Biomedical subjects

M Mizokami

Publications and source records attributed to M Mizokami.

At least 19 recordsLinked to original sources

PubMed2001

Mucosal immunization against hepatitis B virus by intranasal co-administration of recombinant hepatitis B surface antigen and recombinant cholera toxin B subunit as an adjuvant.

Recombinant cholera toxin B subunit (rCTB) produced by Bacillus brevis carrying pNU212-CTB has been previously found to be a potent mucosal adjuvant to aluminium-non-adsorbed tetanus toxoid (nTT) and diphtheria toxoid (nDT) co-administered intranasally, and the possibility of needle-free inoculation of these vaccines with rCTB has been suggested. In this paper we examined the potentiality of rCTB as a mucosal adjuvant to aluminium-non-adsorbed yeast-derived recombinant hepatitis B surface antigen (rHBs) being a particulate antigen when administered intranasally with rCTB. In-house ELISA showed that a mixture of rHBs (1 or 5 microg) and rCTB (10 microg) elevated not only systemic responses but also mucosal immune responses at the nasal cavity, the lung, the saliva, the small intestine and the vagina against rHBs, and these could be further increased with higher doses of antigen. With antibody isotypes of IgG, there were equally high levels of serum HBs-specific IgG1, IgG2a and IgG2b antibodies and induction of mixed Th1- and Th2-type responses was considered to occur in combination of rHBs and rCTB. Serum anti-HBs titres in almost all mice obtained from sandwich EIA using a commercial kit were higher than 1000 milli-international units ml(-1) (mIU ml(-1)). These results show that rCTB is also very effective as a mucosal adjuvant for a particulate antigen like rHBs, as well as soluble antigens like nTT and nDT reported previously, suggesting the possibility of intranasal immunization with rHBs plus rCTB in humans.

Adjuvants, Immunologic↗

PubMed2001

Determination of hepatitis B virus genotype G by polymerase chain reaction with hemi-nested primers.

Hepatitis B virus (HBV) has been classified into six genotypes designated A-F by sequence divergence in the entire genome exceeding 8%. Very recently, the seventh genotype was reported and named genotype G. HBV genotype G is distinct from genomes of the other six genotypes in that it possesses an insertion of 36 nucleotides in the core gene, and has been found so far in France and the United States. A method for determining HBV genotype G was developed by polymerase chain reaction (PCR) with primers deduced from the 36-nucleotide (nt) insertion in five isolates of HBV genotype G the sequences of which have been deposited in DNA databases. The validity of this method, for specifically detecting HBV genotype G, was verified on a panel consisting of 142 HBV isolates of six major genotypes and four of genotype G. A total of 540 sera containing HBV in Japan covering symptom free carriers and patients with a spectrum of chronic liver disease were tested by this method, but not a single HBV genotype G sample was found. A possible method for serological determination of hepatitis B surface antigen of genotype G is suggested, without amplification or sequencing nucleotides, which would expand epidemiological and clinical researches on HBV genotype G.

Amino Acid Sequence↗

PubMed2001

Biochemical response to interferon therapy correlates with interferon sensitivity-determining region in hepatitis C virus genotype 1b infection.

Biochemical responders maintain normal alanine aminotransferase levels after interferon (IFN) therapy despite persistent presence of hepatitis C virus (HCV) RNA in their sera. There have been few reports on predictive factors for biochemical response. A region associated with sensitivity to IFN was identified in the nonstructural protein 5 A of genotype 1b [aa 2209-2248; IFN sensitivity-determining region (ISDR)]. The substitutions in ISDR correlate with sustained response to IFN. In this report, we assessed the association of ISDR with biochemical response. The sequences of ISDR were determined in 62 patients with HCV genotype 1b treated by IFN in two randomized controlled trials. 30 patients had wild ISDR (identical to HCV-J), 20 intermediate ISDR (1-3 amino acid substitutions compared with HCV-J), and 12 mutant ISDR (four or more amino acid substitutions). All 12 patients with mutant ISDR had a sustained response, while only one of those with wild or intermediate ISDR had a sustained response (P < 0.0001). In the 49 patients other than sustained responders, the patients with intermediate ISDR obtained biochemical response significantly more frequently (52.6%, 10/19) than those with wild-type ISDR (20.0%, 6/30) (P < 0.05). Multivariate analysis indicated the number of substitutions in ISDR as the most important predictor for biochemical response (discriminant coefficient=1.08, P < 0.05) and sustained response (discriminant coefficient=6.13, P < 0.0001). In phylogenetic analysis, clustering of sustained responders and biochemical responders was observed. These results demonstrate that the substitutions in ISDR are the most important predictor for biochemical response to IFN in patients infected with genotype 1b as well as for sustained response.

