POGLUT1, the putative effector gene driven by rs2293370 in primary biliary cholangitis susceptibility locus chromosome 3q13.33.

Primary biliary cholangitis (PBC) is a chronic and cholestatic autoimmune liver disease caused by the destruction of intrahepatic small bile ducts. Our previous genome-wide association study (GWAS) identified six susceptibility loci for PBC. Here, in order to further elucidate the genetic architecture of PBC, a GWAS was performed on an additional independent sample set, then a genome-wide meta-analysis with our previous GWAS was performed based on a whole-genome single nucleotide polymorphism (SNP) imputation analysis of a total of 4,045 Japanese individuals (2,060 cases and 1,985 healthy controls). A susceptibility locus on chromosome 3q13.33 (including ARHGAP31, TMEM39A, POGLUT1, TIMMDC1, and CD80) was previously identified both in the European and Chinese populations and was replicated in the Japanese population (OR = 0.7241, P = 3.5 × 10-9). Subsequent in silico and in vitro functional analyses identified rs2293370, previously reported as the top-hit SNP in this locus in the European population, as the primary functional SNP. Moreover, e-QTL analysis indicated that the effector gene of rs2293370 was Protein O-Glucosyltransferase 1 (POGLUT1) (P = 3.4 × 10-8). This is the first study to demonstrate that POGLUT1 and not CD80 is the effector gene regulated by the primary functional SNP rs2293370, and that increased expression of POGLUT1 might be involved in the pathogenesis of PBC.

Importance of the Average Glucose Level and Estimated Glycated Hemoglobin in a Diabetic Patient with Hereditary Hemolytic Anemia and Liver Cirrhosis.

Glycated hemoglobin (HbA1c) is a widely used marker of glycemic control but can be affected by hemolytic anemia. Glycated albumin (GA) is also affected in patients with liver cirrhosis. We herein report the assessment of glycemic control in a 41-year-old man with dehydrated hereditary stomatocytosis and a PIEZO1 gene mutation complicated by diabetes mellitus and liver cirrhosis due to hemochromatosis. The estimated HbA1c calculated from the average glucose level obtained by continuous glucose monitoring or by self-monitoring of blood glucose was useful for evaluating the glycemic control in this patient, as HbA1c and GA were unreliable due to the coexisting conditions.

Serum IFN-λ3 Levels Correlate with Serum Hepatitis C Virus RNA Levels in Symptomatic Patients with Acute Hepatitis C.

BACKGROUND: Recent genome-wide association studies demonstrated that 2 single nucleotide polymorphisms (SNPs), upstream of the interferon-λ (IFNL) 3 gene, are associated with the spontaneous clearance of hepatitis C virus (HCV) in symptomatic patients with acute hepatitis C (AHC). Although these 2 SNPs, rs8099917 and rs12979860, have established their significant roles in the innate immunity response to spontaneously clear HCV in patients with AHC, the detailed mechanisms of their roles remain largely unknown. AIM: This study is aimed at clarifying the factors affecting IFNL3 production and assessing the roles of IFNL3 in AHC. MATERIALS AND METHODS: A total of 21 AHC patients who visited the hospital within 10 days after symptom onset were assessed. As controls, 23 healthy volunteers (HVs) were examined. Serum IFNL3 levels were quantified using an in-house, IFNL3-specific chemiluminescence enzyme immunoassay (CLEIA) kit. Serum IFNL1, IFN-α, IFN-ß, and IFN-γ induced protein-10 (IP-10) levels were assayed using commercial enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: At baseline, serum IFNL3 levels were higher in AHC patients than in HVs (p < 0.0001). The higher levels in AHC patients did not differ between patients with the rs8099917 TT genotype and those with the non-TT (TG/GG) genotype (p = 0.546). Serial measurement of serum IFNL3 levels did not predict the outcome of conventional AHC. However, serum IFNL3 levels at baseline correlated positively with the HCV RNA levels (p = 0.005). Following HCV eradication, serum IFNL3 levels reduced to within the range obtained for HVs. Baseline serum IFNL1 levels did not differ significantly between AHC patients and HVs (p = 0.284). Serum levels of IFNL1 and IFNL3 at baseline also showed no correlative power (p = 0.288). Serum IFN-α and IFN-ß were detected together with remarkably high serum IFNL3 levels in only one patient who progressed to acute liver failure (ALF). CONCLUSION: These findings indicate that serum IFNL3 levels at baseline are higher in AHC patients regardless of the rs8099917 polymorphism, and primary HCV infection triggers the production of IFNL3. As a first line of defense in the innate immune system against invading HCV, increased IFNL3 levels play an important role, but serum IFNL3 levels are not the principal determinant of the clinical course of conventional AHC.

