Robert S. Britton

Targeted mutagenesis of the murine transferrin receptor-2 gene produces hemochromatosis

Hereditary hemochromatosis (HH) is a common genetic disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, particularly liver. The vast majority of individuals with HH are homozygous for mutations in the HFE gene. Recently a second transferrin receptor (TFR2) was discovered, and a previously uncharacterized type of hemochromatosis (HH type 3) was identified in humans carrying mutations in the TFR2 gene. To characterize the role for TFR2 in iron homeostasis, we generated mice in which a premature stop codon (Y245X) was introduced by targeted mutagenesis in the murine Tfr2 coding sequence. This mutation is orthologous to the Y250X mutation identified in some patients with HH type 3. The homozygous Tfr2Y245Xmutant mice showed profound abnormalities in parameters of iron homeostasis. Even on a standard diet, hepatic iron concentration was several-fold higher in the homozygous Tfr2Y245Xmutant mice than in wild-type littermates by 4 weeks of age. The iron deposition in the mutant mice was predominantly hepatocellular and periportal. The mean splenic iron concentration in the homozygous Tfr2Y245Xmutant mice was significantly less than that observed in the wild-type mice. The homozygous Tfr2Y245Xmutant mice also demonstrated elevated transferrin saturations. There were no significant differences in parameters of erythrocyte production including hemoglobin levels, hematocrits, erythrocyte indices, and reticulocyte counts. Heterozygous Tfr2Y245Xmice did not differ in any measured parameter from wild-type mice. This study confirms the important role for TFR2 in iron homeostasis and provides a tool for investigating the excess iron absorption and abnormal iron distribution in iron-overload disorders.

Iron Toxicity and Chelation Therapy

Iron is an essential mineral for normal cellular physiology, but an excess can result in cell injury. Iron in low-molecular-weight forms may play a catalytic role in the initiation of free radical reactions. The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates; the result is wide-ranging impairment in cellular function and integrity. The rate of free radical production must overwhelm the cytoprotective defenses of cells before injury occurs. There is substantial evidence that iron overload in experimental animals can result in oxidative damage to lipids in vivo, once the concentration of iron exceeds a threshold level. In the liver, this lipid peroxidation is associated with impairment of membrane-dependent functions of mitochondria and lysosomes. Iron overload impairs hepatic mitochondrial respiration primarily through a decrease in cytochrome C oxidase activity, and hepatocellular calcium homeostasis may be compromised through damage to mitochondrial and microsomal calcium sequestration. DNA has also been reported to be a target of iron-induced damage, and this may have consequences in regard to malignant transformation. Mitochondrial respiratory enzymes and plasma membrane enzymes such as sodium-potassium-adenosine triphosphatase (Na++K+-ATPase) may be key targets of damage by non-transferrin-bound iron in cardiac myocytes. Levels of some antioxidants are decreased during iron overload, a finding suggestive of ongoing oxidative stress. Reduced cellular levels of ATP, lysosomal fragility, impaired cellular calcium homeostasis, and damage to DNA all may contribute to cellular injury in iron overload. Evidence is accumulating that free-radical production is increased in patients with iron overload. Iron-loaded patients have elevated plasma levels of thiobarbituric acid reactants and increased hepatic levels of aldehyde-protein adducts, indicating lipid peroxidation. Hepatic DNA of iron-loaded patients shows evidence of damage, including mutations of the tumor suppressor gene p53. Although phlebotomy therapy is effective in removing excess iron in hereditary hemochromatosis, chelation therapy is required in the treatment of many patients who have combined secondary and transfusional iron overload due to disorders in erythropoiesis. In patients with β-thalassemia who undergo regular transfusions, deferoxamine treatment has been shown to be effective in preventing iron-induced tissue injury and in prolonging life expectancy. The use of the oral chelator deferiprone remains controversial, and work is continuing on the development of new orally effective iron chelators.

