Inherited microcytic anemias.

Inherited microcytic anemias can be broadly classified into 3 subgroups: (1) defects in globin chains (hemoglobinopathies or thalassemias), (2) defects in heme synthesis, and (3) defects in iron availability or iron acquisition by the erythroid precursors. These conditions are characterized by a decreased availability of hemoglobin (Hb) components (globins, iron, and heme) that in turn causes a reduced Hb content in red cell precursors with subsequent delayed erythroid differentiation. Iron metabolism alterations remain central to the diagnosis of microcytic anemia, and, in general, the iron status has to be evaluated in cases of microcytosis. Besides the very common microcytic anemia due to acquired iron deficiency, a range of hereditary abnormalities that result in actual or functional iron deficiency are now being recognized. Atransferrinemia, DMT1 deficiency, ferroportin disease, and iron-refractory iron deficiency anemia are hereditary disorders due to iron metabolism abnormalities, some of which are associated with iron overload. Because causes of microcytosis other than iron deficiency should be considered, it is important to evaluate several other red blood cell and iron parameters in patients with a reduced mean corpuscular volume (MCV), including mean corpuscular hemoglobin, red blood cell distribution width, reticulocyte hemoglobin content, serum iron and serum ferritin levels, total iron-binding capacity, transferrin saturation, hemoglobin electrophoresis, and sometimes reticulocyte count. From the epidemiological perspective, hemoglobinopathies/thalassemias are the most common forms of hereditary microcytic anemia, ranging from inconsequential changes in MCV to severe anemia syndromes.

Idiopathic copper toxicosis: is abnormal copper metabolism a primary cause of this disease?

Idiopathic copper toxicosis (ICT) is characterized by marked copper deposition, Mallory-Denk body (MDB) formation and severe hepatic injury. Although the characteristics are apparently different from Wilson disease, large amounts of copper accumulate in the liver of the patients. We extensively treated a patient with ICT to reduce the body copper, however, the patient needed liver transplantation. Previous liver biopsy revealed high copper content. But extirpated liver contained an extremely small amount of copper, although MDBs and severe inflammation remained. These phenomena suggest abnormal copper metabolism is not the principle cause of ICT but some other abnormality must exist.

Metabolic crisis after trivial head trauma in late-onset isolated sulfite oxidase deficiency: Report of two new cases and review of published patients.

BACKGROUND: Isolated sulfite oxidase deficiency (ISOD) is a rare autosomal recessively inherited inborn error of metabolism, caused by mutation in SUOX gene. ISOD has two kind of presentation; early and late-onset. The late-onset form is extremely rare and only 10 cases have been reported. METHODS: We report two new cases of late-onset ISOD with biochemical and genetic confirmation. We did a review of the previously published cases of late-onset ISOD. RESULTS: Together with the presented two cases, 12 cases were available for analysis. The median age at symptom onset and at diagnosis was 8.5 and 23 months respectively. Almost all children had acute regression of milestones followed by slow recovery. The common presenting signs and symptoms were movement disorders, seizures, ectopia lentis and hypertonia. Five children had antecedent events. Trivial trauma precipitating the metabolic crisis was unique to the two cases we report. The most common MRI feature was globus pallidi changes followed by cerebellar white matter changes, vermian hypoplasia and thinned out corpus callosum. Diffusion weighted sequence was performed in 3 children and all had diffusion restriction in the affected area. CONCLUSION: Trivial trauma can precipitate metabolic crisis in late-onset ISOD. Low plasma homocysteine and involvement of globus pallidi with diffusion restriction on the MRI are important diagnostic clues. Early diagnosis and intervention with special diet may be effective in preventing long term neurodisability.

Mild phenotype in Molybdenum cofactor deficiency: A new patient and review of the literature.

