Serum Hepcidin Hormone Level and Its Genes Polymorphism

Abstract

This chapter sheds light on hepcidin, historical view of hepcidin, and the time of its discovery in the first section. Then this chapter gives information about the genetic aspect and the importance of gene knowledge of hepcidin in explaining many disorders in human beings, supported by illustration figures. The regulation of iron in the human body as an essential function of hepcidin is discussed in this chapter. Examples of the genes of hepcidin (HAMP and HFE) are highlighted in detail as they are essential in regulating iron as well as discussing the genetic mutations that occur in these genes and their medical and clinical impacts for many diseases such as thalassemia. Finally, the inherited disorders related to hepcidin that lead to genetic diseases are discussed.

1. Introduction

Hepcidin is presently regarded as the key to the iron balance regulator. The balance of intracellular iron is preserved by proteins that regulate iron. Hepcidin, encoded by the HAMP gene is a 25 amino acid peptide that has been lately found [1]. Several mutations in the HAMP gene have been reported. The G71D mutation is probable to be linked to reduced hepcidin activity [2]. Mutations in iron-regulating proteins cause the disorder (HFE, TfR2, and HAMP) genes. Fekri et al. showed that H63D mutation of the HFE gene could play some role in disease evolution. In iron homeostasis, the HFE gene plays a very significant role by regulating iron absorption [3]. HFE mutations are currently referred to as the reason for decreased absorption of iron, iron overload, and hereditary hemochromatosis [4]. Many types of research have shown that patients with HFE mutations in beta-thalassemia are likely to create hemochromatosis that will require early chelation of iron even in heterozygous conditions [5]. Among the mutations discovered most frequently are the three missense mutations (SNPs), which are found in the HFE gene. The most prevalent mutation within the HFE gene exon 4, leading in a shift of cysteine-to-tyrosine amino acid at position 282 (C282Y), 60% of hereditary hemochromatosis instances in Mediterranean populations accounted for this mutation. H63D is also a mutation leading to the replacement of histidine with aspartic acid during a C-G shift at nucleotide 187 of exon 2 of the HFE gene. In combination with the C282Y allele (C282Y/H63D), hemochromatosis is most pronounced. The HFE gene’s third mutation is a substitute for 193AT in exon a pair of with an ensuing serine to cysteine replacement in amino acid position 65 (S65C) [6]. The interaction concerning the mutations over genes influencing blood homeostasis including thalassemia may want to hold a synergistic result, increasing the iron storage [7].

2. Hepcidin

2.1 Discovery of hepcidin

The hepcidin molecule (“hep” hepatic origin, “cidin” antimicrobial activity) was described in the year 2000; it is an antimicrobial peptide that acts in parts in innate immunity and iron metabolism [8]. It is a peptide hormone the liver produces, and it works as a regulator of iron [9]. Hepcidin is a regulator of iron homeostasis. Its production is increased by iron excess and inflammation and decreased by hypoxia and anemia. Hepcidin inhibits the flow of iron into the plasma from duodenal enterocytes that absorb dietary iron, macrophages that recycle iron from senescent erythrocytes, and iron-storing hepatocytes. Iron-loading anemias are diseases in which hepcidin is controlled by ineffective erythropoiesis and concurrent iron overload impacts [10]. Hepcidin was isolated from the human urine and blood, especially from plasma after filtration [8]. Hepcidin was produced by macrophages, adipocytes, neutrophils, lymphocytes, renal cells, and β-cells [11].

Hepcidin is produced by macrophages, adipocytes, neutrophils, lymphocytes, renal cells, and β-cells [11]. In the studies of experiment on mice used for the determination of hepcidin regulation, the expression, function, and structure showed that severe iron overload is occurring due to the gene responsible for hepcidin production, and the gene has the role of iron regulation. Hepcidin has several functions such as inflammation, hypoxia, hypoxia, and iron stores [12]. Hepcidin reacts with ferroportin, and the ferroportin is found in spleen, duodenum, and placenta. If the ferroportin decreases, it results in reduced iron intake and macrophage release of iron and using of the iron, which is stored in the liver [13].