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

Mouse strain differences determine severity of iron accumulation in Hfe knockout model of hereditary hemochromatosis

Hereditary hemochromatosis (HH) is a common disorder of iron metabolism caused by mutation in HFE, a gene encoding an MHC class I-like protein. Clinical studies demonstrate that the severity of iron loading is highly variable among individuals with identical HFE genotypes. To determine whether genetic factors other than Hfe genotype influence the severity of iron loading in the murine model of HH, we bred the disrupted murine Hfe allele onto three different genetically defined mouse strains (AKR, C57BL/6, and C3H), which differ in basal iron status and sensitivity to dietary iron loading. Serum transferrin saturations (percent saturation of serum transferrin with iron), hepatic and splenic iron concentrations, and hepatocellular iron distribution patterns were compared for wild-type (Hfe +/+), heterozygote (Hfe +/−), and knockout (Hfe −/−) mice from each strain. Although the Hfe −/− mice from all three strains demonstrated increased transferrin saturations and liver iron concentrations compared with Hfe +/+ mice, strain differences in severity of iron accumulation were striking. Targeted disruption of the Hfe gene led to hepatic iron levels in Hfe −/− AKR mice that were 2.5 or 3.6 times higher than those of Hfe −/− C3H or Hfe −/− C57BL/6 mice, respectively. The Hfe −/− mice also demonstrated strain-dependent differences in transferrin saturation, with the highest values in AKR mice and the lowest values in C3H mice. These observations demonstrate that heritable factors markedly influence iron homeostasis in response to Hfe disruption. Analysis of mice from crosses between C57BL/6 and AKR mice should allow the mapping and subsequent identification of genes modifying the severity of iron loading in this murine model of HH.

Oxidant injury to hepatic mitochondria in patients with Wilson's disease and Bedlington terriers with copper toxicosis

BACKGROUND/AIMS Copper overload leads to liver injury in humans with Wilsons disease and in Bedlington terriers with copper toxicosis; however, the mechanisms of liver injury are poorly understood. This study was undertaken to determine if oxidant (free radical) damage to hepatic mitochondria is involved in naturally occurring copper toxicosis. METHODS Fresh liver samples were obtained at the time of liver transplantation from 3 patients with Wilsons disease, 8 with cholestatic liver disease, and 5 with noncholestatic liver disease and from 8 control livers. Fresh liver was also obtained by open liver biopsy from 4 copper-overloaded and 4 normal Bedlington terriers and from 8 control dogs. Hepatic mitochondria and microsomes (humans only) were isolated, and lipid peroxidation was measured by lipid-conjugated dienes and thiobarbituric acid-reacting substances. In humans, liver alpha-tocopherol content was measured. RESULTS Lipid peroxidation and copper content were significantly increased (P < 0.05) in mitochondria from patients with Wilsons disease and copper-overloaded Bedlington terriers. More modest increases in lipid peroxidation were present in microsomes from patients with Wilsons disease. Mitochondrial copper concentrations correlated strongly with the severity of mitochondrial lipid peroxidation. Hepatic alpha-tocopherol content was decreased significantly in Wilsons disease liver. CONCLUSIONS These data suggest that the hepatic mitochondrion is an important target in hepatic copper toxicity and that oxidant damage to the liver may be involved in the pathogenesis of copper-induced injury.

Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis

The protein defective in hereditary hemochromatosis, called HFE, is similar to MHC class I-type proteins and associates with β2-microglobulin (β2M). Its association with β2M was previously shown to be necessary for its stability, normal intracellular processing, and cell surface expression in transfected COS cells. Here we use stably transfected Chinese hamster ovary cell lines expressing both HFE and β2M or HFE alone to study the effects of β2M on the stability and maturation of the HFE protein and on the role of HFE in transferrin receptor 1 (TfR1)-mediated iron uptake. In agreement with prior studies on other cell lines, we found that overexpression of HFE, without overexpressing β2M, resulted in a decrease in TfR1dependent iron uptake and in lower iron levels in the cells, as evidenced by ferritin and TfR1 levels measured at steady state. However, overexpression of both HFE and β2M had the reverse effect and resulted in an increase in TfR1-dependent iron uptake and increased iron levels in the cells. The HFE-β2M complex did not affect the affinity of TfR1 for transferrin or the internalization rate of transferrin-bound TfR1. Instead, HFE-β2M enhanced the rate of recycling of TfR1 and resulted in an increase in the steady-state level of TfR1 at the cell surface of stably transfected cells. We propose that Chinese hamster ovary cells provide a model to explain the effect of the HFE-β2M complex in duodenal crypt cells, where the HFE-β2M complex appears to facilitate the uptake of transferrin-bound iron to sense the level of body iron stores. Impairment of this process in duodenal crypt cells leads them to be iron poor and to signal the differentiating enterocytes to take up iron excessively after they mature into villus cells in the duodenum of hereditary hemochromatosis patients.