Selection of regimens for patients with hepatitis C virus genotype/serotype discrepancy.

In the present study, the usefulness of the resistance-associated variant (RAV) analysis to select direct acting antiviral (DAA) drugs for patients with hepatitis C virus (HCV) genotype/serotype discrepancy was evaluated. The core-genotype and serotype were determined in the 559 patients recruited in the study. The RAV analysis and NS5B-genotype determination were performed in the eight patients who exhibited a genotype/serotype discrepancy. One of these patients exhibited a core-genotype 1b/serotype 2, and detection by RAV analysis was possible in this patient. The other seven patients demonstrated a core-genotype 2/serotype 1, and detection using the RAV analysis was possible in four of them. The NS5B-genotype was 1b in all patients in whom detection using the RAV analysis was possible and was other than 1b in patients in whom detection using the RAV analysis was impossible. The RAV analysis could detect RNA sequences specific to genotype 1b in the NS5A region. Therefore, in patients with genotype/serotype discrepancy in whom detection using the RAV analysis is possible, the treatment regimens should be selected based on the assumption that HCV with genome that is highly homologous to genotype 1b is present in the NS5A region.

Genome-wide association studies identify PRKCB as a novel genetic susceptibility locus for primary biliary cholangitis in the Japanese population.

A previous genome-wide association study (GWAS) performed in 963 Japanese individuals (487 primary biliary cholangitis [PBC] cases and 476 healthy controls) identified TNFSF15 (rs4979462) and POU2AF1 (rs4938534) as strong susceptibility loci for PBC. In this study, we performed GWAS in additional 1,923 Japanese individuals (894 PBC cases and 1,029 healthy controls), and combined the results with the previous data. This GWAS, together with a subsequent replication study in an independent set of 7,024 Japanese individuals (512 PBC cases and 6,512 healthy controls), identified PRKCB (rs7404928) as a novel susceptibility locus for PBC (odds ratio [OR] = 1.26, P = 4.13 × 10-9). Furthermore, a primary functional variant of PRKCB (rs35015313) was identified by genotype imputation using a phased panel of 1,070 Japanese individuals from a prospective, general population cohort study and subsequent in vitro functional analyses. These results may lead to improved understanding of the disease pathways involved in PBC, forming a basis for prevention of PBC and development of novel therapeutics.

Long-term outcomes of add-on adefovir dipivoxil therapy to ongoing lamivudine in patients with lamivudine-resistant chronic hepatitis B.

