John K. Olynyk

A population-based study of the clinical expression of the hemochromatosis gene

BACKGROUND AND METHODS Hereditary hemochromatosis is associated with homozygosity for the C282Y mutation in the hemochromatosis (HFE) gene on chromosome 6, elevated serum transferrin saturation, and excess iron deposits throughout the body. To assess the prevalence and clinical expression of the HFE gene, we conducted a population-based study in Busselton, Australia. In 1994, we obtained blood samples for the determination of serum transferrin saturation and ferritin levels and the presence or absence of the C282Y mutation and the H63D mutation (which may contribute to increased hepatic iron levels) in 3011 unrelated white adults. We evaluated all subjects who had persistently elevated transferrin-saturation values (45 percent or higher) or were homozygous for the C282Y mutation. We recommended liver biopsy for subjects with serum ferritin levels of 300 ng per milliliter or higher. The subjects were followed for up to four years. RESULTS Sixteen of the subjects (0.5 percent) were homozygous for the C282Y mutation, and 424 (14.1 percent) were heterozygous. The serum transferrin saturation was 45 percent or higher in 15 of the 16 who were homozygous; in 1 subject it was 43 percent. Four of the homozygous subjects had previously been given a diagnosis of hemochromatosis, and 12 had not. Seven of these 12 patients had elevated serum ferritin levels in 1994; 6 of the 7 had further increases in 1998, and 1 had a decrease, although the value remained elevated. The serum ferritin levels in the four other homozygous patients remained in the normal range. Eleven of the 16 homozygous subjects underwent liver biopsy; 3 had hepatic fibrosis, and 1, who had a history of excessive alcohol consumption, had cirrhosis and mild microvesicular steatosis. Eight of the 16 homozygous subjects had clinical findings that were consistent with the presence of hereditary hemochromatosis, such as hepatomegaly, skin pigmentation, and arthritis. CONCLUSIONS In a population of white adults of northern European ancestry, 0.5 percent were homozygous for the C282Y mutation in the HFE gene. However, only half of those who were homozygous had clinical features of hemochromatosis, and one quarter had serum ferritin levels that remained normal over a four-year period.

Nomenclature of the finer branches of the biliary tree: Canals, ductules, and ductular reactions in human livers

The work of liver stem cell biologists, largely carried out in rodent models, has now started to manifest in human investigations and applications. We can now recognize complex regenerative processes in tissue specimens that had only been suspected for decades, but we also struggle to describe what we see in human tissues in a way that takes into account the findings from the animal investigations, using a language derived from species not, in fact, so much like our own. This international group of liver pathologists and hepatologists, most of whom are actively engaged in both clinical work and scientific research, seeks to arrive at a consensus on nomenclature for normal human livers and human reactive lesions that can facilitate more rapid advancement of our field. (HEPATOLOGY 2004; 39:1739–1745.)

Iron-overload-related disease in HFE hereditary hemochromatosis.

BACKGROUND Most persons who are homozygous for C282Y, the HFE allele most commonly asssociated with hereditary hemochromatosis, have elevated levels of serum ferritin and transferrin saturation. Diseases related to iron overload develop in some C282Y homozygotes, but the extent of the risk is controversial. METHODS We assessed HFE mutations in 31,192 persons of northern European descent between the ages of 40 and 69 years who participated in the Melbourne Collaborative Cohort Study and were followed for an average of 12 years. In a random sample of 1438 subjects stratified according to HFE genotype, including all 203 C282Y homozygotes (of whom 108 were women and 95 were men), we obtained clinical and biochemical data, including two sets of iron measurements performed 12 years apart. Disease related to iron overload was defined as documented iron overload and one or more of the following conditions: cirrhosis, liver fibrosis, hepatocellular carcinoma, elevated aminotransferase levels, physician-diagnosed symptomatic hemochromatosis, and arthropathy of the second and third metacarpophalangeal joints. RESULTS The proportion of C282Y homozygotes with documented iron-overload-related disease was 28.4% (95% confidence interval [CI], 18.8 to 40.2) for men and 1.2% (95% CI, 0.03 to 6.5) for women. Only one non-C282Y homozygote (a compound heterozygote) had documented iron-overload-related disease. Male C282Y homozygotes with a serum ferritin level of 1000 mug per liter or more were more likely to report fatigue, use of arthritis medicine, and a history of liver disease than were men who had the wild-type gene. CONCLUSIONS In persons who are homozygous for the C282Y mutation, iron-overload-related disease developed in a substantial proportion of men but in a small proportion of women.

