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The quasispecies nature of the hepatitis C virus (HCV) genome is central to the transmission, persistence and pathogenesis of the infection. Heteroduplex mobility analysis (HMA) is a simple and an inexpensive technique for the qualitative and quantitative analysis of genetic variation of viral quasispecies. An original HMA for the HVR1 region of HCV was developed, based on a semi-automated, non-radioactive capillary electrophoresis system, which allows the processing of large numbers of samples in short times, the accurate measure of mobility shifts and the quantitation of heteroduplexes. A set of 120 HVR1 clones of known sequence was used to develop the assay, which was tested on HVR1 sequences amplified directly from sera of 17 HCV-infected patients. HVR1 sequence divergence directly correlated with the heteroduplex mobility ratio (HMR) of hybrid molecules between six and 40 mismatches. Heteroduplexes between one and six mismatches were resolved, although HMRs were not proportional to base changes, likely due to an effect of type and position of the substitutions. The assay sensitivity was 1% of the total sample size. This assay may allow the application of quasispecies analysis to a wider range of clinical and basic investigations.

Cancer risk can be influenced by the exposure to endogenous or environmental toxins. Polymorphic enzymes involved in the metabolic activation/detoxification of carcinogens may account for individual variations of risk. We studied the polymorphisms of five enzymes of the P450 superfamily, CYP1A1, CYP1A2, CYP2D6, CYP2E1 and CY3A4, as risk factors for liver disease progression and cancer in hepatitis C virus-infected patients. CYP genotyping was performed by polymerase chain reaction (PCR) restriction fragment length polymorphism or allele-specific PCR. Different stages of disease were considered, as follows: 90 asymptomatic carriers and 87 chronic hepatitis, 92 cirrhosis and 91 hepatocellular carcinoma (HCC) cases. Reference allele frequencies were obtained from 99 blood donors. Allele distributions among categories were compared using the chi(2) test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to express relative risks. Independent associations were modeled by correspondence analysis and logistic regression. Frequencies of the CYP1A1 highly inducible alleles, MspI m2 and Val, were increased in liver disease patients compared with carriers; no specific association with HCC was found. The high-activity CYP2E1 c2 allele was underrepresented among HCC patients with respect to other HCV categories, including cirrhosis. CYP2D6 poor metabolizer (PM) genotypes were significantly more frequent in healthy subjects (7.1%) and carriers (11.1%) than in hepatitis/cirrhosis (4.6%) and HCC (1.2%) patients. This was confirmed by multivariable analysis. PM genotypes protected against progressive disease as ORs reduced proportionally to stage. The age at diagnosis for HCC was anticipated in non-PM individuals. No differences were seen for CYP1A2 and CYP3A4 genes. Polymorphic variants of CYP genes may contribute to the progression of liver disease and HCC risk in HCV-infected subjects.

Factors influencing the progression of liver disease and the development of hepatocellular carcinoma (HCC) in chronic hepatitis C virus (HCV) infection are poorly understood. Inherited variations of drug-metabolizing enzyme (DME) activities may affect liver damage and cancer risk by modifying individual susceptibility to endogenous or exogenous toxic compounds. We investigated the association of liver disease severity with common alleles of microsomal epoxide hydrolase (mEH), an enzyme involved in the metabolism of highly reactive epoxide intermediates. Three polymorphisms (Tyr113His, His139Arg, and −613C/T) were analyzed by polymerase chain reaction (PCR) restriction fragment length polymorphisms (RFLPs) in 394 patients at different stages of disease, including 92 asymptomatic carriers, 109 patients with chronic hepatitis, 100 patients with cirrhosis, and 93 patients with HCC. Reference allele frequencies were obtained from 99 healthy blood donors. Allele distributions between categories were compared using the [Chi ]2 test; odds ratios (ORs) and 95% CI were calculated to express relative risks. Allele frequencies among 99 healthy controls were as follows: 15.1% for 113His/His, 4.0% for 139Arg/Arg, and 46.5% for −613C/T. mEH 113His/His homozygotes were overrepresented in advanced stages of disease, in particular among HCC patients (27.9%; P = .03; OR, 2.2; 95% CI, 1.0-4.6). Differences were more pronounced among men and between extreme patient categories. When mEH genotypes were combined to express a metabolic phenotype, very slow metabolizers were highly prevalent among cirrhotic and HCC patients (18% vs. 3.3% in carriers; P < .001). In conclusion, mEH gene polymorphisms were significantly associated with HCV-related liver disease severity and HCC risk. Men were at higher risk than women; this might be explained by hormonal regulation of gene expression or by differential exposure to environmental toxins. (HEPATOLOGY 2002;36:195-201.)