AIM: Add-on adefovir dipivoxil (ADV) therapy has been a standard rescue treatment for patients with lamivudine (LAM)-resistant chronic hepatitis B, but the overall benefits of long-term add-on ADV therapy are still limited. The aim of this study was to evaluate the long-term efficiency of add-on ADV treatment and to explore predictive factors associated with it. METHODS: A total of 158 patients with LAM-resistant chronic hepatitis B were included in this retrospective, multicenter, nationwide study in Japan. After confirming LAM resistance, ADV was added to LAM treatment. Three types of events were considered as outcomes: virological response, hepatitis B e antigen (HBeAg) clearance and alanine aminotransferase (ALT) normalization. Virological response was defined as serum hepatitis B virus (HBV) DNA levels of less than 3 log copies/mL. Baseline factors contributing to these outcomes were examined by univariate and multivariate analyses. RESULTS: The median total duration of ADV treatment was 41 months (range, 6-84). The rate of virological response was 90.8% at 4 years of treatment; HBeAg clearance and ALT normalization were achieved by 34.0% and 82.7%, respectively, at the end of follow up. Each outcome had different predictive factors: baseline HBV DNA and albumin level were predictive factors for virological response, history of interferon therapy and ALT level for HBeAg clearance, and sex and baseline albumin level for ALT normalization. CONCLUSION: Long-term add-on ADV treatment was highly effective in LAM-resistant chronic hepatitis B patients in terms of virological and biochemical responses. Lower HBV replication and lower albumin level at baseline led to better outcomes.

Hepatitis B virus strains of subgenotype A2 with an identical sequence spreading rapidly from the capital region to all over Japan in patients with acute hepatitis B.

OBJECTIVE: To examine recent trends of acute infection with hepatitis B virus (HBV) in Japan by nationwide surveillance and phylogenetic analyses. METHODS: During 1991 through 2009, a sentinel surveillance was conducted in 28 national hospitals in a prospective cohort study. Genotypes of HBV were determined in 547 patients with acute hepatitis B. Nucleotide sequences in the preS1/S2/S gene of genotype A and B isolates were determined for phylogenetic analyses. RESULTS: HBV genotype A was detected in 137 (25% (accompanied by genotype G in one)) patients, B in 48 (9%), C in 359 (66%), and other genotypes in the remaining three (0.5%). HBV persisted in five with genotype A including the one accompanied by genotype G; another was co-infected with HIV type 1. The genotype was A in 4.8% of patients during 1991-1996, 29.3% during 1997-2002, and 50.0% during 2003-2008 in the capital region, as against 6.5%, 8.5% and 33.1%, respectively, in other regions. Of the 114 genotype A isolates, 13 (11.4%) were subgenotype A1, and 101 (88.6%) were A2, whereas of the 43 genotype B isolates, 10 (23.3%) were subgenotype B1, 28 (65.1%) were B2, two (4.7%) were B3, and three (7.0%) were B4. Sequences of 65 (64%) isolates of A2 were identical, as were three (23%) of A1, and five (18%) of B2, but none of the B1, B3 and B4 isolates shared a sequence. CONCLUSIONS: Acute infection with HBV of genotype A, subgenotype A2 in particular, appear to be increasing, mainly through sexual contact, and spreading from the capital region to other regions in Japan nationwide. Infection persisted in 4% of the patients with genotype A, and HBV strains with an identical sequence prevailed in subgenotype A2 infections. This study indicates the need for universal vaccination of young people to prevent increases in HBV infection in Japan.

Genetic polymorphisms in CTLA4 and SLC4A2 are differentially associated with the pathogenesis of primary biliary cirrhosis in Japanese patients.