BACKGROUND: Molybdenum cofactor deficiency (MoCD) is a rare autosomal-recessive disorder that results in the combined deficiency of molybdenum-dependent enzymes. Four different genes are involved in Molybdenum cofactor biosynthesis: MOCS1, MOCS2, MOCS3, and GEPH. The classical form manifests in the neonatal period with severe encephalopathy, including intractable seizures, MRI changes that resemble hypoxic-ischemic injury, microcephaly, and early death. To date, an atypical phenotype with late-onset has been reported in the literature in 13 patients. METHODS: We describe a late-onset and a relatively mild phenotype in a patient with MOCS2 homozygous mutation. RESULTS: Pyramidal and extrapyramidal signs are recognized in those patients, often exacerbated by intercurrent illness. Expressive language is usually compromised. Neurological deterioration is possible even in adulthood, probably due to accumulation of sulfite with time. CONCLUSION: Sulfite inhibition of mitochondrial metabolism could be responsible for the ischemic lesions described in patients with MoCD or alternatively could predispose the brain to suffer an ischemic damage through the action of other insults, for instance intercurrent illness. It is possible that sulfite accumulation together with other external triggers, can lead to neurological deterioration even in adulthood. The role of other factors involved in clinical expression should be investigated to establish the reason for phenotypic variability in patients with the same mutation.

A mild case of molybdenum cofactor deficiency defines an alternative route of MOCS1 protein maturation.

Molybdenum cofactor deficiency is an autosomal recessive inborn error of metabolism, which results from mutations in genes involved in Moco biosynthesis. Moco serves as a cofactor of several enzymes, including sulfite oxidase. MoCD is clinically characterized by intractable seizures and severe, rapidly progressing neurodegeneration leading to death in early childhood in the majority of known cases. Here we report a patient with an unusual late disease onset and mild phenotype, characterized by a lack of seizures, normal early development, a decline triggered by febrile illness and a subsequent dystonic movement disorder. Genetic analysis revealed a homozygous c.1338delG MOCS1 mutation causing a frameshift (p.S442fs) with a premature termination of the MOCS1AB translation product at position 477 lacking the entire MOCS1B domain. Surprisingly, urine analysis detected trace amounts (1% of control) of the Moco degradation product urothione, suggesting a residual Moco synthesis in the patient, which was consistent with the mild clinical presentation. Therefore, we performed bioinformatic analysis of the patient's mutated MOCS1 transcript and found a potential Kozak-sequence downstream of the mutation site providing the possibility of an independent expression of a MOCS1B protein. Following the expression of the patient's MOCS1 cDNA in HEK293 cells we detected two proteins: a truncated MOCS1AB protein and a 22.4 kDa protein representing MOCS1B. Functional studies of both proteins confirmed activity of MOCS1B, but not of the truncated MOCS1AB. This finding demonstrates an unusual mechanism of translation re-initiation in the MOCS1 transcript, which results in trace amounts of functional MOCS1B protein being sufficient to partially protect the patient from the most severe symptoms of MoCD.

S-sulfocysteine/NMDA receptor-dependent signaling underlies neurodegeneration in molybdenum cofactor deficiency.

Molybdenum cofactor deficiency (MoCD) is an autosomal recessive inborn error of metabolism characterized by neurodegeneration and death in early childhood. The rapid and progressive neurodegeneration in MoCD presents a major clinical challenge and may relate to the poor understanding of the molecular mechanisms involved. Recently, we reported that treating patients with cyclic pyranopterin monophosphate (cPMP) is a successful therapy for a subset of infants with MoCD and prevents irreversible brain damage. Here, we studied S-sulfocysteine (SSC), a structural analog of glutamate that accumulates in the plasma and urine of patients with MoCD, and demonstrated that it acts as an N-methyl D-aspartate receptor (NMDA-R) agonist, leading to calcium influx and downstream cell signaling events and neurotoxicity. SSC treatment activated the protease calpain, and calpain-dependent degradation of the inhibitory synaptic protein gephyrin subsequently exacerbated SSC-mediated excitotoxicity and promoted loss of GABAergic synapses. Pharmacological blockade of NMDA-R, calcium influx, or calpain activity abolished SSC and glutamate neurotoxicity in primary murine neurons. Finally, the NMDA-R antagonist memantine was protective against the manifestation of symptoms in a tungstate-induced MoCD mouse model. These findings demonstrate that SSC drives excitotoxic neurodegeneration in MoCD and introduce NMDA-R antagonists as potential therapeutics for this fatal disease.

