Iron Storage Disease Research Articles

Preliminary investigation of bottlenose dolphins (Tursiops truncatus) for hfe gene-related hemochromatosis.

Hemochromatosis (iron storage disease) has been reported in diverse mammals including bottlenose dolphins (Tursiops truncatus). The primary cause of excessive iron storage in humans is hereditary hemochromatosis. Most human hereditary hemochromatosis cases (up to 90%) are caused by a point mutation in the hfe gene, resulting in a C282Y substitution leading to iron accumulation. To evaluate the possibility of a hereditary hemochromatosis-like genetic predisposition in dolphins, we sequenced the bottlenose dolphin hfe gene, using reverse transcriptase-PCR and hfe primers designed from the dolphin genome, from liver of affected and healthy control dolphins. Sample size included two case animals and five control animals. Although isotype diversity was evident, no coding differences were identified in the hfe gene between any of the animals examined. Because our sample size was small, we cannot exclude the possibility that hemochromatosis in dolphins is due to a coding mutation in the hfe gene. Other potential causes of hemochromatosis, including mutations in different genes, diet, primary liver disease, and insulin resistance, should be evaluated.

Deadly outbreak of iron storage disease (ISD) in Italian birds of the family Turdidae.

ABSTRACTA widespread deadly outbreak occurred in captive birds belonging to the family Turdidae in Italy. The present study was performed on 46 dead birds coming from 3 small decoy-bird breeders in central Italy. Only Turdus pilaris, Turdus iliacus, Turdus philomelos and Turdus merula were affected. No other species of bird held by these breeders died. A change of diet before the hunting season was reported from all breeders. Full necropsy of the animals and histological investigations of representative tissue samples were performed. Microscopical examination showed marked iron deposits in liver samples. Bacteriological investigations and molecular analysis to exclude bacterial and viral diseases were carried out. Contamination of food pellet samples by mycotoxins and analysis to detect heavy metal contaminants in food pellet samples were considered. An interesting result was the high iron content found in food pellets. It was higher than that considered suitable for birds, especially for species susceptible to development iron storage disease (ISD). Taken together, the results suggested an outbreak of ISD caused by the high iron content of food given to the birds before the hunting season. The high mortality recorded only in species belonging to the family Turdidae suggests a genetic predisposition in the affected birds.

Relationship of Lipid Profile and Serum Ferritin levels with Acute Myocardial Infarction.

National Health and Nutrition Examination Survey (NHANES III), 1988-1994, first time reported a significant, positive association in iron storage and heart disease risk. Thereafter several researchers have found an association between iron overload, serum ferritin (SF) and MI. No such Indian study was available in the literature and so we decided to find out the relation of lipid profile and Serum Ferritin with myocardial infarction (MI). Fifty indian patients of AMI (study group) and fifty indian healthy volunteers (control group) were included for the present study. Lipid profile including TC, HDL-c, LDL-c, VLDL-c & TG and SF levels were estimated in all subjects. Mean ± SD of TC level was 250.64 ± 25.61, of HDL-c was 36.52 ± 2.86, of LDL-c was 165.69 ± 26.80, of VLDL-c was 42.35 ± 8.53 and of TG was 211.83 ± 42.65 in study group while these values were 174.46±47.68, 43.2±12.52, 98.37±41.13, 32.88±21.45 and 164.42±107.29 respectively in control group. All the parameters were found not only raised in patients of acute myocardial infarction (AMI) but were also statistically significant when compared with control group (p=<0.01). Mean ± SD of SF levels was 268.43±30.17 ng/ml in study group and 110.96±56.5 ng/ml in control group; this level was found not only raised in patients of AMI but were also statistically significant when compared with control group (p=<0.01). TC, LDL-c, VLDL-c, TG and SF levels were raised in patients of AMI and found to be statistically significant; while HDL-c levels were reduced in such patients and is also statistically significant. It can be concluded that there exists an association in lipid profile and SF with AMI therefore dyslipidemia and raised SF levels are the features of AMI.

Panhypopituitarism Due to Hemochromatosis

Hemochromatosis is an iron storage disease characterized by iron deposition in parenchymal cells due to increased intestinal iron absorption. Iron overload leads to tissue damage and dysfunction particularly in the liver, pancreas, heart, joints, and pituitary gland. Panhypopituitarism is a clinical condition in which the anterior pituitary hormones are deficient. Herein, we report a rare case of hemochromatosis.