BACKGROUND: Anti-gp210 and anti-centromere antibodies are different risk factors for the progression of primary biliary cirrhosis (PBC). In order to dissect the genetic basis for the production of these autoantibodies, as well as the development and progression of PBC in Japanese patients, we examined single nucleotide polymorphisms (SNPs) in cytotoxic T-lymphocyte antigen 4 (CTLA4) and solute carrier family 4 anion exchanger, member 2 (SLC4A2), which have been associated with the pathogenesis of PBC in Caucasian patients. METHODS: Four SNPs for both CTLA4 and SLC4A2 were genotyped, using the polymerase chain reaction-restriction fragment length polymorphism method and TaqMan assay, in 450 Japanese PBC patients and 371 sex-matched healthy controls. RESULTS: The CTLA4 rs231775, rs3087243, and rs231725 SNPs were significantly associated with PBC susceptibility. The CTLA4 rs231725 SNP was significantly associated with progression to late-stage disease. The CTLA-4 haplotype 1 (rs231775 G, rs231777 C, rs3087243 G, rs231725 A; GCGA) was a risk factor for PBC susceptibility but a protective factor for PBC progression. Conversely, the CTLA-4 haplotype 2 (ACAG) was a protective and risk factor, respectively, for PBC susceptibility and progression. In addition, the CTLA4 rs231777 SNP and haplotype 3 (ATGG) was significantly associated with anti-gp210 antibody production, while SLC4A2 haplotype 4 (rs2069443 A, rs2303933 G, rs2303937 A, rs2303941 T; AGAT) and haplotype 3 (AAGC) were significantly associated with PBC susceptibility and anti-centromere antibody production, respectively. CONCLUSIONS: CTLA4 and SLC4A2 genetic polymorphisms are differentially associated with PBC development and progression, as well as anti-gp210 or anti-centromere antibody production, in Japanese PBC patients.

[A case of epithelioid hemangioendothelioma of the liver: detection by FDG-PET].

A 57 year-old woman was admitted for focal accumulation of 18F-fluorodeoxyglucose (FDG) in the liver detected by positron emission tomography (PET). A 25- mm hypovascular tumor was detected by computed tomography. Tumor biopsy revealed many atypical cells with positive staining for factor VIII-related-antigen in sinusoids. Right lobectomy was performed and the tumor was diagnosed as epithelioid hemangioendothelioma (EHE) pathologically. We demonstrated that FDG-PET was useful for the diagnosis of EHE and making deciding on therapeutic strategy.

Index of the systemic balance of end products of glucocorticoid metabolism in fresh urine from humans.

OBJECTIVE: Dysregulation of tissue-specific intracellular glucocorticoid reactivation is implicated in obesity and related metabolic diseases in humans. The ratio of end products of glucocorticoid metabolism in fresh urine sample, tetrahydrocortisol (THF) + allo-tetrahydrocortisol (allo-THF) vs. tetrahydrocortisone (THE), i.e., the urinary ratio is regarded as an index of the systemic balance underlying intracellular glucocorticoid metabolism, where the enzymes, 11ß-hydroxysteroid dehydrogenase type 1 and type 2 as well as 5α- and 5ß-reductase are involved in a tissue-specific manner. METHODS: To explore the clinical implications of the urinary ratio in obesity and related metabolic diseases, the urinary ratio was determined by gas chromatography and mass spectrometry. RESULTS: The urinary ratio was shown to be constant and reproducible in the same individuals. The ratio was found to inversely correlate with BMI (P < 0.01), waist circumference (P < 0.01), and liver transaminase (P < 0.05) in a large cohort of ∼200 Japanese subjects. This finding suggests that the systemic balance underlying intracellular glucocorticoid reactivation was suppressed in obesity and liver dysfunction. Consistent with this notion, the ratio was decreased in patients with non-alcoholic steatohepatitis (P < 0.01). The urinary ratio was not altered in patients with type 2 diabetes on a 2-month mild calorie restriction. In contrast, the ratio was significantly reduced in patients who responded to the anti-diabetic pioglitazone (P < 0.01). CONCLUSION: The present study provides novel evidence that the urinary ratio reflects the facet of adipose tissue and liver function in humans, thereby offering a unique opportunity to evaluate obesity-related diseases.

Genome-wide semiquantitative microsatellite analysis of human hepatocellular carcinoma: discrete mapping of smallest region of overlap of recurrent chromosomal gains and losses.

