This article from Trends in Molecular Medicine appeals to my long-standin interest in Iron Metabolism. It will only appeal to molecular Biologists and those affected by iron-related diseases. But it adds to the sum of my knowledge as the field moves on.
Iron must enter the body from the diet through absorption in the proximal small intestine. Although some plasma iron is derived from iron absorption, most is derived from the recirculation of hemoglobin-derived iron from senescent red blood cells. This process is carried out by macrophages. Iron that enters the plasma is bound by transferrin and distributed around the body to sites of utilization and storage. Transferrin delivers its iron to cells via an interaction with transferrin receptor 1 (TFR1) found on the plasma membrane of most cells. Iron in excess of immediate cellular needs is stored within ferritin. The coordinated regulation of TFR1 and ferritin is mediated by RNA binding proteins known as iron regulatory proteins. Systemically, iron entry into the plasma is controlled by the liver-derived peptide hepcidin, which is secreted by hepatocytes and acts through its effect on ferroportin, the only known iron export protein. Hepcidin expression is increased by high plasma iron and decreased by low plasma iron. HFE, TFR2 (transferrin receptor 2) and HJV (hemojuvelin) are positive stimuli of hepcidin expression, whereas matriptase-2 is a negative stimulus. In its free form, iron is compatible neither with its transport in the plasma (it would precipitate) nor with the intracellular milieu where it would exert immediate toxicity. Therefore, the human body has developed ways for enabling both iron transport (through transferrin) and intracellular deposition (through ferritin).
Approximately 10% of dietary iron content is absorbed every day in the duodenum, corresponding to 1–2 mg. It is delivered, through the ferroportin channel, to the blood where it is taken up by circulating transferrin which delivers iron, via TFR1, mainly to the bone marrow to contribute to the build-up of red blood cells. Red blood cell degradation within the spleen, and to a lesser degree within the liver, occurs 120 days after production and quantitatively provides the main source of iron to the plasma. Iron of splenic origin is taken up by transferrin and a new iron cycle begins. Almost all cells are able to take up iron from transferrin but in a much smaller amount than erythroid cells. Nevertheless, this small amount of iron is essential to serve in many enzymatic reactions, particularly within the respiratory chain. The total amount of body iron approximates 4 g, with 70% within red blood cells and the remainder within liver, spleen and muscles. Circulating iron represents only 0.05% of total body iron but is functionally of major importance. The amount of iron leaving the body each day (mainly in the feces and urine) is equivalent to the amount entering (i.e. 1–2 mg).
Hepcidin (encoded by the HAMP gene) is the hormone that regulates iron metabolism. It is a 25 amino acid peptide primarily produced by hepatocytes. Released into the blood stream, it mainly interacts with enterocytes of the duodenum and macrophage in the spleen by targeting ferroportin. Ferroportin, once bound to hepcidin, is internalized and then degraded. Iron egress from the cell is subsequently hampered by decreased ferroportin activity. It should be noted that the hepcidin control of iron egress from duodenal cells might also involve modulation of DMT1 degradation. The overall result is an inhibition of intestinal and recycled macrophage iron release to circulating transferrin that occurs particularly in response to increased body iron load to compensate for this increase. The reverse mechanism occurs in cases of body iron deficiency (i.e. increased release of iron from the cell related to increased export activity of ferroportin). Hepcidin regulation is mainly characterized as a multifactorial transcriptional process. A major factor that increases hepcidin levels, in addition to increased iron stores, is inflammation. In addition to reduced iron stores, hypoxia, anemia and increased erythropoiesis are hepcidin-reducing factors. The molecular regulators of hepcidin expression are numerous. The bone morphogenetic proteins (BMPs) signaling pathway is implicated in responding to levels of iron stores. Schematically, BMP6 activates its receptors (BMPRI and II) in the presence of hemojuvelin (BMPRI–II coreceptor), which activates the SMAD (Son of Mothers against Decapentaplegic) proteins 1, 5 and 8. A complex is then formed with SMAD4, leading to translocation to the nucleus and activation of hepcidin transcription. The HFE–TFR1–TFR2 complex is an iron sensor for plasma diferric transferrin, leading to hepcidin transcription by a yet not fully identified pathway. TMPRSS6 (transmembrane protease serine 6) encodes matriptase-2, a negative regulator of hemojuvelin expression that decreases hepcidin expression. The STAT3 signaling pathway is involved in IL-6-dependent hepcidin expression. IL-6, produced during inflammatory processes, interacts with its receptor and leads to phosphorylation of the STAT3 protein, which is then translocated into the nucleus where it interacts with the hepcidin promoter.