2.2 The hepcidin antimicrobial peptide (HAMP) gene and the structure of hepcidin

Hepcidin was initially identified as a liver-expressed antimicrobial peptide (LEAP1) with direct antimicrobial activity against a number of bacterial and fungal species [14]. It was soon discovered that hepcidin plays a major role in the regulation of iron homeostasis, being overexpressed in the liver with an induced (dietary or parenteral) iron overload [15]. Hepcidin acts as a negative regulator of iron stores; in response to increased iron levels, the liver increases hepcidin synthesis which then acts on the sites of absorption (enterocytes of the duodenum), storage (primarily hepatocytes of the liver), or recycling (macrophages of the reticuloendothelial system) leading to a decrease in the release of iron from these tissues. Hepcidin exerts its influence by binding to and inducing the internalization and degradation of ferroportin (FPN), the only known exporter of iron [16].

Gene of human hepcidin is carried out by chromosome 19q13.1. It consists of a (2637) nucleated base [17]. HAMP gene was founded in the liver cells in the brain, trachea, heart, tonsils, and lung [18]. HAMP gene encodes preprohepcidin, which consists of (84) amino acids; hepcidin has three forms: 25 aa, 22 aa, and 20 aa peptides. All the types are founded in urine, while 25 and 20 are founded in human serum [19]. Figure 1 shows the structure of hepcidin-25, which consists of (8) cysteine linked by disulfide link [21]. Nuclear magnetic resonance spectroscopy (NMR) is used for the analysis of the structure of hepcidin; it has four disulfide bonds [22].

Figure 1.

Molecule structure of human synthetic hepcidin-25. Front: an overview of the structure of hepcidin-25. Distorted β-sheets are shown as gray arrows, and the peptide backbone is colored gray. The disulfide bonds are colored yellow, highlighting the position of an unusual vicinal bond between adjacent cysteines at the hairpin turn. Positive residues of arginine (Arg) and lysine (Lys) are pictured in blue, the negative residue of aspartic acid (asp) in red, and the histidine containing amino-terminal Cu2 ± Ni2 + (ATCUN)-binding motif in the N terminal region is colored green. Background: Hepcidin-25 molecule displayed with a solvent-accessible surface that illustrates the amphipathic structure of the molecule [20].

2.3 Iron regulation by hepcidin

The iron content in biological fluids is strictly regulated in all organisms to provide iron as required and to prevent toxicity, as excess iron can contribute to reactive oxygen species production. Iron homeostasis in mammals is controlled at the level of intestinal absorption, as iron is not excreted. Hepcidin, a circulating peptide hormone, is the master systemic iron homeostasis regulator that combines iron use and processing with the iron acquisition [22].

This hormone is released primarily by hepatocytes and is a negative regulator of plasma iron entry (Figure 2).

Figure 2.

Hepcidin internalization and degradation [22].

Hepcidin works by binding to ferroportin, an iron carrier present in intestinal duodenum cells, macrophages, and placenta cells. Hepcidin binding leads to the internalization and degradation of the ferroportin [16]. Cell surface ferroportin deficiency prevents iron from entering the plasma. Reduced iron entry into plasma results in low transferrin saturation, and less iron is delivered to the production of erythroblast [23]. In addition, reduced hepcidin expression results in increased ferroportin cell surface and increased iron absorption. Hepcidin dysregulation leads to iron disorders [24].

Overexpression of hepcidin results in chronic disease anemia, whereas low production of hepcidin results in hereditary hemochromatosis with consequent accumulation of iron in vital organs [25] (Figure 2). Most inherited iron disorders arise from inadequate development of hepcidin relative to the degree of accumulation of tissue iron. Impaired hepcidin expression was found to result from mutations in any of four genes: transferrin receptor 2 (TFR2), hemochromatosis (HFE), type 2 hemochromatosis (HFE2), and antimicrobial hepcidin peptide (HAMP) [26]. Mutations in HAMP, the gene encoding hepcidin, lead to iron overload disease because the absence of hepcidin allows for constitutively high absorption of iron.