Oval Cell Numbers in Human Chronic Liver Diseases Are Directly Related to Disease Severity

The risk of developing hepatocellular carcinoma is significantly increased in patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C infection. The precise mechanisms underlying the development of hepatocellular carcinoma in these conditions are not well understood. Stem cells within the liver, termed oval cells, are involved in the pathogenesis of hepatocellular carcinoma in animal models and may be important in the development of hepatocellular carcinoma in human chronic liver diseases. The aims of this study were to determine whether oval cells could be detected in the liver of patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C, and whether there is a relationship between the severity of the liver disease and the number of oval cells. Oval cells were detected using histology and immunohistochemistry in liver biopsies from patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. Oval cells were not observed in normal liver controls. Oval cell numbers increased significantly with the progression of disease severity from mild to severe in each of the diseases studied. We conclude that oval cells are frequently found in subjects with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. There is an association between severity of liver disease and increase in the number of oval cells consistent with the hypothesis that oval cell proliferation is associated with increased risk for development of hepatocellular carcinoma in chronic liver disease.

NAFLD as a risk factor for the development of diabetes and the metabolic syndrome: an eleven-year follow-up study.

OBJECTIVES:Non-alcoholic fatty liver disease (NAFLD) uncommonly results in cirrhosis and liver-related death; however, its impact on the development of metabolic complications remains unclear. We sought to determine whether NAFLD with elevated aminotransaminase (ALT) levels was a risk factor for incident diabetes or the metabolic syndrome (MS) over an 11-year period.METHODS:Adult residents of Busselton, Western Australia underwent assessment in 1994–1995 as part of the Busselton Health Survey. NAFLD was diagnosed on the basis of a raised ALT (>40 IU/l) after the exclusion of alcohol, viral, metabolic, and autoimmune liver disease. NAFLD and non-NAFLD subjects were reassessed in 2005 for liver complications, diabetes, and the MS.RESULTS:A total of 358 subjects, 68% male (109 NAFLD, 249 non-NAFLD), mean age (s.d.) 59.9 (11.6) years, attended follow-up 11.1 years after the initial assessment. After excluding subjects with diabetes at baseline, those with NAFLD were more likely to have developed diabetes on follow-up (20/106, 18.9% vs. 15/246, 6.1%; P<0.001). After excluding subjects with MS at baseline, those with NAFLD were more likely to have developed MS at follow-up (27/81, 33.3% vs. 51/226, 22.6%; P=0.056). However, in multivariate logistic regression models, NAFLD was no longer a significant independent predictor of the development of diabetes or MS after adjusting for baseline waist circumference, hypertension, and insulin resistance. None of the subjects developed liver complications.CONCLUSIONS:Subjects with NAFLD and elevated ALT levels are at an increased risk of developing diabetes and the MS. This may be because of the presence of associated metabolic risk factors.

Hepatic iron concentration as a predictor of response to interferon alfa therapy in chronic hepatitis C

BACKGROUND/AIMS It has been reported that hepatic iron concentration (HIC) may influence response to therapy in chronic viral hepatitis. The aim of this study was to determine the relationship between HIC and response to interferon alfa therapy in patients with chronic hepatitis C. METHODS HIC was measured in liver biopsy specimens from 58 patients with chronic hepatitis C treated at three centers. Three patients had mild chronic hepatitis C, 35 had moderate to severe chronic hepatitis C, and 20 had active cirrhosis. Serum ferritin levels were measured in 51 of these 58 patients. Response to therapy was defined as normalization of alanine aminotransferase levels at the end of treatment. RESULTS Twenty-four patients (41%) responded to therapy. HICs were generally within the normal range (< 1500 micrograms/g). The mean HIC in nonresponders (860 +/- 100 micrograms/g; range, 116-2296 micrograms/g) was significantly higher than in responders (548 +/- 85 micrograms/g; range, 29-1870 micrograms/g) (P < 0.05). Eighty-eight percent of patients with an HIC of > 1100 micrograms/g and 87% of patients with an elevated serum ferritin concentration did not respond to interferon alfa therapy. CONCLUSIONS HIC seems to influence response to interferon alfa therapy among patients with chronic hepatitis C. A subgroup of patients with chronic hepatitis C has been identified for which an HIC of > 1100 micrograms/g predicted nonresponse in 88% of patients.