Inherited Disorders of Manganese Metabolism.

While the neurotoxic effects of manganese were recognized in 1837, the first genetic disorder of manganese metabolism was described only in 2012 when homozygous mutations in SLC30A10 were reported to cause manganese-induced neurotoxicity. Two other genetic disorders of manganese metabolism have now been described - mutations in SLC39A14 cause manganese toxicity, while mutations in SLC39A8 cause manganese and zinc deficiency. Study of rare genetic disorders often provides unique insights into disease pathobiology, and the discoveries of these three inherited disorders of manganese metabolism are already transforming our understanding of manganese homeostasis, detoxification, and neurotoxicity. Here, we review the mechanisms by which mutations in SLC30A10, SLC39A14, and SLC39A8 impact manganese homeostasis to cause human disease.

Drosophila melanogaster Models of Metal-Related Human Diseases and Metal Toxicity.

Iron, copper and zinc are transition metals essential for life because they are required in a multitude of biological processes. Organisms have evolved to acquire metals from nutrition and to maintain adequate levels of each metal to avoid damaging effects associated with its deficiency, excess or misplacement. Interestingly, the main components of metal homeostatic pathways are conserved, with many orthologues of the human metal-related genes having been identified and characterized in Drosophila melanogaster. Drosophila has gained appreciation as a useful model for studying human diseases, including those caused by mutations in pathways controlling cellular metal homeostasis. Flies have many advantages in the laboratory, such as a short life cycle, easy handling and inexpensive maintenance. Furthermore, they can be raised in a large number. In addition, flies are greatly appreciated because they offer a considerable number of genetic tools to address some of the unresolved questions concerning disease pathology, which in turn could contribute to our understanding of the metal metabolism and homeostasis. This review recapitulates the metabolism of the principal transition metals, namely iron, zinc and copper, in Drosophila and the utility of this organism as an experimental model to explore the role of metal dyshomeostasis in different human diseases. Finally, a summary of the contribution of Drosophila as a model for testing metal toxicity is provided.

Iron homeostasis: transport, metabolism, and regulation.

PURPOSE OF REVIEW: Iron is essential for normal cellular function and many diseases result from disturbances in iron homeostasis. This review describes some of the recent key advances in iron transport and its regulation, how this relates to iron-related disorders, and emerging therapies for these diseases. RECENT FINDINGS: The iron-regulatory hormone hepcidin and its target, the iron exporter ferroportin (FPN), play central roles in iron homeostasis. Recent studies have expanded our understanding of how hepcidin is regulated in response to stimulated erythropoiesis and have added some new players to the complex network of factors that influences hepcidin expression. Novel structural insights into how FPN transports iron have been an important addition to the field, as has the recognition that some zinc transporters such as ZIP14 can transport iron. Investigations into cardiac iron homeostasis have revealed a key role for FPN, and transferrin receptor 1, which is essential for cellular iron uptake, has been shown to be critical for normal immune function. SUMMARY: The increased understanding of mechanisms of iron homeostasis that has resulted from recent research has greatly improved our ability to diagnose and manage iron-related disorders, and has offered new therapies for this important class of human diseases.

Canine Models for Copper Homeostasis Disorders.

Copper is an essential trace nutrient metal involved in a multitude of cellular processes. Hereditary defects in copper metabolism result in disorders with a severe clinical course such as Wilson disease and Menkes disease. In Wilson disease, copper accumulation leads to liver cirrhosis and neurological impairments. A lack in genotype-phenotype correlation in Wilson disease points toward the influence of environmental factors or modifying genes. In a number of Non-Wilsonian forms of copper metabolism, the underlying genetic defects remain elusive. Several pure bred dog populations are affected with copper-associated hepatitis showing similarities to human copper metabolism disorders. Gene-mapping studies in these populations offer the opportunity to discover new genes involved in copper metabolism. Furthermore, due to the relatively large body size and long life-span of dogs they are excellent models for development of new treatment strategies. One example is the recent use of canine organoids for disease modeling and gene therapy of copper storage disease. This review addresses the opportunities offered by canine genetics for discovery of genes involved in copper metabolism disorders. Further, possibilities for the use of dogs in development of new treatment modalities for copper storage disorders, including gene repair in patient-derived hepatic organoids, are highlighted.