Adult↗

PubMed2001

Effect of interferon therapy on Japanese chronic hepatitis C virus patients with anti-liver/kidney microsome autoantibody type 1.

AIM: The aim of this study was to determine the prevalence of anti-liver/kidney microsome autoantibody type 1 (anti-LKM-1) among hepatitis C virus (HCV)-infected Japanese patients at various stages (chronic hepatitis, liver cirrhosis and hepatocellular carcinoma), and to assess the influence of anti-LKM-1 on interferon therapy. METHODS: A total of 390 serum samples from 215 HCV-infected patients with chronic hepatitis (HCV-CH), 81 HCV-infected patients with liver cirrhosis (HCV-LC), and 94 HCV-HCC infected patients were subjected to examination. Ninety-one HBsAg-positive patients and 137 healthy subjects served as controls. Anti-liver/kidney microsome autoantibody type 1 was determined by using a newly developed ELISA using recombinant cytochrome P450 IID6 as the antigen. RESULTS: Anti-liver/kidney microsome autoantibody type 1 was detected in six of the 390 (1.5%) chronic HCV-infected patients (four were HCV-CH and two were HCV-LC); in contrast, it was not detected in control groups. Among the 110 HCV-CH patients treated with interferon (IFN), four were positive for anti-LKM-1. No change in anti-LKM-1 immunoreactivity from negative to positive during interferon therapy was observed. Moreover, no increase in the serum alanine aminotransferase level was observed in these four patients with anti-LKM-1. CONCLUSION: Our study indicates that: (i) anti-LKM-1 does not aggravate the liver disease associated with HCV infection; and (ii) no change in anti-LKM-1 immunoreactivity from negative to positive or no aggravations of liver dysfunction were observed among HCV-CH patients during the IFN therapy for Japanese patients with liver disease.

Autoantibodies↗

PubMed2001

A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV Genotype Research Group.

Clinical and molecular virological differences were evaluated in 50 Japanese patients chronically infected with HBV of genotype B and C who were matched for age and sex as well as the severity of liver disease in a case-control study. Hepatitis B e antigen (HBeAg) was significantly less frequent (16% vs. 42%, P <.01), whereas antibody to HBeAg (anti-HBe) was significantly more common (84% vs. 56%, P <. 01) in genotype B than C patients. The predominance of mutants with G-to-A mutation at nucleotide (nt) 1896 in the precore region (A1896) over the wild-type was comparable between genotype B and C patients (60% and 62%, respectively), and it correlated with anti-HBe. The double mutation in the basic core promoter (A-to-T at nt 1762 and G-to-A at nt 1764), however, was significantly more frequent in genotype C than B patients (58% vs. 16%, P <.01), and it did not correlate with anti-HBe or HBeAg. By the multiple logistic regression analysis, the double mutation in the basic core promoter (T1762/A1764) was significantly associated with genotype C [odds ratio (OR), 9.3; 95% confidence interval (CI), 3.4-25.1]], age > or = 35 years (OR, 5.5; CI, 1.5-20.5), and more advanced liver disease (OR, 4.1; CI, 1.6-10.2), but it was not associated with sex, HBeAg, HBV DNA, or the precore mutation (A1896). These results suggest a role of the double mutation in the basic core promoter in association with genotype C and a longer duration of infection in the aggravation of chronic hepatitis B.

Adult↗

PubMed2001

Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan.

The geographic distribution of hepatitis B virus (HBV) genotypes in Japan and its clinical relevance are poorly understood. We studied 731 Japanese patients with chronic HBV infection. HBV genotype was determined by the restriction fragment length polymorphism (RFLP) method after polymerase chain reaction (PCR). Of the 720 patients with positive PCR, 12 (1.7%) were HBV genotype A, 88 (12.2%) were genotype B, 610 (84.7%) were genotype C, 3 (0.4%) were genotype D, and 7 (1.0%) were of mixed genotype. Over 94% of patients on the Japanese mainland had genotype C, while 60% of the patients on Okinawa, the most southern islands, and 22.9% in the Tohoku area, the northern part of the mainland, harbored genotype B. Compared with genotype C patients, genotype B patients were older (53.6 to 42.2 years; P <.01), had a lower rate of positive hepatitis B e antigen (HBeAg) (18.4% to 50.6%; P <.01), and a lower level of serum HBV DNA (5.02 to 5.87 log genome equivalents (LGE)/mL; P <.01). The mean age of the genotype B patients with hepatocellular carcinoma was 70.1 +/- 9.2 years, compared with 55.2 +/- 9.7 of genotype C patients (P <.01). These results indicate that genotypes C and B are predominant in Japan, and there are significant differences in geographic distribution and clinical characteristics among the patients with the different genotypes.

