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The mammalian multicopper ferroxidases (MCFs) ceruloplasmin (CP), hephaestin (HEPH) and zyklopen (ZP) comprise a family of conserved enzymes that are essential for body iron homeostasis. Each of these enzymes contains six biosynthetically incorporated copper atoms which act as intermediate electron acceptors, and the oxidation of iron is associated with the four electron reduction of dioxygen to generate two water molecules. CP occurs in both a secreted and GPI-linked (membrane-bound) form, while HEPH and ZP each contain a single C-terminal transmembrane domain. These enzymes function to ensure the efficient oxidation of iron so that it can be effectively released from tissues via the iron export protein ferroportin and subsequently bound to the iron carrier protein transferrin in the blood. CP is particularly important in facilitating iron release from the liver and central nervous system, HEPH is the major MCF in the small intestine and is critical for dietary iron absorption, and ZP is important for normal hair development. CP and HEPH (and possibly ZP) function in multiple tissues. These proteins also play other (non-iron-related) physiological roles, but many of these are ill-defined. In addition to disrupting iron homeostasis, MCF dysfunction perturbs neurological and immune function, alters cancer susceptibility, and causes hair loss, but, despite their importance, how MCFs co-ordinately maintain body iron homeostasis and perform other functions remains incompletely understood.

Background & aimsMulticopper ferroxidases (MCFs) facilitate intestinal iron absorption and systemic iron recycling, likely by a mechanism involving the oxidization of Fe2+ from the iron exporter ferroportin 1 for delivery to the circulating Fe3+ carrier transferrin. Hephaestin (HEPH), the only MCF known to be expressed in enterocytes, aids in the basolateral transfer of dietary iron to the blood. Mice lacking HEPH in the whole body (Heph-/- ) or intestine alone (Hephint/int ) exhibit defects in dietary iron absorption but still survive and grow. Circulating ceruloplasmin (CP) is the only other known MCF likely to interact with enterocytes. Our aim was to assess the effects of combined deletion of HEPH and CP on intestinal iron absorption and homeostasis in mice.MethodsMice lacking both HEPH and CP (Heph-/-Cp-/- ) and mice with whole-body knockout of CP and intestine-specific deletion of HEPH (Hephint/intCp-/- ) were generated and phenotyped.ResultsHeph-/-Cp-/- mice were severely anemic and had low serum iron, but they exhibited marked iron loading in duodenal enterocytes, the liver, heart, pancreas, and other tissues. Hephint/intCp-/- mice were moderately anemic (similar to Cp-/- mice) but were iron loaded only in the duodenum and liver, as in Hephint/int and Cp-/- mice, respectively. Both double knockout models absorbed iron in radiolabeled intestinal iron absorption studies, but the iron was inappropriately distributed, with an abnormally high percentage retained in the liver.ConclusionsThese studies indicate that HEPH and CP, and likely MCFs in general, are not essential for intestinal iron absorption but are required for proper systemic iron distribution. They also point to important extra-intestinal roles for HEPH in maintaining whole-body iron homeostasis.

Background & aimsMulticopper ferroxidases (MCFs) facilitate intestinal iron absorption and systemic iron recycling, likely by a mechanism involving the oxidization of Fe2+ from the iron exporter ferroportin 1 for delivery to the circulating Fe3+ carrier transferrin. Hephaestin (HEPH), the only MCF known to be expressed in enterocytes, aids in the basolateral transfer of dietary iron to the blood. Mice lacking HEPH in the whole body (Heph-/- ) or intestine alone (Hephint/int ) exhibit defects in dietary iron absorption but still survive and grow. Circulating ceruloplasmin (CP) is the only other known MCF likely to interact with enterocytes. Our aim was to assess the effects of combined deletion of HEPH and CP on intestinal iron absorption and homeostasis in mice.MethodsMice lacking both HEPH and CP (Heph-/-Cp-/- ) and mice with whole-body knockout of CP and intestine-specific deletion of HEPH (Hephint/intCp-/- ) were generated and phenotyped.ResultsHeph-/-Cp-/- mice were severely anemic and had low serum iron, but they exhibited marked iron loading in duodenal enterocytes, the liver, heart, pancreas, and other tissues. Hephint/intCp-/- mice were moderately anemic (similar to Cp-/- mice) but were iron loaded only in the duodenum and liver, as in Hephint/int and Cp-/- mice, respectively. Both double knockout models absorbed iron in radiolabeled intestinal iron absorption studies, but the iron was inappropriately distributed, with an abnormally high percentage retained in the liver.ConclusionsThese studies indicate that HEPH and CP, and likely MCFs in general, are not essential for intestinal iron absorption but are required for proper systemic iron distribution. They also point to important extra-intestinal roles for HEPH in maintaining whole-body iron homeostasis.

Migraine is a common neurological disorder that affects approximately 12-20% of the general adult population. Migraine pathogenesis is complex and not wholly understood. Molecular genetic investigations, imaging and biochemical studies, have unveiled a number of interconnected neurological pathways which seem to have a cause and effect component integral to its cause. Much weight of migraine attack initiation can be placed on the initial trigger and the pathways involved in its neuronal counter reaction. Ion channels play a large role in the generation, portrayal and mitigation of the brains response to external triggers. Several genetic studies have identified and implicated a number of ion channelopathy genes which may contribute to this generalised process. This review will focus on the genetics of migraine with particular emphasis placed on the potentially important role genes HEPH (responsible for iron transport and homeostasis) and KCNK18 (important for the transport and homeostasis of potassium) play in migraine cause.

The development of targeted next-generation sequencing (NGS) applications now promises to be a clinically viable option for the diagnosis of rare disorders. This approach is proving to have significant utility where standardized testing has failed to identify the underlying molecular basis of disease. We have developed a unique targeted NGS panel for the systematic sequence-based analysis of atypical iron disorders. We report the analysis of 39 genes associated with iron regulation in eight cases of atypical iron dysregulation, in which five cases we identified the definitive causative mutation, and a possible causative mutation in a sixth. We further provide a molecular and cellular characterization study of one of these mutations (TFR2, p.I529N) in a familial case as proof of principle. Cellular analysis of the mutant protein indicates that this amino acid substitution affects the localization of the protein, which results in its retention in the endoplasmic reticulum and thus failure to function at the cell surface. Our unique NGS panel presents a rapid and cost-efficient approach to identify the underlying genetic cause in cases of atypical iron homeostasis disorders.