William J.H. Griffiths

Ferroportin disease: A systematic meta-analysis of clinical and molecular findings

Background & Aims Classical ferroportin disease is characterized by hyperferritinemia, normal transferrin saturation, and iron overload in macrophages. A non-classical form is characterized by additional hepatocellular iron deposits and a high transferrin saturation. Both forms demonstrate autosomal dominant transmission and are associated with ferroportin gene (SLC40A1) mutations. SLC40A1 encodes a cellular iron exporter expressed in macrophages, enterocytes, and hepatocytes. The aim of the analysis is to determine the penetrance of SLC40A1 mutations and to evaluate in silico tools to predict the functional impairment of ferroportin mutations as an alternative to in vitro studies. Methods We conducted a systematic review of the literature and meta-analysis of the biochemical presentation, genetics, and pathology of ferroportin disease. Results Of the 176 individuals reported with SLC40A1 mutations, 80 were classified as classical phenotype with hyperferritinemia and normal transferrin saturation. The non-classical phenotype with hyperferritinemia and elevated transferrin saturation was present in 53 patients. The remaining patients had normal serum ferritin or the data were reported incompletely. Despite an increased hepatic iron concentration in all biopsied patients, significant fibrosis or cirrhosis was present in only 11%. Hyperferritinemia was present in 86% of individuals with ferroportin mutations. Bio-informatic analysis of ferroportin mutations showed that the PolyPhen score has a sensitivity of 99% and a specificity of 67% for the discrimination between ferroportin mutations and polymorphisms. Conclusions In contrast to HFE hemochromatosis, ferroportin disease has a high penetrance, is genetically heterogeneous and is rarely associated with fibrosis. Non-classical ferroportin disease is associated with a higher risk of fibrosis and a more severe overload of hepatic iron.

Co-localization of the Mammalian Hemochromatosis Gene Product (HFE) and a Newly Identified Transferrin Receptor (TfR2) in Intestinal Tissue and Cells

Mutations in the HFE gene and a newly identified second transferrin receptor gene, TfR2, cause hemochromatosis. The cognate proteins, HFE and TfR2, are therefore of key importance in human iron homeostasis. HFE is expressed in small intestinal crypt cells where transferrin-iron entry may determine subsequent iron absorption by mature enterocytes, but the physiological function of TfR2 is unknown. Using specific peptide antisera, we examined the duodenal localization of HFE and TfR2 in humans and mice, with and without HFE deficiency, by confocal microscopy. We also investigated potential interactions of these proteins in human intestinal cells in situ. Duodenal expression of HFE and TfR2 (but not TfR1) in wild-type mice and humans was restricted to crypt cells, in which they co-localized. HFE deficiency disrupted this interaction, altering the cellular distribution of TfR2 in human crypts. In human Caco-2 cells, HFE and TfR2 co-localized to a distinct CD63-negative vesicular compartment showing marked signal enhancement on exposure to iron-saturated transferrin ligand, indicating that HFE preferentially interacts with TfR2 in a specialized early endosomal transport pathway for transferrin-iron. This interaction occurs specifically in small intestinal crypt cells that differentiate to become iron-absorbing enterocytes. Our immunohistochemical findings provide evidence for a novel mechanism for the regulation of iron balance in mammals.

Identification of Mutations in SLC40A1 That Affect Ferroportin Function and Phenotype of Human Ferroportin Iron Overload

BACKGROUND & AIMS Patients with ferroportin iron overload due to loss-of-function mutations in SLC40A1 have macrophage iron overload, hyperferritinemia, and normal transferrin saturation. In contrast, hepatocellular iron storage, hyperferritinemia, and increased saturation of transferrin are a distinct clinical presentation of ferroportin iron overload that results from SLC40A1 mutations that confer resistance of ferroportin to hepcidin-mediated inactivation. METHODS SLC40A1 was sequenced in patients from 2 independent pedigrees affected by hepatic iron overload unrelated to HFE. Functions of the ferroportin variants were tested in vitro. RESULTS A patient heterozygous for the variant p.W158C in SLC40A1 presented with macrophage iron overload, hyperferritinemia, and normal transferrin saturation. A patient with hepatocellular iron storage, hyperferritinemia, and increased transferrin saturation was heterozygous for p.H507R. Expression of the p.W158C form of ferroportin in 293T cells resulted in defective trafficking to the plasma membrane and reduced iron export activity; the iron export activity of cells that expressed the p.H507R form of ferroportin did not differ from cells that expressed ferroportin without this mutation. The p.H507R of ferroportin localizes normally to the plasma membrane but is resistant to hepcidin-mediated inactivation. Addition of a synthetic peptide derived from ferroportin without these mutations (amino acids 500-518) decreased the inhibitory activity of hepcidin in cells, whereas a peptide from the same region, with p.H507R, had no effect on hepcidin activity. CONCLUSIONS The variant p.W158C in SLC40A1 impairs intracellular trafficking of ferroportin, resulting in reduced iron export. The variant p.H507R does not bind hepcidin in vitro and results in apparent hepcidin resistance.