Adult↗

PubMed2001

Evaluation of accumulation of hepatitis C virus mutations in a chronically infected chimpanzee: comparison of the core, E1, HVR1, and NS5b regions.

Four hepatitis C virus genome regions (the core, E1, HVR1, and NS5b) were amplified and sequenced from yearly samples obtained from a chronically infected chimpanzee over a 12-year span. Nucleotide substitutions were found to accumulate in the core, E1, and HVR1 regions during the course of chronic infection; substitutions within the NS5b region were not detected for the first 8 years and were found to be minimal during the last 4 years. The rate of accumulation of mutations in the core and E1 regions, based on a direct comparison between the first 1979 sequence and the last 1990 sequence, was 1.120 x 10(-3), while phylogenetic ancestral comparison using the 12 yearly sequences showed a rate of 0.816 x 10(-3) bases per site per year. Temporal evaluation of the sequences revealed that there appeared to be periods in which substitutions accumulated and became fixed, followed by periods with relative stasis or random substitutions that did not persist. Synonymous and nonsynonymous substitutions within the core, E1, and HVR1 regions were also analyzed. In the core and E1 regions, synonymous substitutions predominated and gradually increased over time. However, within the HVR1 region, nonsynonymous substitutions predominated but gradually decreased over time.

Animals↗

PubMed2001

Hepatitis B virus genotype distribution among chronic hepatitis B virus carriers in Shanghai, China.

OBJECTIVE: Hepatitis B virus (HBV) genotype distribution is still unclear in China, where a high prevalence of HBV infection exists, although it is well known that HBV can be classified into six genotypes based on intergroup divergence. The aim of this study was to investigate the epidemiological distribution of HBV genotypes and to clarify further the genotype-related differences in the pathogenicity of HBV. METHODS: Seminested PCR and restriction fragment length polymorphism analysis were conducted in 97 asymptomatic HBV carriers (ASC) and 46 chronic hepatitis (CH), 37 liver cirrhosis (LC) and 44 hepatocellular carcinoma (HCC) patients in Shanghai, China. RESULTS: Two hundred and twenty samples (98.2%) were positive for HBV DNA, and of these, 3 (1.4%), 38 (17.2%) and 179 (81.4%) were classified as genotype A, B and C, respectively. There was a statistically significant difference in the distribution of genotypes B and C among various categories of liver diseases (p < 0.01). The distribution of genotype C showed an increasing trend from ASC, CH and LC to the HCC group; in contrast, the distribution of genotype B showed a decreasing trend in the same order. HBeAg positivity was higher in genotype C than in genotype B in all the subjects or in the ASC group alone (p < 0.05, p < 0.01, respectively). More severe liver damage and a higher mean age were observed in genotype C than in genotype B (p < 0.01, p < 0.05, respectively). CONCLUSIONS: These results indicate the following: (1) genotypes A, B and C of HBV exist in Shanghai, China; (2) genotype C is the major genotype in this area; (3) genotype C is associated with the development of severe liver diseases, and (4) genotype B has a relatively good prognosis.

Adolescent↗

PubMed2001

Risk factors and seroprevalence of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infection in uzbekistan.