Recurrent chromosomal gains at 1q, 6p, 8q, and 17q, or losses at 1p, 4q, 6q, 8p, 9p, 13q, 16q, and 17p are common features of human hepatocellular carcinoma (HCC). For precise determination of the shortest region of overlap (SRO), 49 HCC obtained at the time of surgery or autopsy were subjected to comprehensive microsatellite analysis by using 400 markers distributed at almost equal distances throughout the 22 autosomes and X chromosomes. Each allele showing imbalance was subjected to comparative duplex polymerase chain reaction using a retained allele as an internal control to determine whether the imbalance was the result of chromosomal gain or loss. The following SRO of recurrent chromosomal gains and losses were determined: -1p36.22 approximately p36.33, D1S450-D1S2893, 5.0 mega-base pairs (Mbp); +1q23.3 approximately q25.3, D1S2878-D1S2619, 16.9 Mbp; -4q21.2 approximately q24, D4S2964-D4S1572, 23.0 Mbp; -6q23.3 approximately qter, D6S292-qter, 34.7 Mb; -8p22 approximately p23.1, D8S549-D8S550, 4.8 Mbp; +8q12.2 approximately q24.13, D8S260-D8S514, 61.8 Mbp; -13q13.3 approximately q22.1, D13S218-D13S156, 35.6 Mbp; -16q22.1 approximately qter, D16S503-qter, 26.7 Mbp; and -17p12 approximately pter, D17S921-pter, 14.2 Mbp. Contrary to our initial expectations, many HCC showed major deletions or additions of chromosome arms, so that a number of genes were included in the SRO. Although some putative oncogenes or tumor suppressor genes mapped in these SRO may be important, relative copy number changes of numerous other genes may affect pathogenesis of HCC.

Interferon-alpha improves bone resorption and osteopenia in patients with chronic hepatitis C.

BACKGROUND: Interferon (IFN)-beta is known to be involved in the regulation of bone homeostasis. As IFN-alpha and -beta share the same receptor complex and signaling pathway, we speculated that treatment with IFN-alpha for chronic hepatitis C (CHC) may provide a beneficial effect on bone loss. METHODS: Urinary deoxypyridinoline (uDPD) of 41 patients with CHC who had been receiving IFN-alpha for 24 weeks was examined during the period of observation. Among them, eight patients showed a bone mineral density (BMD) of less than 0.850g/cm(2) before IFN therapy and they were examined a BMD again after completion of IFN administration. Relationships between the percentage difference of uDPD after discontinuation of IFN and various factors related to CHC were also examined. RESULTS: A mean uDPD of 7.1+/-3.4nM/mM creatinine before IFN therapy decreased to 4.5+/-2.4 in the 4th week and 4.2+/-2.7 in the 24th week of IFN therapy, respectively (p<0.0001). The reduction in uDPD was more prominent in cases with a lower viral load (p=0.0266). The BMD of the eight patients, which was less than 0.850g/cm(2) before IFN therapy, showed significant increase after the end of therapy (p=0.0172). CONCLUSION: IFN-alpha can improve bone resorption in CHC patients, especially in those with a lower viral load, and increased BMD. These effects are thought to be a result of direct action of IFN on bone homeostasis.

Obesity and NASH in Japan.

BACKGROUND: NASH is expected to be a major target disease in near future, although we are too short in information to realize NASH. In this study, we revealed the prevalence of fatty liver, which is a background disease of NASH, in Kyoto. Then we made histological diagnosis of NASH from chronic liver disorder with fatty liver. MATERIALS AND METHODS: By using the data obtained from medical check-up with more than 1000 subjects per year in Kyoto, the prevalence of fatty liver on ultrasound, serum ALT, body mass index (BMI) were compared between 1994 and 2004. Next 32 patients with elevated ALT and bright liver on ultrasound were biopsied in order to diagnose them as NASH or not. Biochemical, serological and hormonal parameters as well as adipocytokine profiles were also studied. Lastly, liver tissues from those patients were examined to compare the expression of peroxisome proliferator activator receptor (PPAR) alpha and gamma. RESULTS: It was revealed that the prevalence of fatty liver increased from 12.9 to 34.7% over 10 years. The mean ALT and BMI also increased in this period. From 32 patients, 6 cases were excluded for alcohol, autoimmune hepatitis, and drug-induced liver injury, leaving 26 cases to enter the study. Among them, 12 and 8 patients showed grade 1 and grade 2 steatohepatitis, which were consistant with NASH. Various data were compared among simple steatosis, grade 1 steatohepatitis, and grade 2 steatohepatitis, but highly sensitive CRP was the sole parameter with statistical significance. PPAR alpha seems to be expressed higher in patients with steatohepatitis, although PPAR alpha did not show any significance. CONCLUSION: We found the prevalence of fatty liver was increasing over 10 years. The histological examination showed more patients with grade 1 or grade 2 steatohepatitis than expected, which means we might have much more NASH cases undiagnosed. We were unable to propose reliable diagnostic markers to diagnose NASH from this study.