HFE-related iron overload disease is, by far, the most frequent form of genetic iron overload disorders. Type 1 hemochromatosis is a recessive disease that is linked to mutations of the HFE gene located on chromosome 6. Homozygosity for the p.Cys282Tyr (C282Y) mutation explains more than 90% of type 1 hemochromatosis. The p.Cys282Tyr mutation inhibits the molecular cascade that results in decreased hepatic production of hepcidin. As a consequence, increased plasma iron leads to increased transferrin saturation, which is associated with the appearance of non-transferrin bound iron (NTBI). NTBI is avidly taken up by parenchymal cells in the liver, the pancreas and the heart, leading to excess iron in these organs. In addition, when transferrin saturation is over 75%, NTBI is considered labile plasma iron (LPI), which might generate radical oxygen species and represents the potentially toxic form of circulating iron. The damaging effect of LPI contributes to the clinical expression of chronic iron overload disorders. The liver increases in volume (hepatomegaly), releases more transaminases into the plasma due to hepatocytic iron-related damage and can develop scarring called fibrosis that can lead to cirrhosis and, later on, to hepatocellular carcinoma. Excessive iron deposition in the pancreas, endocrine glands (pituitary, gonads) and heart can lead to insulin-dependent diabetes, hypogonadism (with impotence in males) and cardiac failure, respectively. Moreover, bone and joints can be affected, leading to osteoporosis and chronic arthritis. The skin becomes hyperpigmented (bronzed) and chronic fatigue is a major symptom.
Of Celtic origin (and not Viking origin), as suggested by the mutation date before 4000 BC, the p.Cys282Tyr mutation is highly prevalent in this population, affecting one allele in more than 10% of Caucasian individuals. At least 1 person in 1000 is homozygous for this mutation, and the usual frequency is approximately 3 people in 1000, with even greater frequency in some areas, such as Brittany (France) and Ireland. Penetrance is partial, but if severe clinical presentation is rare, biochemical penetrance is high. Current research efforts are targeting identification of other genes and polymorphisms that modulate HFE function and the interaction between the C282Y genetic background and environmental factors.
Diagnosis of type 1 hemochromatosis is based on a three-step strategy: the first step is recognizing clinical symptoms, the second is confirming excess iron and the third is molecular diagnosis by genetic testing. Clinical symptoms are numerous, including, more or less combined, chronic fatigue, impotence, joint pains, osteoporosis, mild elevated plasma transaminases, hepatomegaly, cirrhosis, diabetes, cardiac rhythm disturbances, heart failure and skin pigmentation. It should be pointed out, however, that many patients, including those with advanced iron overload, have few or no symptoms and that many symptoms lack specificity.
Confirming iron excess is the diagnostic cornerstone. Plasma iron and transferrin saturation are increased in patients, reflecting the basic metabolic dysregulation of the disease. Increased plasma ferritin concentration reflects intracellular iron overload and is the most frequent surrogate marker for tissue iron overload. Magnetic resonance imaging (MRI) is a valuable tool for confirming hepatic iron overload as it shows a characteristic hyposignal that is correlated with the degree of iron excess. By contrast, no iron overload is observed in the spleen. In addition, the T2-star MRI technique has been developed for the assessment of cardiac iron overload. Liver biopsy to evaluate iron excess is performed less and less, considering the combined value of the ferritin assay and MRI. The main use of biopsy is to evaluate possible cofactors of hepatotoxicity (fatty infiltration or signs of alcoholism) and to search for cirrhosis which exposes the patient to the development of hepatocellular carcinoma, especially when associated to iron-free foci. Deducing iron overload from the amount of withdrawn iron required to reach iron depletion remains a valuable, although retrospective, method.
The third step is molecular diagnosis via genetic tests that detect the presence of p.C282Y homozygosity in the HFE gene. It should be emphasized that p.C282Y heterozygosity cannot be considered, alone, to be responsible for clinically significant body iron excess. This is true even for HFE compound heterozygosity, which is commonly the association of heterozygosity for p.Cys282Tyr and heterozygosity for the other frequent mutation, p.His63Asp (p.H63D), which can only lead to a limited increase in plasma transferrin saturation and ferritin. Likewise, p.H63D homozygosity, despite some experimental data and rare case reports, is not generally considered to account for significant iron excess. In practice, the search for the p.H63D mutation is no longer recommended in France and should be confined to clinical research. The same holds true for other HFE protein mutations, such as p.Ser65Cys (S65C), p.Val59Met (V59 M), p.Arg66Cys (R66C), p.Gly93Arg (G93R), p.Ile105Thr (I105T), p.Arg224Gly (R224G) and p.Val295Ala (V295A). Whenever a p.Cys282Tyr mutation in the heterozygous state is associated with significant visceral iron overload (hepatic iron concentration more than 3-fold the upper normal limit), one must evoke an associated rare HFE mutation , a homozygous deletion of HFE, or associated non-HFE mutations. In practice, testing for mutations other than p.CysC282Tyr (and p.His63Asp) remains confined to specialized laboratories.