A population-based study of the biochemical and clinical expression of the H63D hemochromatosis mutation

BACKGROUND & AIMS Two major mutations are defined within the hemochromatosis gene, HFE. Although the effects of the C282Y mutation have been well characterized, the effects of the H63D mutation remain unclear. We accessed a well-defined population in Busselton, Australia, and determined the frequency of the H63D mutation and its influence on total body iron stores. METHODS Serum transferrin saturation and ferritin levels were correlated with the H63D mutation in 2531 unrelated white subjects who did not possess the C282Y mutation. RESULTS Sixty-two subjects (2.1%) were homozygous for the H63D mutation, 711 (23.6%) were heterozygous, and 1758 (58.4%) were wild-type for the H63D mutation. Serum transferrin saturation was significantly increased in male and female H63D homozygotes and heterozygotes compared with wild-types. Serum ferritin levels within each gender were not influenced by H63D genotypes. Elevated transferrin saturation > or = 45% was observed in a greater proportion of male H63D carriers than male wild-types. Male H63D homozygotes (9%) and heterozygotes (3%) were more likely to have both elevated transferrin saturation and elevated ferritin > or = 300 ng/mL than male wild-types (0.7%). Homozygosity for H63D was not associated with the development of clinically significant iron overload. CONCLUSIONS Presence of the H63D mutation results in a significant increase in serum transferrin saturation but does not result in significant iron overload. In the absence of the C282Y mutation, the H63D mutation is not clinically significant.