Prevalence and risk factors for liver involvement in individuals with PiZZ-related lung disease.

RATIONALE α1-Antitrypsin deficiency is one of the most common heritable human diseases, predisposing to liver and lung injury. Significant heterogeneity in phenotypic expression is well documented, but less is known of the prevalence, severity, and correlates of chronic liver disease among individuals presenting with lung disease. OBJECTIVES To determine the frequency of and risk factors for severe liver fibrosis and cirrhosis among individuals with PiZZ-related lung disease. METHODS A well-characterized cohort of 57 PiZZ adults attending a tertiary referral respiratory clinic was screened prospectively for clinical, laboratory, radiologic, and (when appropriate) histologic evidence of chronic liver disease. MEASUREMENTS AND MAIN RESULTS Thirty-six (63.2%) of 57 had a history or clinical findings suggestive of liver disease; or had one or more abnormalities of liver function, or liver ultrasound, and 24 of these underwent liver biopsy. Ten (17.5%) had evidence of severe fibrosis or cirrhosis and were more likely to have higher body mass index (P = 0.04), alanine transaminase (P = 0.0001), alkaline phosphatase (P = 0.0009), prothrombin time (P = 0.0005), and maximal vital capacity (VCmax) (P = 0.04); lower platelet count (P = 0.007); abnormal liver echogenicity (P < 0.001); and splenomegaly (P = 0.001) at ultrasound. Screening with liver ultrasound provided a sensitivity and negative predictive value for severe fibrosis or cirrhosis of 100%, as were the specificity and positive predictive value for platelet count less than or equal to 174,000 per mm(3) and splenomegaly. Among individuals undergoing liver biopsy, fibrosis stage correlated with increasing VCmax (P = 0.02) and % predicted VCmax (P = 0.05), and decreasing residual volume/total lung capacity (TLC) (P = 0.02) and % predicted residual volume/TLC (P = 0.05). CONCLUSIONS Significant chronic liver disease is common in PiZZ individuals with lung disease and can be screened effectively by a combination of conventional tests of liver function, platelet count, and liver ultrasound.

Clinical Presentation and Molecular Pathophysiology of Autosomal Dominant Hemochromatosis Caused by a Novel Ferroportin Mutation

Mutations in the SLC40A1 gene, which encodes ferroportin, are associated with autosomal dominant hemochromatosis. Ferroportin is inhibited directly by hepcidin, a key iron‐regulatory peptide, and functional consequences of SLC40A1 mutations account for observed phenotypic differences in patients with ferroportin disease. We describe a large pedigree with a novel SLC40A1 mutation and, through in vitro analysis, elucidate the associated molecular mechanism of iron overload. The entire coding sequence of the SLC40A1 gene was sequenced in a pedigree, presenting with autosomal dominant hyperferritinemia. The functional effects of a novel SLC40A1 mutation were studied by overexpression of wild‐type and mutant ferroportin fusion proteins in human embryonic kidney cells. Iron export was studied in these cells using 59Fe transport assays; subcellular localization of ferroportin was examined by way of confocal microscopy. A novel SLC40A1 mutation p.R489K segregated with iron overload in a family with clinical and histopathological signs of macrophage‐type ferroportin disease. Human embryonic kidney cells overexpressing p.R489K ferroportin showed decreased iron export capacity when compared with wild‐type ferroportin overexpressing cells. Subcellular localization studies demonstrated that p.R489K ferroportin was retained abnormally within an intracellular compartment. Conclusion: We report a novel pathological SLC40A1 variant associated with abnormal cell surface expression of ferroportin due to intracellular retention of the mutant protein. These findings predict macrophage‐type ferroportin disease, the phenotype observed in this kindred. Study of the molecular cell biology of ferroportin and its mutants is key to understanding the pathogenesis of this increasingly recognized form of hemochromatosis, which responds poorly to conventional therapy. (HEPATOLOGY 2009.)

Biliary atresia and survival into adulthood without transplantation: a collaborative multicentre clinic review

Biliary atresia is a progressive biliary injury which occurs only in infants.