OBJECTIVES: The aim of this study was to elucidate the seroprevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) infection in Uzbekistan and to explore whether there is a correlation between those blood-borne agents and socioeconomic risk factors. METHODS: One thousand nine hundred and eighteen subjects were studied. The subjects were divided into a low-risk group, a high-risk group and a patient group. Sera were tested for HBV surface antigen (HBsAg), anti-HCV, and anti-HIV. RESULTS: The seroprevalence of HBsAg, anti-HCV, and anti-HIV in the general population was 13.3, 13.1 and 0%, respectively. The anti-HCV infection rate was significantly higher in intravenous drug users (62.7%) than in prostitutes (9.2%), homosexuals (11.1%), and medical laboratory employees (12.5%) (p < 0.01). In the low-risk group, positivity for anti-HCV increased with age from 2.2% in the 15- to 20-year-olds up to the highest rate of 17.6% in the 31- to 40-year-olds; the positivity then decreased to 0% in the group over 60 years of age. In the high-risk group, the positivity for anti-HCV in the age groups under 40 years was approximately 30% and significantly higher than in the low-risk group (p < 0.01). Risk factors for transmission of HCV were medical treatment in the low-risk group, drug abuse in the high-risk group, and both in the patient group. CONCLUSIONS: This study demonstrates that the seroprevalence of HBV and HCV infection is high, whereas HIV infection is yet uncommon in Uzbekistan.

Adolescent↗

PubMed2000

Genomic and molecular evolutionary analysis of a newly identified infectious agent (SEN virus) and its relationship to the TT virus family.

A new group of transmissible single-stranded (ss) DNA viruses (SENV) distantly related to the large TT virus (TTV) family was recently identified. Eight different SENV isolates have been found, some with an association with posttransfusion hepatitis. A phylogenetic analysis of near-complete open-reading frame 1, including conserved motifs and excluding recombinant regions, was performed. The analysis used TTV-like minivirus as an outgroup, to determine a root of the phylogenetic tree, and compared 8 SENV isolates, 6 prototype TTV isolates, and 7 TTV variants (including SANBAN, TUS01, PMV, and YONBAN). Four distinct clusters separated by a bootstrap value of 100% were observed. YONBAN isolates formed a distinct outer group, representing the earliest recognized phylogenetic divergence (group 1). Prototype TTV formed group 2, PMV formed group 3, and SENV, SANBAN, and TUS01 isolates formed group 4, the most recently evolved group. This taxonomic classification suggests that these circular ssDNA viruses probably evolved from a common ancestor virus.

Amino Acid Sequence↗

PubMed2000

Molecular evolutionary analysis of the complete nucleotide sequence of hepatitis B virus (HBV) in a case of HBV infection acquired through a needlestick accident.

To elucidate needlestick transmission of hepatitis B virus (HBV), strains isolated from 1 physician who acquired HBV infection through a needlestick accident and 3 patients with chronic hepatitis B (donor patients A, B, and C) were tested using molecular evolutionary analysis based on full-length HBV genomic sequences. Nucleotide sequences of these isolates were aligned with 55 previously reported full-length genomic sequences. Genetic distances were estimated using the 6-parameter method, and phylogenetic trees were constructed using the neighbor-joining method. Strains isolated from patient A and the recipient pair were clustered within a closer range of evolutionary distances than were strains recovered from the recipient pair and patients B and C. Furthermore, strains from patient A and the recipient were also clustered on the S gene sequences of HBV. These results demonstrated that patient A alone was the source of direct transmission to the recipient. This approach can be used to investigate the transmission route of HBV.

Adult↗

PubMed2000

Hepatitis B virus infection among residents of a nursing home for the elderly: seroepidemiological study and molecular evolutionary analysis.

A seroepidemiological study of HBV infection was carried out to investigate the seroprevalence of hepatitis B surface antigen (HBsAg) and the transmission routes of hepatitis B virus (HBV) infection among residents of a nursing home for the elderly. HBV serum markers were examined in 119 residents and 71 healthcare workers in the institution, as also in 1330 healthy subjects from the same geographical area, as the control group. HBsAg was detected in 6 (5%), 0 and 20 (1.5%) residents, healthcare workers and healthy subjects, respectively. Four residents (A-D) who had HBV-DNA in the serum were studied by molecular evolutionary analysis. The strains derived from residents A, B and D were clustered together within a close range of evolutionary distances. Residents B and D, who were not positive for HBsAg at the time of admission to the institution, subsequently became HBsAg-positive asymptomatic carriers. These results suggested intrainstitutional transmission of HBV in the nursing home for the elderly, and confirmed that the source of transmission of HBV to residents B and D was resident A who was positive for HBsAg. Residents in a nursing home for the elderly should be considered as being a high-risk group for HBV infection, and vaccination against HBV of these groups is recommended.

Aged↗

PubMed2000

Identification of a novel 23kDa protein encoded by putative open reading frame 2 of TT virus (TTV) genotype 1 different from the other genotypes.