Molybdenum cofactor deficiency.

Molybdenum cofactor deficiency (MoCD) is a severe autosomal recessive inborn error of metabolism first described in 1978. It is characterized by a neonatal presentation of intractable seizures, feeding difficulties, severe developmental delay, microcephaly with brain atrophy and coarse facial features. MoCD results in deficiency of the molybdenum cofactor dependent enzymes sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase and mitochondrial amidoxime reducing component. The resultant accumulation of sulfite, taurine, S-sulfocysteine and thiosulfate contributes to the severe neurological impairment. Recently, initial evidence has demonstrated early treatment with cyclic PMP can turn MoCD type A from a previously neonatal lethal condition with only palliative options, to near normal neurological outcomes in affected patients. We review MoCD and focus on describing the currently published evidence of this exciting new therapeutic option for MoCD type A caused by pathogenic variants in MOCD1.

Functional understanding of the versatile protein copper metabolism MURR1 domain 1 (COMMD1) in copper homeostasis.

Copper is an important cofactor in numerous biological processes in all living organisms. However, excessive copper can be extremely toxic, so it is vital that the copper level within a cell is tightly regulated. The damaging effect of copper is seen in several hereditary forms of copper toxicity in humans and animals. At present, Wilson's disease is the best-described and best-studied copper-storage disorder in humans; it is caused by mutations in the ATP7B gene. In dogs, a mutation in the COMMD1 gene has been found to be associated with copper toxicosis. Using a liver-specific Commd1 knockout mouse, the biological role of Commd1 in copper homeostasis has been confirmed. Yet, the exact mechanism by which COMMD1 regulates copper homeostasis is still unknown. Here, we give an overview of the current knowledge and perspectives on the molecular function of COMMD1 in copper homeostasis.

Zinc transferred through breast milk does not differ between appropriate- and small-for-gestational-age, predominantly breast-fed Bangladeshi infants.

Information is needed on breast milk zinc concentration and amount of zinc transferred to the infant in relation to the time since birth for both appropriate-for-gestational-age (AGA) and small-for-gestational-age (SGA) infants. Breast milk zinc concentration and total milk volume were measured among mothers of AGA and SGA infants, using deuterium oxide "dose-to-mother" tracer technique and plasma zinc concentrations of mothers and infants. Forty-six mother-infant pairs (20 AGA and 26 SGA infants) were recruited from a low-income community in Bangladesh. Each mother-infant pair was studied 3 times: at 4, 12, and 24 wk postpartum. During each round, 2-wk studies of breast milk transfer were performed, using deuterium oxide dose-to-mother tracer technique. Breast milk samples were collected on days 1 and 5 of each round to determine milk zinc concentration. Mean ± SD birth weight and length were 3.02 ± 0.2 kg and 0.482 ± 0.012 m, respectively, for AGA and 2.34 ± 0.20 kg and 0.462 ± 0.011 m, respectively, for SGA infants, and both variables were different between groups (birth weight, P < 0.001; length, P < 0.001). Breast milk intake increased gradually with time postpartum in both groups. Breast milk zinc concentration decreased with increasing infant age (P < 0.001) but did not differ for mothers of AGA and SGA infants. Breast milk zinc transfer decreased significantly with age in both groups (P < 0.001) but did not differ by birth-weight category. Breast milk zinc concentration among Bangladeshi mothers and patterns of change at 24 wk were similar to those of wealthier countries, and there was no relation between infant birth-weight category and milk zinc concentration or transfer. This trial was registered at www.clinicaltrials.gov as NCT01728766.

Impact of copper deficiency in humans.