Hepcidin-mediated iron homeostasis regulation. As in condition with high levels of hepcidin in the bloodstream result in the iron exporter ferroportin which is being internalized and degraded. Loss of ferroportin cell surface results in the loading of macrophage iron, low levels of plasma iron, and decreased erythropoiesis due to decreased iron-bound transferrin. Decreased erythropoiesis causes chronic disease anemia and regulates the level of iron imports into plasma, normal transferrin saturation, and normal levels of erythropoiesis in response to iron demand (Figure 3). Hemochromatosis, or iron overload, results from the insufficient levels of hepcidin, resulting in increased plasma iron imports, high transferrin saturation, and excess liver iron deposition [28].

Figure 3.

Regulation of iron balance [27].

2.4 The HAMP gene mutation

Mutation in the HAMP gene will produce a change in the hepcidin function. HAMP gene consists of exon 3, the last exon is encoded proteins, and it is considered the most important and largest area in the gene and is contained on many polymorphisms [29]. Polymorphisms are in the HFE gene more than in the HAMP gene. There are about 16 types of single nucleotide polymorphism founded in different studies [30]. Many reports detected (8) mutations in the gene. The persons who carry mutations in the HAMP gene show juvenile hemochromatosis, which occurs at 10–30 years [31]. The microsatellite marker probes are used as the first genetic alteration in the HAMP gene [32]. In some time, the mutation occurs in c.233G > A after exchanging of some amino acids’ inactive peptide or substitution of C78 by a tyrosine, C78T. The mutation makes possible the bisulfite bonds binding of ferroportin to hepcidin, which results in the accumulation absorbed of iron [33].

Mutation of C70R causes malformation in the bisulfite bond of the cysteines. The arginine becomes an alternative for the cysteine that its exchanging does not allow the production of bisulfite bridge between (3) and (6) in hepcidin peptide [34]. Alteration of C to T was occurring at position (166) (166C-T) of the HAMP gene and alteration of arginine at position (56) for a stopping codon (R56X), 193A to (T). As well as the ferroportin also does not bind to hepcidin, producing more iron. While defect in guanine exon 2 at position 93 leads to a mutation in RNA [35], defect in Met50del from exon 2 causes a disorder in the expression of the active peptide and causes changes in reading frames. Another mutation, G71D, alters to amino acid 71, which is between (3–4) cysteine, which prevents linking with ferroportin [36].

HFE-H63D variant is associated with the HAMP-G71D variant in sickle cell disease patients and increases iron overload [37]. The polymorphism (G to A) at the +14 position of the 5′-UTR regions produce new initiation codon, inducing yielding of a new abnormal protein and change in the reading frame. Unstable protein will be generated, which is analyzed after the translation of the mRNA [38]. The haplotype of the HAMP gene is caused by the linked of polymorphisms NC-582A > G and NC-1010C > T, with ferritin concentration more than 300 μg/L [39].

The association of HFE gene polymorphisms and HAMP is common. In some clinical cases, there are several mixed clinical signs, with iron overload. The variants C-582A > G and C-153C > T decrease hepcidin expression, but the mechanism of action of peptide stays the same without transferrin saturation and increasing ferritin [40]. The body organs contain iron at large amounts such as the heart and liver, and it will be affected and damaged [41]. Any change in the HAMP gene may cause a defective hepcidin protein, and it became no action. Accumulation of iron and ferritin in the organs helps to develop diseases in different organs such as coronary diseases, diabetes mellitus, HIV, HBV, and HCV [42]. It was noticed that some neurodegenerative diseases are associated with a high level of hepcidin in plasma such as Alzheimer, Parkinson, and sclerosis [43].

2.5 Homeostatic iron regulation gene (HFE) human

Human homeostatic iron regulator protein, also known as the HFE protein (HighmFE2+), is a protein that in humans is encoded by the HFE gene. The HFE gene is located on the short arm of chromosome 6 at location 6p21.3 [44]. Simon and colleagues, in the 1970s, noted that hemochromatosis is relatively common, associated with markers of human leukocyte antigen (HLA), and transmitted as an autosomal recessive trait [45]. In 1996, Feder and colleagues used positional cloning to classify HFE, the gene of hemochromatosis, associated with the main chromosome 6p histocompatibility complex (MHC) [46]. The HFE membrane protein is similar to the proteins of the MHC class I and binds beta-2 microglobulin (β2M) [46]. HFE binds the extracellular α1-α2 domain to the transferrin receptor (TFRC) [47]. HFE is needed for normal hepatic synthesis regulation of hepcidin, the principal iron metabolism controller [25].