HFE Genotype in Patients with Hemochromatosis and Other Liver Diseases

Hereditary hemochromatosis is a common inherited disorder of iron metabolism that affects between 1 in 200 and 1 in 400 persons of northern European descent (1). With early diagnosis and appropriate treatment, survival of patients is normal (1, 2). Recently, researchers identified a novel MHC class 1-like gene, HFE, which contains two missense mutations (3). Eighty-three percent of 178 typical patients with hemochromatosis were homozygous for one of these mutations (Cys282Tyr [C282Y]) (3). Subsequent studies from the United States, Australia, France, and Italy showed homozygosity for the C282Y mutation in 64% to 100% of patients with hemochromatosis (4-7). Heterozygosity for the second mutation (His63Asp [H63D]) is seen in 15% to 20% of the general population; this mutation is not believed to cause the same extent of progressive iron loading (3-7). The estimated allelic frequency of these mutations in white populations is 0.04 for the C282Y mutation and 0.14 for the H63D mutation (1). The presence of C282Y homozygosity and direct (elevated hepatic iron concentration) or indirect (elevated transferrin saturation or ferritin level) evidence of increased iron stores constitute the current gold standard for a definitive diagnosis of hemochromatosis (1, 8). Approximately 40% to 50% of patients with alcoholic liver disease (9), chronic viral hepatitis (10), and nonalcoholic steatohepatitis (11) have abnormal results on blood iron studies. About 5% to 10% of these patients have a modestly increased hepatic iron concentration, but not to the degree seen in typical patients with hemochromatosis. Clinicians have suspected that some of these patients are heterozygous for hemochromatosis; however, without pedigree studies (using HLA haplotyping) of a family with hemochromatosis, this interpretation has been only speculative (10, 11). A high prevalence of C282Y heterozygosity was found in patients with nonalcoholic steatohepatitis (12, 13). In patients with hepatitis C (14-16) and patients with alcoholic liver disease (17), researchers have found an incidence of C282Y heterozygosity equivalent to that in control populations. Recently, a genetic test for hemochromatosis that analyzes the C282Y and H63D mutations has become available. This test allows genotyping of patients who have typical hemochromatosis and those who have liver disease with or without abnormal results on iron studies. We evaluated the contribution of HFE genotyping to the diagnosis of hemochromatosis and determined the prevalence of HFE mutations in a group of patients with liver disease. Methods Patients with Hemochromatosis Between September 1990 and September 1997, clinical hemochromatosis was newly diagnosed in 66 patients by using one of two criteria: 1) a compatible result on liver biopsy (iron deposits of 2+, 3+, or 4+, predominantly in hepatocytes) and a hepatic iron index greater than 1.9 mmol/kg per year [18-22] or 2) HLA identity to a proband. A subset of these 66 patients (n=44) was included in a previous study (3) that identified the HFE gene. Patients with Liver Disease Between January 1996 and September 1997, we performed HFE genotyping on 132 patients with various liver diseases for whom we had also obtained a hepatic iron concentration. Nineteen of these patients were referred for suspected hemochromatosis on the basis of abnormal results on iron studies. The cause of liver disease was thoroughly evaluated in these 132 patients by examination of history of alcohol consumption, viral serologic studies for hepatitis B and C, autoimmune markers (antinuclear antibody, anti-smooth-muscle antibody, and antimitochondrial antibody), and markers of inherited metabolic diseases (transferrin saturation, ferritin level, ceruloplasmin level, and 1-antitrypsin level and protein typing). Laboratory Studies To aid in initial diagnosis, fasting transferrin saturation (reference range, 0.16 to 0.5), ferritin level (reference range, 15 to 200 g/L in women and 30 to 300 g/L in men), routine chemistry panel, and complete blood count were obtained for all patients. Specific serologic studies were also done, as appropriate. All studies were performed at routine clinical laboratories. After obtaining informed consent from all patients, we performed standard percutaneous liver biopsy. Sections of liver tissue were prepared in a routine manner. Staining was done with hematoxylin and eosin, the periodic acid-Schiff test after diastase digestion, Masson trichrome stain, Sweet reticulin stain, and Perls Prussian blue stain. Iron deposits in hepatocytes were graded from 0 to 4+ (23). Hepatic iron concentration was measured on a portion of the liver biopsy sample, as described by Torrance and Bothwell (24). The upper limit of normal used in our laboratory is 26.8 mmol/kg dry weight (1500 g/g dry weight). Hepatic iron index was calculated as hepatic iron concentration (mmol/kg) divided by age (years) (18). HFE genotyping for the C282Y and H63D mutations was performed by oligonucleotide ligation assays on polymerase chain reaction-amplified genomic samples of DNA taken from each patient (3). After obtaining informed consent, we drew blood for HFE genotyping from all patients with liver disease and from all patients with hemochromatosis (before the test became commercially available) by using protocols approved by the institutional review board of Saint Louis University. Statistical Analysis Data are presented as the median and range. Statistical comparisons among the cumulative distributions of the groups were performed by using the exact Komolgorov-Smirnov two-sample test. A P value less than 0.01 was considered statistically significant. Role of the Funding Sources The funding sources had no role in the collection, analysis, or interpretation of the data or in the decision to submit the paper for publication. Results Genotype by Patient Group Of the 66 patients who had clinically diagnosed hemochromatosis, 60 (91%) were C282Y homozygotes, 2 (3%) were compound heterozygotes, 1 (1.5%) was a C282Y heterozygote, 2 (3%) were H63D heterozygotes, and 1 (1.5%) was negative for both mutations. Of the 132 patients with liver disease, 80 had chronic hepatitis C, 19 had abnormal results on serum iron studies and had been referred for evaluation of iron overload or suspected hemochromatosis, 17 had nonalcoholic steatohepatitis, 4 had primary biliary cirrhosis or primary sclerosing cholangitis, and 12 had other liver disorders. Of these 132 patients, 6 (5%) were C282Y homozygotes, 8 (6%) were compound heterozygotes, 6 (5%) were C282Y heterozygotes, 5 (4%) were H63D homozygotes, 20 (15%) were H63D heterozygotes, and 87 (66%) were negative for both mutations. In the group of 19 patients with abnormal results on iron studies who were referred for evaluation of iron overload, 5 (26.5%) were C282Y homozygotes, 1 (5%) was a C282Y heterozygote, 1 (5%) was an H63D homozygote, 5 (26.5%) were H63D heterozygotes, and 7 (37%) were negative for both mutations. Iron Status by HFE Genotype To define and analyze the iron status of all patients for whom genotyping had been performed, we grouped patients with hemochromatosis and patients with liver disease together and divided them according to genotype (Table 1). Additional information about patients with certain genotypes is presented in Tables 2, 3, and 4. Table 1. HFE Genotype, Age, Serum Iron Studies, and Hepatic Iron Studies in 198 Patients with Hemochromatosis and Other Liver Diseases C282Y Homozygotes Of the 66 patients who were homozygous for the C282Y mutation (C282Y/C282Y), 40 were men and 26 were women. Median age at diagnosis was 45 years and 49 years, respectively. At diagnosis, men and women had similar transferrin saturations (P>0.2) and ferritin levels (P=0.07). The hepatic iron concentration (median, 159.1 mmol/kg dry weight [8910 g/g dry weight]; P<0.01) and the hepatic iron index (median, 3.25 mmol/kg per year; P<0.01) were higher in C282Y homozygotes than in the groups of patients with other genotypes (Figure 1). The hepatic iron concentration and the hepatic iron index were similar in male and female C282Y homozygotes (P>0.2 [data not shown]). On biopsy, all patients had hepatic iron deposition of grade 2+ to 4+. Three women and 9 men had substantial fibrosis or cirrhosis on liver biopsy; 6 of these patients had alanine aminotransferase levels or aspartate aminotransferase levels above the upper limit of normal. The median age of patients with fibrosis was 54 years (range, 40 to 72 years). The youngest patient with cirrhosis was a 40-year-old man with concomitant chronic hepatitis C. The youngest patients with cirrhosis or substantial fibrosis who were homozygous for the C282Y mutation and did not have contributing factors were a 47-year-old man and a 47-year-old woman. Figure 1. Hepatic iron concentration ( top ) and hepatic iron index ( bottom ) according to HFE genotype. Ten of 66 patients (15% [CI, 7.5% to 26%]) who were C282Y homozygotes had a hepatic iron index less than 1.9 mmol/kg per year. Specific findings of these patients are detailed in Table 2. Six of these 10 patients were men; 1 was a blood donor. Four of these patients (patients 1, 2, 5, and 8) were identified by HLA haplotyping in family studies; the other 6 were identified from the group of patients with liver disease who were not suspected of having hemochromatosis at the time of initial evaluation. These patients were thought to be heterozygotes or to have concomitant nonalcoholic steatohepatitis. Nine of the 10 patients had a transferrin saturation of at least 45%, and all 10 patients had a hepatic iron concentration above the upper limit of normal. All patients had hepatic iron deposition of grade 2+ to 4+, and only 1 had minimal fibrosis on biopsy. Table 2. Characteristics of C282Y Homozygotes with a Hepatic Iron Index Less Than 1.9 mmol/kg per Year When C282Y homozygosity is used as the gold standard for the diagnosis of hemochromatosis, the sensitivity