Characterization of the hepcidin gene in eight species of bats

Hemochromatosis, or iron storage disease, has been associated with significant liver disease and mortality in captive Egyptian fruit bats (Rousettus aegyptiacus). The physiologic basis for this susceptibility has not been established. In humans, a deficiency or resistance to the iron regulatory hormone, hepcidin has been implicated in the development of hereditary hemochromatosis. In the present study, we compared the coding sequence of the hepcidin gene in eight species of bats representing three distinct taxonomic families with diverse life histories and dietary preferences. Bat hepcidin mRNA encoded a 23 amino acid signal peptide, a 34 or 35 amino acid pro-region, and a 25 amino acid mature peptide, similar to other mammalian species. Differences in the sequence of the portion of the hepcidin gene that encodes the mature peptide that might account for the increased susceptibility of the Egyptian fruit bat to iron storage disease were not identified. Variability in gene sequence corresponded to the taxonomic relationship amongst species.

Iron storage disease in Asia‐Pacific populations: The importance of non‐HFE mutations

Hereditary hemochromatosis (HH) is a widely recognized and well-studied condition in European populations. This is largely due to the high prevalence of the C282Y mutation of HFE. Although less common than in Europe, HH cases have been reported in the Asia-Pacific region because of mutations in both HFE and non-HFE genes. Mutations in all of the currently known genes implicated in non-HFE HH (hemojuvelin, hepcidin, transferrin receptor 2, and ferroportin) have been reported in patients from the Asia-Pacific region. This review discusses the molecular basis of HH and the genes and mutations known to cause non-HFE HH with particular reference to the Asia-Pacific region. Challenges in the genetic diagnosis of non-HFE HH are also discussed and how new technologies such as next generation sequencing may be informative in the future.

AVIAN IRON STORAGE DISEASE: VARIATIONS ON A COMMON THEME?

Many frugivorous avian species kept in captivity develop iron storage disease (ISD) as indicated by high concentrations of hepatic iron and hemosiderin deposits in hepatocytes or phagocytes. In several susceptible species fed diets containing moderate levels of iron, ISD develops because of an inability to match rates of iron absorption to tissue needs. Evidence suggests that the pathophysiologic basis of excess iron absorption is due to high levels of expression of divalent metal transporter-1 that transports iron into enterocytes in the proximal intestine, and ferroportin that exports iron to the circulation. The regulatory basis for this inability to sufficiently down-regulate iron absorption is unknown, but disruptions in the hepcidin-ferroportin axis are likely candidates based on recent research in humans and laboratory rodents. It is likely that ISD-susceptible avian species evolved on foods that were very low in bioavailable iron, so there was strong selection pressure for the efficient capture of the small amount of dietary iron but low selection pressure for preventing iron toxicities. Thus, the transporters and regulatory networks for iron absorption seem to be heavily skewed toward iron storage even when food items that are high in iron are consumed. Infections, trauma and neoplasias that trigger an acute phase response may exacerbate ISD in susceptible species and may be the primary cause in species that are normally resistant to ISD (i.e., those that are normally able to shut down intestinal iron absorption when iron stores are replete). The evolutionary basis that resulted in some avian species to be susceptible to ISD (e.g., dietary cause) seems to differ from many inherited ISD disorders in humans that are thought to have evolved to bolster protection against infectious diseases. However the evolutionary basis of ISD in other mammalian species might be more similar to that in ISD-susceptible avian species.

IRON STORAGE DISORDERS IN CAPTIVE WILD MAMMALS: THE COMPARATIVE EVIDENCE

Excessive burden of iron, or iron storage disease (ISD), has been reported in a large variety of captive mammal species, including browsing rhinoceroses; tapirs; fruit bats; lemurs; marmosets and some other primates; sugar gliders; hyraxes; some rodents and lagomorphs; dolphins; and some carnivores; including procyonids and pinnipeds. This report collates the comparative evidence for species' susceptibility, recognizing that the data for mammal species are limited. Differences reported in the occurrence of ISD between facilities, or within facilities over periods that span management changes, have been reported in individual cases but are underused in ISD research. Given the species composition, the hypothesis that evolutionary adaptations to the iron content and availability in the natural diet determine a species' susceptibility to ISD (in the face of deviating iron content and availability in diets offered in captivity) seems plausible in many cases. But exceptions, and additional species putatively susceptible based on this rationale, should be investigated. Whereas screening for ISD should be routine in zoo animal necropsy, screening of live individuals may be implemented for valuable species, to decide on therapeutic measures such as chelator application or phlebotomy. Whatever the reasons for ISD susceptibility, reducing dietary iron levels to maintenance requirements of the species in question seems to be a logical, preventive measure.