Genotype stability and clonal evolution of hepatocellular carcinoma assessed by autopsy-based genome-wide microsatellite analysis.

It is widely accepted that chromosomal instability is an essential feature of cancer cells including hepatocellular carcinoma (HCC) cells. For an accurate characterization of clonal evolution of HCC cells, we studied chromosomal alterations in various metastatic lesions in an autopsy case of HCC. Tissues from the main tumor, which consisted of 2 macroscopically distinct portions, and from intrahepatic metastasis, portal vein thrombus, epiploic lymph node metastasis, and pulmonary metastasis as well as from the non-tumorous liver were analyzed with comprehensive microsatellite analysis. Alleles showing imbalance of the main tumor were further subjected to comparative duplex PCR, with use of a retained allele as an internal control, to determine whether the imbalance was the result of chromosomal gain or loss. A striking finding was that allelic imbalances detected in the main tumor and metastatic lesions were almost identical, showing -1p, +1q, -4q, -7, -8p, +8q, +9q, +10, -13q, -17p, +19p, -19q, and -X. Additional alterations of +2q and -16q were detected in one portion of the main tumor and the portal vein thrombus. In conclusion, clonal evolution of the HCC cells during metastatic progression seems rare, in contrast to many recurrent chromosomal aberrations that may have accumulated before the clinical manifestation.

Discrete breakpoint mapping and shortest region of overlap of chromosome arm 1q gain and 1p loss in human hepatocellular carcinoma detected by semiquantitative microsatellite analysis.

Recurrent chromosomal gain at 1q is one of the most common features of human hepatocellular carcinoma (HCC), but how the gain at 1q contributes to hepatocarcinogenesis is still unclear. To identify the target genes, precise determination of the shortest region of overlap (SRO) and of breakpoints is necessary. Similarly, the role of loss at 1p, which is also a major cytogenetic aberration in HCC, needs to be determined. Fifty HCCs were examined with the aid of 59 microsatellite markers distributed throughout both arms of chromosome 1. To detect allelic gain effectively, the cutoff value of the allelic imbalance index was set at 0.70. Alleles showing imbalance were subjected to multiplex PCR, using a retained allele as an internal control, to determine whether the imbalance was the result of chromosomal gain or loss. The SRO of the gains was defined as D1S2878-D1S2619 (1q23.-q25.3, 16.9 Mb), which involved 36 cases (72%). Gains in the number of copies of certain oncogenes within this region seemed to be critical for the pathogenesis of HCC. In contrast, the centromeric breakpoints of these gains varied, but they tended to occur mainly in the pericentromeric region (26 of 50 cases, 52%). Rearrangement of specific genes associated with the gains is unlikely. On the other hand, the SRO of deletion was defined as D1S2893-D1S450 (1p36.32-p36.22, 5.1 Mb). Four known putative tumor-suppressor genes (TP73, RIZ1, NBL1/DAN, and CDKN2C) were outside the SRO, suggesting the presence of other candidate genes with critical roles in hepatocarcinogenesis.

Alteration of the p14(ARF) gene and p53 status in human hepatocellular carcinomas.