Treatment for type 1 hemochromatosis consists of repeated venesections on the principle that removing red blood cells, which are iron-rich, obliges the body to compensate for this erythrocyte loss by mobilizing iron from its storage locations. Provided this therapy is started before the development of severe complications (cirrhosis, insulin-dependent diabetes or cardiomyopathy), the efficacy is excellent, and patients recover both an overall good quality of life (except for arthritis) and a normal life expectancy. Oral chelation might be used, particularly in the case of contraindications of phlebotomies, such as poor venous access or patient unwillingness. Future therapy might consist of normalizing hepcidin levels to prevent absorption of additional quantities of excess iron and excessive iron release from macrophages.
Non-HFE-related genetic iron overload diseases are rare but are distributed worldwide. Type 2 hemochromatosis (juvenile hemochromatosis) is linked to mutations in the hemojuvelin gene (HJV, located on chromosome 1 or the hepcidin gene (HAMP, located on chromosome 19, which correspond to hemochromatosis types 2A and 2B, respectively. Type 2 hemochromatosis produces massive iron overload, specifically targeting the heart and the endocrine glands, and is mainly expressed, in individuals under 30 years old, as cardiac failure and/or hypopituitary hypogonadism. Repeated phlebotomies are the mainstay of treatment, possibly combined with oral iron chelation. Genetic testing, which is not routinely available, would find hemojuvelin or hepcidin mutations, either as homozygous mutations (for HAMP and most often for HJV but also as compound heterozygosity. The hepcidin promoter is also studied by sequencing, as mutations have been described in this region.
Type 3 hemochromatosis is due to mutations in the transferrin receptor 2 (TFR2) gene. When TFR2 is mutated, hepcidin production by the liver is decreased, leading, in turn, to body iron excess. The clinical picture mimics type 1 hemochromatosis; however, cases have also been reported in young patients that resemble juvenile hemochromatosis. Molecular diagnosis, if available, would identify homozygous TFR2 mutations, but compound heterozygosity has also been reported. Genetic testing is performed as for HJV and HAMP.
Type 4 hemochromatosis is due to mutations of the ferroportin gene (SLC40A1) and is also called ferroportin disease. It has a dominant pattern of transmission and incomplete penetrance. Typical ferroportin disease (‘loss-of-function’ or type A), the most frequent form of non-HFE genetic iron overload, is due to mutations affecting iron export capacity. The ‘loss-of-function’ profile is that of a predominantly macrophagic iron overload with normal or low plasma iron (and transferrin saturation). Ferritin levels are often high (>1000 μg/l) contrasting with normal or low plasma iron and transferrin saturation levels. MRI shows marked splenic iron excess and relatively less pronounced hepatic iron overload. Clinical symptoms are rare and this disease does not seem to have, at the time of diagnosis, significant morbidity in the absence of acquired or genetic cofactors. Genetic testing is not routinely available, but identified mutations are mainly located on the cytoplasmic portion or transmembrane segments of ferroportin [including p.Val162del, p.Asp157Gly (D157G), p.Gly80ser (G80S) and p.Gly490Asp (G490D). The p.Gln248His (Q248H) mutation might be associated with iron overload in male African Americans. Phlebotomies remain the basis for treatment but, given the impaired iron recycling process, are less well tolerated than in the case of hepcidin deficiency related iron overload (hemochromatosis types 1, 2 and 3). Atypical ferroportin disease (‘gain-of-function’ or type B) is rare, and by generating a hepcidin resistance phenotype it mimics type 1 hemochromatosis. Several mutations linked to this phenotype have been reported: p.Asn144Asp/Thr (N144D/T), p.Tyr64Asn (Y64N) and particularly p.Cys326Ser/Tyr (C326S/Y).
Hereditary aceruloplasminemia is a rare but widely distributed recessive disease arising from mutation of the ceruloplasmin gene located on chromosome 3. Plasma ceruloplasmin, through its ferroxidase activity, is involved in cellular iron egress. It is required for oxidizing ferrous iron, the redox iron species that crosses the membranes, into ferric iron, the iron species transported by circulating transferrin. In the absence of plasma ceruloplasmin, which is related to mutations in the ceruloplasmin gene, the process of cellular iron release is impaired because of subsequent alterations in ferroportin functioning. Therefore, cellular iron overload develops in a way similar to that observed in ferroportin disease, namely iron trapping within the cells associated with decreased iron release into the plasma, accounting for hyposideremia and low transferrin saturation levels. However, aceruloplasminemia is more severe than ferroportin disease, with marked anemia and brain involvement, suggesting that further mechanisms not yet fully elucidated are involved. The clinical picture is peculiar, combining, in an adult individual, anemia with low plasma iron and transferrin saturation, high plasma ferritin levels and neurological symptoms. Iron overload is found by MRI not only in the liver and spleen but also within the basal ganglia. The diagnosis rests first on biochemical assessment of plasma ceruloplasmin and plasma ferroxidase activity, which show no detectable activities in affected individuals. Genetic testing, if available, serves for diagnostic confirmation in a given individual and as a diagnostic marker for family members, and mutations are predominantly identified in the homozygous state. Phlebotomies are contraindicated because of pre-existing anemia, and treatment is therefore primarily based on iron chelation, with uncertain results on the brain iron load.