BROWSE DIVERSITY AND IRON LOADING IN CAPTIVE SUMATRAN RHINOCEROSES (DICERORHINUS SUMATRENSIS): A COMPARISON OF SANCTUARY AND ZOOLOGICAL POPULATIONS

Iron storage disease (ISD) is now recognized as a serious clinical disorder acquired by two species of browsing rhinoceroses, the African black (Diceros bicornis) and the Asian Sumatran (Dicerorhinus sumatrensis) rhinoceroses, when displaced from their natural habitats. The most complete knowledge of ISD comes from studies of the black rhinoceros, but the Asian species is also at risk. Sumatran rhinoceroses housed in traditional zoological settings outside of range countries have suffered significant morbidity and mortality potentially related to ISD induced by diet and/or other confinement conditions. With so few animals in captivity, very little information exists on iron loading in the Sumatran rhinoceros. To better characterize the problem, we retrospectively compared captive management conditions of Sumatran rhinoceroses housed under traditional zoological care with those in two native sanctuary environments. In general, zoo rhinoceroses are offered a paucity of plants and browse species compared with their sanctuary and wild counterparts managed in native rainforest habitats. Iron analyte levels and limited histopathologic observations in these populations suggest variable tendencies to overload iron, dependent upon differences in managed diet and individual food preferences. More detailed investigation of these markedly dissimilar ex situ populations is warranted to better understand the role of nutrition and other conditions affecting iron loading in browser rhinoceroses.

IOD IN RHINOS—EPIDEMIOLOGY GROUP REPORT: REPORT FROM THE EPIDEMIOLOGY WORKING GROUP OF THE INTERNATIONAL WORKSHOP ON IRON OVERLOAD DISORDER IN BROWSING RHINOCEROS (FEBRUARY 2011)

The Epidemiology Working Group, a subgroup of the participants of the International Workshop on Iron Storage Disease (renamed iron overload disorder, IOD), identified several areas in which information is lacking regarding IOD in browser rhinoceros. One of the first steps necessary in understanding iron overload disorder (IOD) is to define the parameters by which to identify IOD. Some of the parameters that have been used to evaluate IOD include serum iron, serum iron binding capacity, transferrin saturation, and serum ferritin. Clarification of whether a single parameter or a combination of these parameters is best for evaluating IOD is needed. Once IOD can more clearly be defined using these parameters, it will be necessary to identify whether clinical signs of disease are associated with the presence of IOD. This working group feels that it is necessary to define what is ‘‘normal’’ and what constitutes ‘‘abnormal’’ with regard to IOD. Are all browsing rhinos in captivity affected by IOD, and if not, how many are affected? Is IOD associated with an increased risk of developing disease? PRIORITIES

IRON HOMEOSTASIS AND ITS DISORDERS IN MICE AND MEN: POTENTIAL LESSONS FOR RHINOS

During the last decade, there have been remarkable advances in the understanding of iron homeostasis and its disorders. This review summarizes our presentation at the International Workshop on Iron Storage Disease in Black Rhinos that was held in Orlando, Florida, USA, from 23 to 26 February 2011, and it is directed to veterinarians and nutritional experts who treat rhinoceroses. This review summarizes the current knowledge in humans and mice regarding the physiology and molecular basis of iron overload, and it explores how it can be applied to the problem of iron overload in captive rhino populations.

Hereditary Hemochromatosis Restores the Virulence of Plague Vaccine Strains

Nonpigmented Yersinia pestis (pgm) strains are defective in scavenging host iron and have been used in live-attenuated vaccines to combat plague epidemics. Recently, a Y. pestis pgm strain was isolated from a researcher with hereditary hemochromatosis who died from laboratory-acquired plague. We used hemojuvelin-knockout (Hjv(-/-)) mice to examine whether iron-storage disease restores the virulence defects of nonpigmented Y. pestis. Unlike wild-type mice, Hjv(-/-) mice developed lethal plague when challenged with Y. pestis pgm strains. Immunization of Hjv(-/-) mice with a subunit vaccine that blocks Y. pestis type III secretion generated protection against plague. Thus, individuals with hereditary hemochromatosis may be protected with subunit vaccines but should not be exposed to live-attenuated plague vaccines.

Well-differentiated Hepatocellular Carcinoma in a Ring-tailed Lemur (Lemur catta)

A 16-year-old male ring-tailed lemur (Lemur catta) was presented with severe cachexia and an abdominal mass. The encapsulated, multilobular mass replaced the right medial lobe of the liver and compressed the adjacent gall bladder. Multiple haemorrhages and necrotic foci were found within the mass. Microscopically, neoplastic cells formed cords of moderately pleomorphic, polygonal cells with mild to moderate anaplasia. Immunohistochemical markers used for diagnosis of hepatocellular carcinomas in man were used to characterize the neoplastic cells, which expressed hepatocyte-specific antigen, but not glypican-3 or polyclonal carcinoembryonic antigen. Gross, microscopical and immunohistochemical features of the tumour were most consistent with a well-differentiated hepatocellular carcinoma. Although this tumour is common among prosimians, to the authors' knowledge this is the first documented case in a ring-tailed lemur. Hepatocellular carcinomas have been associated with hepatitis virus infections and excessive hepatic iron in man; however, no association was established between this tumour and viral infection or hepatic iron storage disease in the present case.

