary structure of the wild type was predicted based on the following consensus amino acid sequence of 1336 HCV-1b core, except 70R: MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRAWAQPGYPWPLYGNEGMGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTIPASA.)

Download figure to PowerPoint

In conclusion, eight characteristic mutations in the HCV-1b core gene related to the occurrence of HCC were identified: A028C, G209A, C219U/A, U264C, A271C/U, C378U/A, G435A/C, and G481A. Meanwhile, U303C/A was identified to be significantly associated with the decreased HCC risk. These mutations could bring about four amino acid substitutions: K10Q, R70Q, M91L, and G161S. The structural and functional alterations of core protein due to these mutations and the relationship with the occurrence of HCC need to be further studied.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgments
  6. References

The study was supported by the Global COE Program “Center of Education and Research for Advanced Genome-Based Medicine: For personalized medicine and the control of worldwide infectious diseases”, the Ministry of Education, Culture, Sports, Science and Technology of Japan; by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; by Health and Labor Sciences Research Grants for Research on Hepatitis from the Ministry of Health, Labor and Welfare, Japan; and by a Japan–China Sasakawa Medical Fellowship.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Acknowledgments
  6. References