Humans consume about 1 mg of copper daily, an amount thought adequate for most needs. Genetic, environmental, or physiological alterations can impose a higher copper set point, increasing risk for copper-limited pathophysiology. Humans express about a dozen proteins that require copper for function (cuproenzymes). Limitation in the activity of cuproenzymes can explain the pleiotropic effect of copper deficiency. However, for most of the salient features of human copper deficiency, the precise molecular mechanisms are unknown. This is true for the two most common clinical features, hypochromic anemia and adult onset peripheral neuropathy/ataxia, a condition described frequently in the last decade due to multiple etiologies. The challenge for future scientists will be to identify the mechanisms underlying the pathophysiology of copper deficiency so appropriate screening and treatment can occur. The need for a strong copper biomarker to aid in this screening is critical.

The role of zinc dynamics in growth hormone secretion.

Human growth hormone (GH) causes a variety of physiological and metabolic effects in humans and plays a pivotal role in postnatal growth. In somatotroph cells of the anterior pituitary, GH is stored in concentrated forms in secretory granules to be rapidly released upon GH-releasing hormone stimulation. During the process of secretory granule biogenesis, self-association of GH occurs in the compartments of the early secretory pathway (endoplasmic reticulum and Golgi complex). Since this process is greatly facilitated by the presence of zinc ions, it is of importance to understand the potential role of zinc transporters that participate in the fine-tuning of zinc homeostasis and dynamics, particularly in the early secretory pathway. Thus, the role of zinc transporters in supplying the secretory pathway with the sufficient amount of zinc required for the biogenesis of GH-containing secretory granules is essential for normal secretion. This report, illustrated by a clinical case report on transient neonatal zinc deficiency, focuses on the role of zinc in GH storage in the secretory granules and highlights the role of specific zinc transporters in the early secretory pathway. © 2013 S. Karger AG, Basel.

Compound heterozygous mutations in SLC30A2/ZnT2 results in low milk zinc concentrations: a novel mechanism for zinc deficiency in a breast-fed infant.

Zinc concentrations in breast milk are considerably higher than those of the maternal serum, to meet the infant's requirements for normal growth and development. Thus, effective mechanisms ensuring secretion of large amounts of zinc into the milk operate in mammary epithelial cells during lactation. ZnT2 was recently found to play an essential role in the secretion of zinc into milk. Heterozygous mutations of human ZnT2 (hZnT2), including H54R and G87R, in mothers result in low (>75% reduction) secretion of zinc into the breast milk, and infants fed on the milk develop transient neonatal zinc deficiency. We identified two novel missense mutations in the SLC30A2/ZnT2 gene in a Japanese mother with low milk zinc concentrations (>90% reduction) whose infant developed severe zinc deficiency; a T to C transition (c.454T>C) at exon 4, which substitutes a tryptophan residue with an arginine residue (W152R), and a C to T transition (c.887C>T) at exon 7, which substitutes a serine residue with a leucine residue (S296L). Biochemical characterization using zinc-sensitive DT40 cells indicated that the W152R mutation abolished the abilities to transport zinc and to form a dimer complex, indicating a loss-of-function mutation. The S296L mutation retained both abilities but was extremely destabilized. The two mutations were found on different alleles, indicating that the genotype of the mother with low milk zinc was compound heterozygous. These results show novel compound heterozygous mutations in the SLC30A2/ZnT2 gene causing zinc deficiency in a breast-fed infant.

Hepatocyte divalent metal-ion transporter-1 is dispensable for hepatic iron accumulation and non-transferrin-bound iron uptake in mice.