We report the entire open reading frames (ORFs) sequences of four TT virus (TTV) isolates, one genotype 2 (G2) and three G4 isolates. Despite a DNA virus, TTV possesses high rate of amino acid (aa) substitution: the aa sequence homology of ORF1 and 2 is lower than the nucleotide homology. The partial 'N22' region of ORF1 is suitable for genotyping of 'prototype TTV' isolates, because the phylogenetic tree from partial 'N22' sequence is consistent with that from the entire ORF1. Based on our sequence data, ORF2 from most isolates excluding G1 encode truncated 49 aa (pORF2a) because of an in-frame stop codon, although ORF2s from most G1 isolates encode 202 aa (pORF2ab). Just downstream the stop codon, another ORF encoding a protein of approximately 150 aa (pORF2b) is found, whose homology is quite low among these genotypes. Our in vitro transcription/translation study supports that all G1a and a part of G b without an in-frame stop codon dominantly encode pORF2ab, a novel 23 kDa protein, whereas the other genotypes with an in-frame stop codon encode pORF2b (17 kDa). Our data indicate TTV G1a and a part of G1b should have different characteristics from the other genotypes.

Amino Acid Sequence↗

PubMed2000

TT virus infection in patients with chronic hepatitis C virus infection--effect of primers, prevalence, and clinical significance. Hepatitis Interventional Therapy Group.

BACKGROUND/AIM: A novel DNA virus, TT virus (TTV), was recently identified in patients with post-transfusion non-A-G hepatitis. The aim of this study was to determine the prevalence and clinical significance of TTV infection in patients with chronic hepatitis C virus (HCV) infection. METHODS: We analyzed pretreatment serum samples from 171 United States and European patients who relapsed after interferon-alpha treatment and were recruited into an interferon-alpha-2b/ribavirin combination treatment trial. TTV DNA was detected by PCR using two different set of primers (TTV-A and TTV-B) derived from open reading frames 1 and 2, respectively. RESULTS: TTV was detected in 29.2% of the patients with the TTV-A primer set, 70.8% with the TTV-B primer-set, and 72.5% if positive by either/both sets of the primers. The amplicons generated by primer set A were sequenced and a phylogenetic tree was constructed. The 50 isolates belonged to group la (n=8), 1b (n=17), 2a (n=21), 2b (n=3), and 4 (n=1). There was no difference in demographic (age, sex distribution, estimated duration of HCV infection), biochemical (serum ALT levels), virologic (serum HCV RNA levels, HCV genotype distribution), or histologic scores, and their subsequent response to either interferon-alpha-2b or interferon-alpha-2b/ribavirin combination treatment. CONCLUSIONS: The prevalence of TTV infection reported previously may have been significantly underestimated, based on the primers originally described and used by most studies. Although TTV infection is very common in patients with chronic HCV infection, it has no identifiable clinical significance.

Adult↗

PubMed2000

Prevalence and risk factors of hepatitis C virus infection among Koreans in rural area of Korea.

In a Korean rural area with a high incidence of liver cancer, a molecular epidemiological study was conducted to estimate the prevalence of hepatitis C virus (HCV) infection and to determine the distribution of HCV genotype and transmission routes of HCV infection. The study population is those who volunteered to participate in a health survey in 1993 and were over 10 years of age. Those who were anti-HCV-positive and 20 of their spouses were re-examined in 1995 and tested for HCV genotype. A phylogenetic tree was constructed after sequencing analysis of core and envelope regions from the subjects with genotype 2a without a blood transfusion history. The age- and sex-standardized prevalence of anti-HCV was 5.52%. In a multivariate analysis, transfusion history was not associated with anti-HCV-positivity, but a history of acupuncture (adjusted odds ratio=2.2, 95% confidence interval=1.0, 4.7) and a history of surgical operation (adjusted odds ratio=2.0, 95% confidence interval=1.0, 4.1) were associated. The prevalence of HCV genotype 2a was the highest, and genotype 1b was less frequent. The phylogenetic tree showed strong homology among our samples with Japanese HCV strains. The present study suggests that there is a highly endemic area of HCV infection in Korea and that this endemicity is probably associated with a parenteral source of HCV infection other than blood transfusion. The study also suggests that some of the HCV infection was spread through non-sterilized or non-disposable acupuncture needles in this HCV endemic area.

Journal Article↗

PubMed2000

Clinical, virological and histological implications of GB virus-C/hepatitis G virus infection in patients with chronic hepatitis C virus infection: a multicentre study based on 671 patients.