Outcomes and Diagnostic Challenges Posed by Incidental Cholangiocarcinoma After Liver Transplantation

Background. Liver transplantation in the presence of cholangiocarcinoma (CCA) generally carries a poor prognosis. However, the outcome of patients found to have incidental CCA (iCCA) on explanted liver histology is less clear. We have evaluated the outcomes of iCCA in our liver transplant population. Methods. A retrospective search was made of the transplantation and histopathology databases for patients fulfilling our definition for iCCA. All records, including archived histopathologic slides were retrieved and analyzed. Results. Of 1288 patients undergoing liver transplantation over the 20-year period 1988–2008, nine were found to have iCCA (0.70%). Seven of the nine patients underwent liver transplantation for primary sclerosing cholangitis. Three additional patients who were transplanted for presumed hepatocellular carcinoma that subsequently turned out to be CCA were identified, but excluded from survival analysis. The majority of tumors were early stage (T2 or below), but five (55.6%) had positive biliary transection margins. Median follow-up was 51 months. Five patients (55.6%) developed recurrence of CCA after a median interval of 25.8 months, giving a disease-free survival of 100% at 1 year and 66.7% at 3 years. Three patients have died of recurrence, with a median interval from transplantation of 25 months. The overall 3-year survival was 66.7%. Conclusions. Patients found to have iCCA after liver transplantation have a relatively poor prognosis. Prospective liver transplant recipients, especially those with primary sclerosing cholangitis, should be investigated rigorously to exclude CCA.

Orthotopic Liver Transplantation in an Adult with Cholesterol Ester Storage Disease

Cholesterol ester storage disease (CESD) is a rare autosomal recessive lipid storage disorder associated with mutations of the gene encoding lysosomal acid lipase, manifestations of which include chronic liver disease and early atherosclerosis. Although normally presenting in childhood, severity is variable and the condition can occasionally remain undetected until middle age. Typical presentation is with asymptomatic hepatosplenomegaly and hyperlipidaemia, though the condition is probably underdiagnosed. Treatment is supportive and may include attention to cardiovascular risk factors. Phase I/II trials of enzyme replacement therapy are ongoing, but this approach remains experimental. We present the case of a 42-year-old woman diagnosed with CESD in childhood who ran an indolent course until re-presentation with cirrhotic hydrothorax. She underwent orthotopic liver transplantation but required re-transplantation for hepatic artery thrombosis. She remains well with excellent graft function 2 years later. Although atherosclerosis was apparent at assessment, and may have contributed to hepatic artery thrombosis, partial correction of the metabolic defect and restoration of liver function by transplantation together with ongoing medical therapy should permit reasonable survival over the longer term from both a liver and a vascular perspective. This is the first reported case of orthotopic liver transplantation for CESD in an adult, which was the only available option to improve survival. The case highlights the importance of monitoring patients with CESD through adulthood and suggests that liver replacement at a later stage may yet be indicated and remain of benefit.

Impact of D181V and A69T on the function of ferroportin as an iron export pump and hepcidin receptor

Mutations in the only known mammalian iron exporter ferroportin cause a rare iron overload disorder termed ferroportin disease. Two distinct clinical phenotypes are caused by different disease mechanisms: mutations in ferroportin either cause loss of iron export function or gain of function due to resistance to hepcidin, the peptide hormone that normally downregulates ferroportin. The aim of the present study was to examine the disease mechanisms of the thus far unclassified A69T and D181V ferroportin mutations. We overexpressed wild-type and mutant ferroportin fused to green fluorescent protein in human embryonic kidney cells and used a (59)Fe-assay, intracellular ferritin concentrations, confocal microscopy and flow cytometry to study iron export function, subcellular localization and the responsiveness to hepcidin. While the A69T ferroportin mutation seems not to affect the iron export function it causes dose-dependent hepcidin resistance. We further found that D181V mutated ferroportin is iron export defective and hepcidin resistant, similar to the loss of function mutations A77D and C367X. This indicates that intact iron export might be necessary for hepcidin-induced downregulation of ferroportin. This hypothesis was investigated by studying the hepcidin response under modulation of iron availability. Incubation of wild-type ferroportin overexpressing cells with holo-transferrin increases the hepcidin effect whereas chelating extracellular ferrous iron causes hepcidin resistance. In this study we present data that postulates to classify the D181V ferroportin mutation as loss of function and the A69T mutation as dose-dependent hepcidin resistant and outline a possible causal link between iron export function and the hepcidin effect.

Severe coagulopathy caused by rifampicin in patients with primary sclerosing cholangitis and refractory pruritus.

Pruritus is one of the most disabling clinical manifestations of cholestatic liver disease. Antipruritic agents are effective only in a proportion of patients [1], [2]. Rifampicin is an antituberculous agent recommended for relieving pruritus refractory to bile acid sequestrants [3]–[6]. This is the first report of severe, reversible prolongation of prothrombin time in patients with primary sclerosing cholangitis (PSC), following the introduction of rifampicin.