Common HFE mutations represent approximately 90% of phenotypes of hemochromatosis in Western European descent whites. Feder and colleagues named the gene HLA-H [46] although the name had been published earlier to designate a presumed pseudogene in the HLA class I region [48]. Marsh demanded a more suitable designation [49]. The HFE symbol (H = high; FE = iron) was accepted by both the WHO Nomenclature Committee for HLA System Factors and the HUGO Genome Nomenclature Committee.

2.6 Structure and function of HFE gene human

HFE has seven exons of 12 kb [39]. HFE includes 9.6 kb of DNA on chromosome 6p in the extended region of HLA class I. Histone genes on both sides of HFE are present [50]. Exon 1 corresponds to the peptide of the signal and exons 2–4, respectively, to the domains α1, α2, and α3. For the transmembrane domain, exon 5 accounts. The cytoplasmic tail is encoded by exon 6’s 5′ portion, which includes a native stop codon. The full-length transcript, therefore, represents six exons [51]. HFE is a protein containing 343 amino acids, including a signal peptide, an extracellular transferrin-binding region (α1 and α2), an immunoglobulin-like domain (α3), a transmembrane region, and a short cytoplasmic tail. HFE binds β2 M to form a cell surface heterodimer [46].

HFE is glycosylated at asparagine residues 110, 130, and 234 during transport to the cell membrane. For normal intracellular trafficking and function, glycosylation is important. HFE interacts with TFRC [51, 52]. HFE’s structure revealed that its TFRC ligand binds in a molar ratio of 2:1 TFRC: HFE [47]. There is a peptide-binding groove in most class I MHC molecules. Since the α1 and α2 helices are closer to HFE, the HFE analog site is too narrow to bind peptides [47]. TFRC and HFE bind strongly to the essential pH of cell surfaces but not to the intracellular vesicle acid pH [47]. The structure of a complex between the extracellular portions of HFE and TFRC shows that binding affects both HFE and its ligand configurations. In their domain arrangements and dimer interfaces, the structures of TFRC alone and TFRC complexed with HFE differ [53].

Studies of 293 cells cultivated to express wild-type HFE proteins showed that with TFR, HFE forms stable complexes. The association of HFE protein with TFR was significantly reduced in 293 cells over the expression of HFE C282Y, as shown in Figure 4 [54]. Through inhibiting TFRC: TF-Fe interaction in an experiment using purified proteins and a biosensor chip [47], normal HFE protein decreased the affinity of TFRC to TF. HFE changes the conformation of the Tf-Fe binding site as observed by biosensor assays when HFE binds to TFRC in vitro, decreasing the entry of iron into Chinese ovarian hamster cells [47]. No evidence of binding of HFE and TFR2 was detected in coimmunoprecipitation or surface resonance-based testing experiments using soluble HFE and TFR2 [55].

Figure 4.

The HFE gene diagram. The image was changed after getting permission from the author. Cys282 -> Tyr282 exchanging mutation of C282Y and His63 -> Asp63 exchanging mutation of H63D [54].

The relevance, if any, of these in vitro results to in vivo iron homeostasis is unclear. Many factors, including HFE, act as hepcidin transcription upstream regulators [18]. HAMP expression in untreated patients with hemochromatosis, homozygosity of C282Y, and iron overload was significantly lower than controls [56].

2.7 Mutation of the HFE gene

The three most common mutations of HFE in the coding region are: C282Y (exon 4; c.845GA; rs1800562), H63D (exon 2; c.187CG; rs1799945), and S65C (exon 2; c.193AT; rs1800730) [57]. The C282Y mutation interrupts a critical disulfide bond in HFE’s α3 domain, abrogating its binding to β2 M and limiting its location to the cytoplasm [58]. H63D and S65C affect the α1 binding groove but do not prevent HFE on cell surfaces. HFE C282 is conserved because cysteine 282 is essential to β2M binding and extracellular presentation of HFE. H63 is also conserved. Histidine 63 forms a salt bridge in the α2 domain that binds TFRC, suggesting that the salt bridge is important for HFE function [59].