To determine the prevalence, as well as the clinical, virological and histological implications of GB virus C/hepatitis G virus (GBC-C/HGV) infection in patients with chronic hepatitis C virus (HCV) infection, sera from 671 well-characterized patients with chronic HCV infection were tested for GBV-C/HGV RNA using a sensitive and specific reverse transcription 'nested' polymerase chain reaction (RT-nPCR). GBV-C/HGV RNA was detected in 65 of 671 (9. 7%) patients with chronic HCV infection. Importantly, GBV-C/HGV co-infection was not associated with any changes in indices of liver diseases, including serum alanine transaminase levels, Knodell score or histology activity index (HAI). In this cohort, GBV-C/HGV co-infection was weakly associated with a shorter mean estimated duration of HCV infection and a higher median HCV viraemia level. We conclude that GBV-C/HGV has minimal or no impact on liver disease activity in patients with chronic HCV infection. This data supports the notion that GBV-C/HGV may not be a hepatitis virus.

Adult↗

PubMed2000

Relationship between histological prognosis of chronic hepatitis C and amount of hepatitis C virus core protein in serum.

BACKGROUND: Hepatitis C virus (HCV) viraemia is one of the factors for histological prognosis of chronic hepatitis C. METHODS: One hundred and thirty-five patients who received hepatic biopsies twice at intervals of 5 years or longer were followed up for a mean of 9.7 +/- 4.0 years were studied retrospectively. The amount of HCV viraemia present was measured as the concentration of HCV core protein by using the fluorescence enzyme immunoassay method. RESULTS: Multiple-regression analysis, using deterioration of the histological stage as a dependent variable, showed that greater age (P = 0.041), higher stage of hepatic histology at the start of follow up (P = 0.029), and higher serum concentration of core protein (P < 0.001) were independent factors affecting the deterioration of the liver's histological stage. At follow up, no significant difference in histological stage was seen between patients with serum HCV core protein > or = 100 pg/mL (n = 60) and those with serum core protein < 100 pg/mL (n = 75). The histological grade in patients with high serum core-protein levels tended to be significantly worse and the deterioration rate of the histological stage was significantly higher than in those with low HCV core protein levels (68 vs 35%, P < 0.001). The mutation rate of the HCV envelope-2/non-structural 1 (E2/NS1) nucleotide region was compared in two patients who had high serum concentrations of HCV core protein and whose histological stage had deteriorated with two patients who had low serum concentrations of the core protein and whose histological stages remained unchanged. No significant difference in E2/NS1 mutation was found. CONCLUSIONS: The amount of HCV viraemia was suggested to be a significant factor for determining histological outcome in patients with chronic hepatitis C. The mutation rate in the E2/NS1 region did not seem to be associated with the prognosis of chronic hepatitis C.

Adult↗

PubMed2000

Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis--A comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma.

A study was conducted to examine the significance of genetic instability and aberrant DNA methylation during hepatocarcinogenesis. Genomic DNA was extracted from 196 microdissected specimens of noncancerous liver tissue that showed no marked histologic findings or findings compatible with chronic hepatitis or cirrhosis, and 80 corresponding microdissected specimens of hepatocellular carcinoma (HCC) from 40 patients. Loss of heterozygosity (LOH) and microsatellite instability (MSI) were examined by polymerase chain reaction (PCR) using 39 microsatellite markers, and DNA methylation status on 8 CpG islands was examined by bisulfite-PCR. In noncancerous liver tissues, LOH, MSI, and DNA hypermethylation were found in 15 (38%), 6 (15%), and 33 (83%) of 40 cases, respectively. The incidence of DNA hypermethylation in histologically normal liver was similar to that in chronic hepatitis and cirrhosis, although neither LOH nor MSI was found in histologically normal liver. In cancerous tissues, LOH, MSI, and DNA hypermethylation were found in 39 (98%), 8 (20%), and 40 (100%) of 40 cases, respectively. CpG islands of the p16 gene and methylated in tumor 1, 2, 12, and 31 clones were frequently methylated in cancerous tissues, although neither the thrombospondin-1 nor the human Mut L homologue (hMLH1) gene was methylated. Absence of silencing of the hMLH1 gene by DNA hypermethylation is consistent with the low incidence of MSI in HCCs. The results of this study indicate that LOH and aberrant DNA methylation contribute to hepatocarcinogenesis; DNA hypermethylation in particular, which precedes or may even cause LOH, is as an early event during hepatocarcinogenesis.

Adult↗