Use of deferiprone for the treatment of hepatic iron storage disease in three hornbills

3 hornbills (2 Papua hornbills [Aceros plicatus] and 1 longtailed hornbill [Tockus albocristatus]) were evaluated because of general listlessness and loss of feather glossiness. Because hepatic iron storage disease was suspected, liver biopsy was performed and formalin-fixed liver samples were submitted for histologic examination and quantitative image analysis (QIA). Additional frozen liver samples were submitted for chemical analysis. Birds also underwent magnetic resonance imaging (MRI) under general anesthesia for noninvasive measurement of liver iron content. Serum biochemical analysis and analysis of feed were also performed. Results of diagnostic testing indicated that all 3 hornbills were affected with hepatic iron storage disease. The iron chelator deferiprone was administered (75 mg/kg [34.1 mg/lb], PO, once daily for 90 days). During the treatment period, liver biopsy samples were obtained at regular intervals for QIA and chemical analysis of the liver iron content and follow-up MRI was performed. In all 3 hornbills, a rapid and large decrease in liver iron content was observed. All 3 methods for quantifying the liver iron content were able to verify the decrease in liver iron content. Orally administered deferiprone was found to effectively reduce the liver iron content in these 3 hornbills with iron storage disease. All 3 methods used to monitor the liver iron content (QIA, chemical analysis of liver biopsy samples, and MRI) had similar results, indicating that all of these methods should be considered for the diagnosis of iron storage disease and monitoring of liver iron content during treatment.

Assessment of Iron Overload with T2* Magnetic Resonance Imaging

T2* is a magnetic relaxation property of any tissue and is inversely related to intracellular iron stores. Measurement is simple, quick, and robust and has high reproducibility. The ability to measure ventricular function plus T2* in the heart and liver during the same scan has revolutionized the understanding of iron storage disease and the management of the iron-loaded patient. The early findings using T2* challenged conventional teachings, and both the technique and the findings were initially viewed with scepticism. However, after a decade of work in validating, calibrating, and expanding access to this method, it is now accepted as the method of choice for tissue iron assessment. In the UK, where T2* measurement was first used in the clinical care of patients with thalassemia major, a large and significant fall in mortality has been seen in this patient group.

Diet Manipulation as Treatment for Elevated Serum Iron Parameters in Captive Raggiana Bird of Paradise (Paradisaea raggiana)

Elevated serum iron parameters were lowered through dietary manipulation in captive Raggiana bird of paradise (Paradisaea raggiana). Study birds were part of a captive breeding program consisting of two males and one female, captive born, 3.5-9 yr of age. Serum iron, total iron binding capacity (TIBC), percentage saturation, body weight, albumin, aspartate aminotransferase, and hematocrit were monitored at regular intervals for 2.5 yr. Routine diet consisted of a variety of fruits, vegetables, a multivitamin supplement, and a commercial low iron avian pellet, with a dietary iron content of 55 mg/kg (dry matter basis) or 1.12 mg iron/bird/day. Dietary treatment involved removal of the commercial avian pellet for 30 days at 6-to 12-mo intervals, resulting in an iron content of 42 mg/kg (dry matter basis) or 0.64 mg iron/bird/day. Average serum iron and TIBC were decreased by 75% (TIBC) to 80% (serum iron) ofpretreatment values after one 30-day treatment. Average iron saturation levels were lowered by 10% of pretreatment values after one 30-day treatment. Average hematocrit, albumin, aspartate aminotransferase, and body weight remained unchanged. No adverse effects were noted through the 2.5-yr evaluation period, and breeding behavior was undisturbed. Periodic removal of low iron commercial pellets in the diet of captive bird of paradise is a safe and effective method for lowering serum iron values without need for handling. Periodic application of this technique may be useful as a preventive tool to maintain appropriate serum iron values in avian species susceptible to iron storage disease.