Histidine 116 and 145 and tyrosine 140 are widely conserved. A cluster of four histidine residues (H109, H111, H116, and H145) is associated with Y140 in the α1 domain. This configuration resembles functional sites in other iron-binding proteins [40]. Hereditary hemochromatosis type-1 is caused by HFE gene mutation. Allelic is most prevalent among the individuals of Europe [60]. Single point simple change in exon 4, 845G to 845A in the HFE gene results in an exchange of cysteine by tyrosine. Also, another gene mutation included a change of the allele 187C to 187G, wherever histidine is exchanged. The substitution of serine for cysteine is considered the third mutation of the HFE gene S65C [61]. Also, H63D mutation is present in HFE protein, causing a decrease in the transferrin receptor [62]. When hepcidin protein cannot bind with the transferrin receptor, another factor has a great role in hepcidin protein, therefore, any changes in hepcidin protein help to aggregation iron by inhibiting the transcription of hepcidin and do not allow iron absorption from the intestine [63]. Also, the modifying of the H63D genotype associated between the metabolism of iron and lead, wherever there is increased iron in the body, is associated with a high level in the same body [64]. Iron overload has been identified in individuals with the digenic inheritance of one or more HFE mutations and a non-HFE gene mutation that is also involved in iron metabolism [44]. One example is the development of hemochromatosis in individuals who are double heterozygous for one or more HFE mutations and a hepcidin gene mutation (HAMP) [65].

Iron loading occurred in people with HFE mutation digenic inheritance and either a hemojuvelin gene (HJV) mutation [58] or a TFR2 gene (TFR2) mutation [66].

2.8 Iron overload and genetic alterations

Activation of the HFE gene works with hemochromatosis that includes iron accumulation that occurs with heart failure, cirrhosis, diabetes, and hepatocarcinoma. Interaction between β2-microglobulin and HFE protein is the main hypothesis that explains the development of hemochromatosis in the body. β2-microglobulin and HFE protein react together and form the transferrin-1 receptor, which helps to absorbed iron from the diet [67]. Many reports founded new genes responsible for the iron metabolism [hereditary hemochromatosis (HH)]. It included two types, the first type is HFE hemochromatosis that occurs due to mutation of the HFE gene, and its spread commonly in the Caucasian people. The second type is non-HFE hemochromatosis. Non-HFE hemochromatosis, including HAMP, ferroportin, TFR, and HJV gene, is detected among the diseases. Genetic alterations in ceruloplasmin and ferritin encourage iron accumulation with the occurrence of hyperferritinemia [44, 68]. The accumulation of iron in the tissues interferes with tissues’ function, such as hepatic failure, cardiac problems, cirrhosis, and diabetes. Furthermore, there are pituitary gland diseases and disorder in the function of joints, gonads, abdominal pain, and hyperpigmentation of the skin [69].

Hemochromatosis diagnosis is made by deepening on clinical signs, biochemical markers, genetic examination, and Liver biopsy [70]. Some indicators, such as ferritin, transferrin iron elevated, and liver enzymes, are elevated in hemochromatosis patients. The level of ferritin in serum is greater 300 μg/L in men and 200 μg/L in women, also the same speech on the level of transferrin. If the level of ferritin and transferring are elevated, it should make a genetic analysis of some genes such as HAMP, HJV, HFE, transferrin receptor genes, and ferroportin [71].

3. Conclusions

The knowledge about hepcidin and its genetic structure, as well as a common mutation that occurs in it, is vital to understand the iron metabolism and iron disorders. This chapter helps the reader to get ideas about that.

Acknowledgments

The authors of this chapter wish to thank the workers in the molecular biology department at the Al-Karama Teaching Hospital in Wasit, Iraq, for the credibility and keenness they have achieved in carrying out the work assigned to them.

Conflict of interest

The authors have no conflict of interest.