UNLABELLED: Divalent metal-ion transporter-1 (DMT1) is required for iron uptake by the intestine and developing erythroid cells. DMT1 is also present in the liver, where it has been implicated in the uptake of transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload. To test the hypothesis that DMT1 is required for hepatic iron uptake, we examined mice with the Dmt1 gene selectively inactivated in hepatocytes (Dmt1(liv/liv) ). We found that Dmt1(liv/liv) mice and controls (Dmt1(flox/flox) ) did not differ in terms of hepatic iron concentrations or other parameters of iron status. To determine whether hepatocyte DMT1 is required for hepatic iron accumulation, we crossed Dmt1(liv/liv) mice with Hfe(-) (/) (-) and hypotransferrinemic (Trf(hpx/hpx) ) mice that develop hepatic iron overload. Double-mutant Hfe(-) (/) (-) Dmt1(liv/liv) and Trf(hpx/hpx) ;Dmt1(liv/liv) mice were found to accumulate similar amounts of hepatic iron as did their respective controls. To directly assess the role of DMT1 in NTBI and TBI uptake, we injected (59) Fe-labeled ferric citrate (for NTBI) or (59) Fe-transferrin into plasma of Dmt1(liv/liv) and Dmt1(flox/flox) mice and measured uptake of (59) Fe by the liver. Dmt1(liv/liv) mice displayed no impairment of hepatic NTBI uptake, but TBI uptake was 40% lower. Hepatic levels of transferrin receptors 1 and 2 and ZRT/IRT-like protein 14, which may also participate in iron uptake, were unaffected in Dmt1(liv/liv) mice. Additionally, liver iron levels were unaffected in Dmt1(liv/liv) mice fed an iron-deficient diet. CONCLUSION: Hepatocyte DMT1 is dispensable for hepatic iron accumulation and NTBI uptake. Although hepatocyte DMT1 is partially required for hepatic TBI uptake, hepatic iron levels were unaffected in Dmt1(liv/liv) mice, suggesting that this pathway is a minor contributor to the iron economy of the liver.

Molybdenum cofactor deficiency: metabolic link between taurine and S-sulfocysteine.

Molybdenum cofactor deficiency (MoCD) is a rare inherited metabolic disorder characterized by severe and progressive neurologic damage mainly caused by the loss of sulfite oxidase activity. Elevated urinary levels of sulfite, thiosulfate, and S-sulfocysteine (SSC) are hallmarks in the diagnosis of both MoCD and sulfite oxidase deficiency. Sulfite is generated throughout the catabolism of sulfur-containing amino acids cysteine and methionine. Accumulated sulfite reacts with cystine, thus leading to the formation of SSC, a glutamate analogue, which is assumed to cause N-methyl-D-aspartate receptor-mediated neurodegeneration in MoCD patients. Recently, we described a fast and sensitive HPLC method for diagnostic and treatment monitoring of MoCD patients based on SSC quantification. In this study, we extend the HPLC method to the analysis of hypotaurine and taurine in urine samples and no interference with other compounds was found. Besides the known elevation of SSC and taurine, also hypotaurine shows strong accumulation in MoCD patients, for which the molecular basis is not understood. SSC, hypotaurine, and taurine urinary excretion values from control individuals as well as MoCD patients are reported and over 20-fold increase in taurine urinary excretion was determined for MoCD patients demonstrating a direct link between sulfite toxicity and taurine biosynthesis in MoCD.

Various copper and iron overload patterns in the livers of patients with Wilson disease and idiopathic copper toxicosis.

Wilson disease (WD) is a major type of primary copper toxicosis associated with hypoceruloplasminemia, while idiopathic copper toxicosis (ICT) is a minor type characterized by normoceruloplasminemia. Because ceruloplasmin is the major circulating ferroxidase, iron metabolism may be affected in patients with WD. Biopsied liver specimens obtained from patients with primary copper toxicosis were fixed with glutaraldehyde solution and embedded in epoxy resin. Ultrathin sections that had or had not been stained with uranyl acetate solution were examined under an electron microscope equipped with an energy dispersive X-ray analyzer. A 7-year-old boy with WD was free from any metal overloading at the pre-treatment stage. Pre-treatment liver specimens of another 16 patients showed a variety of copper and iron overload patterns, from isolated copper to evenly distributed combined overloading. A 19-year-old female patient was free from any metal overloading after 7 years of treatment. Post-treatment overloading in another 6 patients ranged between evenly distributed combined patterns and isolated iron patterns. All patients had hypoceruloplasminemia throughout treatment periods. A patient with normoceruloplasminemic ICT continued to display isolated copper overloading after 2.5 years of treatment. In conclusion, these observations support the hypothesis that iron accumulates in patients with hypoceruloplasminemia.