Hereditary atransferrinemia is a rare recessive disease affecting young individuals. It is responsible for severe anemia (the normal fate of transferrin iron is to provide bone marrow for red blood cell production) associated with cellular iron overload, which is related to plasma NTBI because transferrin is absent. The diagnosis rests on the fact that affected individuals show no detectable plasma transferrin. Molecular diagnosis, if performed, will identify either homozygosity or compound heterozygosity and serves both as an individual confirmation and as a family marker.
Divalent metal transporter1 (DMT1)-related iron overload is due to the fact that DMT1protein is not only involved in dietary iron uptake at the apical membrane of duodenal enterocytes but is also involved in the release of iron from acidified endosomes into the cytosol. Therefore, mutations in the SLC11A2 gene encoding DMT1, as first shown in Belgrade rats and mk/mk mice, give rise to a rare recessive disease expressed as microcytic anemia that is present from birth, is refractory to oral supplementation and is associated with visceral iron overload with only mildly elevated plasma ferritin. Molecular diagnosis, showing either homozygosity or compound heterozygosity, is the key diagnostic approach, although it is not feasible in routine practice. Treatment is mainly based on iron chelation therapy, but can be augmented with erythropoietin.
Finally, genetic sideroblastic anemias are characterized by iron accumulation in perinuclear mitochondria of erythroblasts and combine microcytic anemia with iron excess. They encompass two X-linked forms, delta-aminolevulinic synthase 2 acid mutations (in the ALAS2 gene) and ATP-binding cassette B7 deficiency (related to mutations in the ABCB7 gene), and two recessive forms, one related to SLC25A38 mutations and the other due to mutations in the glutaredoxin X5 gene (GLRX5).
Iron deficiency of genetic origin is essentially represented by iron-refractory iron deficiency anemia (IRIDA). It is a recessive disorder due to mutations in the gene TMPRSS6, which encodes the enzyme matriptase-2. The mutations relieve a form of inhibition on the HJV–BMP–SMAD signaling pathway, resulting in chronic elevation of plasma hepcidin levels. This elevation impairs duodenal iron absorption as well as iron release from the spleen. This process is close to that observed in the anemia of chronic disease, where increased plasma hepcidin levels are related to STAT3 pathway activation by interleukin 6 (IL-6). IRIDA is characterized by severe microcytic anemia with very low transferrin saturation and normal or high plasma ferritin. Hepcidin levels are normal or high, which is strongly abnormal in the setting of profound anemia, which, ‘physiologically’, should result in low plasma hepcidin levels. Anemia is more pronounced in children than in adults. Oral iron supplementation is ineffective and parenteral iron is only partially effective as iron recycling from macrophages – the first site of deposition of parenteral iron – is hampered by ferroportin deficiency subsequent to increased plasma hepcidin levels. Decreasing hepcidin levels, through the use of hepcidin antagonists, represents an important area of translational research.
In conclusion, several novel genetic iron-related disorders have been recently identified, corresponding either to primary hepcidin disorders or to disorders implicating iron transport, utilization and recycling. From a diagnostic viewpoint, molecular confirmation is easy for HFE-related hemochromatosis, but for all other genetic entities highly specific laboratories are needed due to the high cost of time-consuming techniques that are used infrequently given the rarity of these diseases, so that, in practice, only countries that have set up Reference Centers are able to provide clinicians with molecular confirmation. From a therapeutic viewpoint, the improved mechanistic understanding of these various disorders opens the way for the development of innovative treatment approaches.
Questions for future research
• What is the precise pathway whereby HFE regulates hepcidin transcription?
• Identification of new candidates for therapeutic targets in iron metabolism diseases among the signal transduction cascade involved in hepcidin transcription.
• Can cellular iron egress be regulated independently of the hepcidin–ferroportin interaction?
• How is iron metabolism controlled in the brain?
* What are the acquired and/or genetic cofactors accounting for phenotypic variability in the various forms of hemochromatosis?
• Evaluation of plasma hepcidin determination as a diagnostic and/or prognostic tool in the management of hemochromatosis and iron related diseases.
• Development of genetic, functional and metabolic tools to optimize quick diagnosis of iron overload diseases.
• Determination of the most appropriate means for normalizing hepcidin levels in patients with hepcidin-deficient hemochromatosis.
• Finding ways to manipulate iron metabolism in the brain.