BACKGROUND: The INK4a/ARF locus encodes p16INK4a and p14ARF, both of which are crucial for two tumor suppressor pathways, retinoblastoma (RB)/p16INK4a and p53/ARF. Inactivation of RB/p16INK4a was frequently reported, but alterations of the p14ARF gene in hepatocellular carcinoma (HCC) in the Japanese population have been insufficiently analyzed. METHODS: To determine the role of p53/ARF alteration in hepatocarcinogenesis, we examined 44 HCCs for mRNA expression, deletion, mutation, and promoter hypermethylation of the p14(ARF) gene; alterations of p53 were also analyzed in the same series of HCCs. RESULTS: Homozygous deletion, spanning from exon 1 beta to exon 2, was found in 1 HCC mutations within exon 2 were found in 2 HCCs, but no promoter hypermethylation was detected. All 3 HCCs with p14(ARF) alteration were well differentiated. Twelve of the 44 HCCs (27.2%) showed immunohistochemical evidence of p53 alteration; however, only 1 of the tumors with p53 alteration was well differentiated. TaqMan polymarase chain reaction (PCR) indicated that the expression of p14(ARF) in HCCs was higher than in that in all but three of the corresponding non-tumorous tissues ( P < 0.0001), and increased expression of p14(ARF) seemed to be associated with poorly differentiated phenotype. Absence of p14(ARF) expression was seen in only one HCC, with homozygous deletion of the p14(ARF) gene. CONCLUSIONS: Compared with p53 alteration, p14(ARF) alteration does not occur frequently, but may play a role in a subset of Japanese HCCs in the early stage of hepatocarcinogenesis. On the other hand, overexpression of p14(ARF) was frequently observed in HCC, especially in poorly differentiated tumors, probably reflecting oncogenic stimuli in these tumors. The INK4a/ARF locus encodes p16(INK4a) and p14(ARF), both of which are crucial for two tumor suppressor pathways, retinoblastoma (RB)/p16(INK4a) and p53/ARF. Inactivation of RB/p16(INK4a) was frequently reported, but alterations of the p14(ARF) gene in hepatocellular carcinoma (HCC) in the Japanese population have been insufficiently analyzed.

Comprehensive allelotyping of well-differentiated human hepatocellular carcinoma with semiquantitative determination of chromosomal gain or loss.

Allelic imbalance (AI), which represents certain chromosomal gains or losses, has been described in human hepatocellular carcinoma (HCC), but the impact of AI on the early stage of hepatocarcinogenesis has not been fully clarified. Moreover, no previous allelotype studies have identified the difference in chromosomal gain and loss that results in AI. To resolve these problems, we examined 18 well-differentiated HCCs with comprehensive allelotyping by using 400 microsatellite markers with semiquantitative assessment of chromosomal gain or loss. To detect allelic gain effectively, the cutoff value of the allelic imbalance index was set at 0.70. Each allele showing imbalance was subjected to multiplex PCR with use of a retained allele as an internal control to determine whether the imbalance was the result of chromosomal gain or loss. High frequencies of chromosomal gains were detected at 1q (D1S196-D1S2785, 56%), 5q (D5S647-D5S2027, 44%), 6p (6pter-D6S309, 33%), 7 (7pter-D7S657, 22%), and 8q (D8S514-qter, 44%), whereas chromosomal losses were frequently observed at 1p (1pter-D1S234, 22%), 8p (8pter-D8S549, 44%), and 17p (17pter-D17S921, 28%). The extent of overall chromosomal aberration was closely related to the maximum tumor diameter (P = 0.002) and the presence of hepatitis B surface antigen (P = 0.03). Recurrent chromosomal losses at 1p and 8p and gains at 1q and 8q, even in HCCs with a minimal extent of aberrant chromosomes, indicate that these alterations were critical in the early stage of hepatocarcinogenesis. On the other hand, deletions of 13q and 16q were infrequent and were seen only in the most aberrant cases, which suggested that these were late events.