Iron Storage Disease in Red Deer ( Cervus elaphus elaphus) is not associated with Mutations in the HFE Gene

Iron storage diseases are rare conditions of dysregulated iron metabolism in man and animals. A genetic basis has been confirmed only for human haemochromatosis. Iron storage disease was diagnosed in six related, 2-year-old male red deer of the same herd. These animals presented with weight loss and rough hair coats. Haematological examination was unremarkable. At necropsy examination, gross lesions were restricted to cachexia. Microscopical examination revealed severe, diffuse hepatocellular necrosis and iron accumulation in hepatocytes, Kupffer cells, cardiac myocytes and renal tubular cells in all affected animals. Four animals also had moderate bridging fibrosis in the liver. Hepatic iron concentrations were increased (1108-2275 mg/kg wet weight; reference range 100-200 mg/kg). Drinking water in rusty iron tubs in the deer park contained eight times more iron than the accepted level for human drinking water. To test for a possible genetic basis of increased iron uptake and storage in red deer, the cervid haemochromatosis gene (HFE) was identified. Sequence comparisons between the six diseased animals and three healthy free-ranging unrelated animals failed to identify differences in the HFE sequences. Furthermore, the disease was not associated with common amino acid substitutions reported in human patients with haemochromatosis, including C282Y and H63D. Polymorphisms in other non-HFE genes involved in iron metabolism may have led to a higher sensitivity to iron and this, together with the high iron content of the drinking water, may have been the cause of the observed iron storage in these red deer.

Effect of tannic acid on iron absorption in straw‐colored fruit bats (Eidolon helvum)

Excessive absorption and subsequent storage of dietary iron has been found in a variety of captively held birds and mammals, including fruit bats. It is thought that feeding a diet that is low in iron can prevent the onset of this disease; however, manufacturing a diet with commonly available foodstuffs that contains a sufficiently low iron concentration is difficult. An alternative is to feed captive animals that may be susceptible to this disease potential iron chelators such as tannins that may bind to iron and block its absorption. Using stable isotope methods established in humans, we measured iron bioavailability in straw-colored fruit bats (Eidolon helvum) and tested whether tannic acid significantly reduced the extent of iron absorption. Regardless of dose, tannic acid significantly reduced iron absorption (by 40%) and in the absence of tannic acid, iron absorption was extensive in this species (up to 30%), more so than in humans. Species susceptible to iron storage disease may efficiently absorb iron in the gut regardless of iron status, and supplementing these species with tannic acid in captivity may provide an alternative or additional means of preventing the development of this disease.

Diagnosis of Hepatic Iron Overload

Recent identification of genetic variants in iron storage disease has changed the classification system and diagnostic algorithms for hemochromatosis. Clinical diagnosis of the disease requires phenotypic evidence of iron overload because the commonly disease-associated HFE genotypes have an incomplete penetrance. Furthermore, approximately 20% of patients with a clinical diagnosis of hemochromatosis have no disease-associated genotype, which underlines the importance of clear phenotypic criteria of hemochromatosis. A diagnosis of hemochromatosis cannot be made even in patients with liver cirrhosis simply on the basis of genetic testing that indicates that iron overload is the cause of the disease and not its consequence. Proper diagnosis requires integration of clinical presentation, family history, and the results of biochemical and histopathologic tests. Here we propose a rational diagnostic algorithm for hepatic iron overload syndromes and illustrate potential pitfalls by presenting a family study in a pedigree with rare HFE variants (H63D and E168Q), in cis on the same chromosome. Although the clinical suspicion of hemochromatosis was confirmed by histology, chemical analysis of liver tissue revealed a normal hepatic iron concentration, which is compatible with the genetic finding of 1 normal and 1 doubly mutated allele. In conclusion, clinical suspicion of hemochromatosis and elevated serum iron parameters should prompt HFE genotyping for C282Y and H63D. Should they be uninformative, further genetic tests should be recommended only if iron overload in liver tissue has been confirmed chemically.

Regulation of Hepcidin and Iron-Overload Disease

Hepcidin, a 25-amino-acid antimicrobial peptide, is the central regulator of iron homeostasis. Hepcidin transcription is upregulated by inflammatory cytokines, iron, and bone morphogenetic proteins and is downregulated by iron deficiency, ineffective erythropoiesis, and hypoxia. The iron transporter ferroportin is the cognate receptor of hepcidin and is destroyed as a result of interaction with the peptide. Except for inherited defects of ferroportin and hepcidin itself, all forms of iron-storage disease appear to arise from hepcidin dysregulation. Studies using multiple approaches have begun to delineate the molecular mechanisms that regulate hepcidin expression, particularly at the transcriptional level. Knowledge of the regulation of hepcidin by inflammation, iron, erythropoiesis, and hypoxia will lead to an understanding of the pathogenesis of primary hemochromatosis, secondary iron overload, and anemia of inflammatory disease.