TWEAK and LTβ signaling during chronic liver disease

Chronic liver diseases (CLD) such as hepatitis B and C virus infection, alcoholic liver disease, and non-alcoholic steatohepatitis are associated with hepatocellular necrosis, continual inflammation, and hepatic fibrosis. The induced microenvironment triggers the activation of liver-resident progenitor cells (LPCs) while hepatocyte replication is inhibited. In the early injury stages, LPCs regenerate the liver by proliferation, migration to sites of injury, and differentiation into functional biliary epithelial cells or hepatocytes. However, when this process becomes dysregulated, wound healing can progress to pathological fibrosis, cirrhosis, and eventually hepatocellular carcinoma. The other key mediators in the pathogenesis of progressive CLD are fibrosis-driving, activated hepatic stellate cells (HSCs) that usually proliferate in very close spatial association with LPCs. Recent studies from our group and others have suggested the potential for cytokine and chemokine cross-talk between LPCs and HSCs, which is mainly driven by the tumor necrosis factor (TNF) family members, TNF-like weak inducer of apoptosis (TWEAK) and lymphotoxin-β, potentially dictating the pathological outcomes of chronic liver injury.

Oval cell‐mediated liver regeneration: Role of cytokines and growth factors

Abstract   In experimental models, which induce liver damage and simultaneously block hepatocyte proliferation, the recruitment of a hepatic progenitor cell population comprised of oval cells is invariably observed. There is a substantial body of evidence to suggest that oval cells are involved in liver regeneration, as they differentiate into hepatocytes and biliary cells. Recently, bone marrow cells were shown to be a source of a stem cells with the capacity to repopulate the liver. Presently, the relationship between bone marrow cells and oval cells is unclear. Investigations will be greatly assisted by the availability of in vitro models based on a knowledge of cytokines that affect oval cells. While the cytokines, which regulate the different hematopoietic lineages, are well characterized, there is relatively little information regarding those that influence oval cells. This review outlines recent developments in the field of oval cell research and focuses on cytokines and growth factors that have been implicated in regulating oval cell proliferation and differentiation.