Hepatic injury in chronic iron overload. Role of lipid peroxidation

In both hereditary hemochromatosis and in the various forms of secondary hemochromatosis, there is a pathologic expansion of body iron stores due mainly to an increase in absorption of dietary iron. Excess deposition of iron in the parenchymal tissues of several organs (e.g. liver, heart, pancreas, joints, endocrine glands) results in cell injury and functional insufficiency. In the liver, the major pathological manifestations of chronic iron overload are fibrosis and ultimately cirrhosis. Evidence for hepatotoxicity due to iron has been provided by several clinical studies, however the specific pathophysiologic mechanisms for hepatocellular injury and hepatic fibrosis in chronic iron overload are poorly understood. The postulated mechanisms of liver injury in chronic iron overload include (a) increased lysosomal membrane fragility, perhaps mediated by iron-induced lipid peroxidation, (b) peroxidative damage to mitochondria and microsomes resulting in organelle dysfunction, (c) a direct effect of iron on collagen biosynthesis and (d) a combination of all of the above.

Determination of hepatic iron concentration in fresh and paraffin-embedded tissue: Diagnostic implications

BACKGROUND/AIMS Determination of hepatic iron concentration (HIC) is essential for the evaluation of hereditary hemochromatosis. Occasionally, only paraffin-embedded liver biopsy specimens are available, or fresh biopsy specimens have been placed in saline for transport. This study aimed to describe a method for extraction of liver tissue from paraffin blocks, determine the accuracy of measurement of HIC in recovered tissue compared with fresh tissue, and determine the effect of immersion in saline on HIC. METHODS HIC was measured in both fresh and deparaffinized liver specimens (n = 41). Accurate measurements were defined as either a normal result in both specimens or a result in the deparaffinized specimen that was within 30% of the fresh measurement. RESULTS Measurements of HIC in fresh and deparaffinized tissue showed an excellent linear relationship (r = 0.95). In deparaffinized samples > or = 0.4 mg, accurate measurements were seen in 24 out of 29 specimens, compared with 6 out of 12 specimens weighing < 0.4 mg (P < 0.01). The hepatic iron index calculated from results in deparaffinized samples > or = 0.4 mg correctly classified all patients. Immersion of fresh biopsy specimens in saline for 1 hour resulted in up to 50% iron loss (P < 0.05). CONCLUSIONS Accurate measurement of HIC in deparaffinized liver biopsy specimens is possible. Calculation of the hepatic iron index from deparaffinized liver tissue can facilitate diagnosis of hemochromatosis when fresh tissue is not available. Samples should not be transported in saline.

Repetitive Self-Limited Acute Pancreatitis Induces Pancreatic Fibrogenesis in the Mouse

The role of repetitive acute injury in the pathogenesis of chronic pancreatitis remains unknown. To determine if repetitive injury induced by pancreatic hyperstimulation would reproduce the characteristic features of human chronic pancreatitis, acute reversible pancreatic injury was induced in mice by twice weekly cerulein treatment, 50 μg/kg/hr × 6 hr, for 10 weeks. Procollagen α1(I) mRNA was markedly increased by week 2. Sirius red staining of interstitial collagen demonstrated progressive accumulation of extracellular matrix surrounding acinar units and in interlobular spaces. Atrophy, transdifferentiation of acinar units to ductlike tubular complexes, and dilatation of intraacinar lumina also developed. Electron microscopy demonstrated the presence of stromal cells in areas of fibrosis with morphologic characteristics of pancreatic stellate cells. These findings demonstrate that, in a murine model, repetitive acute injury to the pancreas by hyperstimulation can reproduce the major morphological characteristics of human chronic pancreatitis.

PATHOPHYSIOLOGY OF IRON TOXICITY

There are several inherited and acquired disorders that can result in chronic iron overload in humans, and the major clinical consequences are hepatic fibrosis, cirrhosis, hepatocellular cancer, cardiac disease, and diabetes. It is clear that lipid peroxidation occurs in experimental iron overload if sufficiently high levels of iron within hepatocytes are achieved. Lipid peroxidation is associated with hepatic mitochondrial and microsomal dysfunction in experimental iron overload, and lipid peroxidation may underlie the increased lysosomal fragility that has been detected in liver samples from both iron-loaded human subjects and experimental animals. Reduced cellular ATP levels, impaired cellular calcium homeostasis, and damage to DNA may all contribute to hepatocellular injury in iron overload. Long-term dietary iron overload in rats can lead to increased collagen gene expression and hepatic fibrosis, perhaps due to activation of hepatic lipocytes. The mechanisms whereby lipocytes are activated in iron overload remain to be elucidated; possible mediators include aldehydic products of iron-induced lipid peroxidation produced in hepatocytes, tissue ferritin, and/or cytokines released by activated Kupffer cells.