Hepatitis C virus (HCV) infection persists for an indefinite length of time in a major proportion of patients, inducing chronic liver lesions that evolve to cirrhosis and hepatocellular carcinoma (HCC) in approximately 20% of cases. We studied HCV viremia and genotypes by reverse transcription-polymerase chain reaction (RT-PCR) in 341 consecutive anti-HCV-positive patients. Of these, 167 patients had persistently normal or near normal alanine aminotransferase (ALT) levels (fluctuations < or = 5 IU above the upper limit of normal); the remaining 174 patients presented with elevated ALT and histological evidence of chronic liver disease. Seventy percent of patients with normal ALT values had circulating HCV RNA despite the absence of biochemical indicators of liver damage and mild histological forms of chronic hepatitis were detected in most patients who underwent liver biopsy. Isolated genotype III infection was significantly more prevalent in this patient group with respect to control patients with abnormal ALT values (70% vs. 39%; P < .001). Conversely, isolated genotype II was more frequently found in patients with elevated ALT values and evidence of chronic liver disease (45% vs. 23%; P < .01) and it was progressively more represented in advanced liver disease, such as cirrhosis and HCC. Virological features of HCV infection might be associated with different clinical manifestations, suggesting a potential prognostic significance on disease outcome.

Chronic hepatitis C virus (HCV) infection is the most frequent cause of progressive liver disease and liver cancer in the West. The p53 tumor supressor gene is known to play an important role in carcinogenesis of different tissues being involved in gene transcription, DNA synthesis and repair and somatic mutations of p53 are common in primary liver cancer. The p53 gene displays a common genetic Arg/Pro polymorphism at codon 72 with functional significance, that has been investigated as risk factor in several cancer models. We analyzed p53 codon 72 polymorphism in a group of 340 HCV-infected subjects at different stages of disease, including 84 hepatocellular carcinoma patients. No association between codon 72 genotypes and disease severity or liver cancer was observed.

Background/Aims : Genetic polymorphisms of enzymes involved in hormone metabolism can influence hormonal activities and risk of hormone-dependent cancers. As progression of chronic hepatitis C and risk of liver cancer is higher in males than in females, we evaluated whether the polymorphisms of three enzymes participating in the pathway of estrogen and androgen biosynthesis and inactivation, 5α-reductase type II (SRD5A2), cytochrome P450c17α (CYP17) and catechol- O -methyltransferase (COMT), might affect the expression of hepatitis C virus (HCV)-related liver disease. Methods : The study included 78 healthy subjects and 387 HCV patients: 100 asymptomatic carriers, 105 hepatitis, 90 cirrhosis and 92 hepatocellular carcinomas (HCC). Variant positions SRD5A2 V89L and A49T, CYP17 (−34)T/C and COMT V108M were analysed by polymerase chain reaction and restriction fragment length polymorphism. A cross-sectional study of association was performed, considering carriers as reference category. Results : The CYP17 (−34)C/C genotype was over-represented in HCC patients as compared to carriers (22.5 vs. 11.2%, odds ratio (OR): 2.29, P : 0.05). Females mostly contributed to this association (OR: 4.95, P : 0.01) and OR values increased in post-menopausal women (OR: 6.00, P : 0.03). No differences were observed for SRD5A2 and COMT gene polymorphisms. Conclusions : CYP17 high-activity alleles associated with increased circulating levels of estrogens and androgens may affect liver cancer risk in HCV-infected women.

Seventy-nine transitional cell carcinomas (TCCs) of the urinary bladder (25 grade 1, 22 grade 2, and 32 grade 3 tumours) were examined for p53 overexpression by immunohistochemistry with a monoclonal antibody and for human papillomavirus (HPV) infection by the polymerase chain reaction (PCR). Positive immunostaining for p53 was detected in 40.5 per cent of the cases; the percentage of positive cases was significantly lower in low-grade (G1 and G2) TCCs than in high-grade (G3) tumours (10.6 per cent vs. 84.4 per cent; P < 0.0001). The overall rate of HPV infection was 32.9 per cent; 20.3 per cent of the cases were positive for HPV 16, 3.8 per cent for HPV 18, and 8.9 per cent for both. Consensus primers as well as type-specific primers for HPV types 6, 11, and 33 failed to detect any additional case with HPV infection. The prevalence of HPV 16 and/or HPV 18 infection was significantly higher in low-grade than in high-grade tumours (44.7 per cent vs. 15.6 per cent; P = 0.0061). p53-positive cases were more common among papillary, deeply infiltrating tumours, and HPV-positive cases among papillary, non-infiltrating lesions. According to these data, p53 overexpression and HPV 16/18 infection are common findings in bladder TCC and there appears to be an inverse correlation of p53 overexpression and of HPV infection with tumour aggressiveness. The possibility of different molecular pathways in superficial low-grade and in invasive high-grade tumours is suggested.