Ai Hattori

AVAQ 594-597 deletion of the TfR2 gene in a Japanese family with hemochromatosis

The majority of Caucasian patients with hemochromatosis are homozygous for C282Y mutation of the HFE gene. In contrast to its high prevalence in Caucasians, hemochromatosis is a rare disorder in Japan. This may be due to the low prevalence of the C282Y mutation of the HFE gene in Japanese. Recent reports suggest that the mutations of transferrin receptor 2 (TfR2) gene may be involved in non-HFE hemochromatosis. Therefore, we investigated the TfR2 gene of 6 sporadic and 5 familiar cases of Japanese hemochromatosis. Three siblings in one family were found to be homozygous for an AVAQ 594-597 deletion. All three had severe iron deposits in the hepatocytes and bile ducts, but none was affected by diabetes mellitus. This mutation was not detected in 100 control individuals. Further study was undertaken to investigate whether the large deletion of the TfR2 gene is the mutation responsible for some of the Japanese hemochromatosis cases.

Increase in P-glycoprotein accompanied by activation of protein kinase Cα and NF-κB p65 in the livers of rats with streptozotocin-induced diabetes

It is known that protein kinase C (PKC) signal transduction is enhanced in a diabetic state, and that PKC activator phorbol esters increase the gene expression of MDR1 in human tumor cells. To clarify the expression of the liver transporters under diabetic conditions and the roles of PKCalpha and the transcription factor NF-kappaB, we investigated the expression levels of Mdr1a, Mdr1b, Mdr2, Mrp2, Bcrp, Bsep, Oct1, Oat2, and Oat3 transporters, PKCalpha, IkappaB, and NF-kappaB in the liver of rats with STZ-induced hyperglycemia. A selective increase in the gene expression of Mdr1b was detected by RT-PCR. Western blotting with C219 antibody revealed an increase in P-glycoprotein. Although the mRNA level of PKCalpha was not affected, translocation of PKCalpha to the microsomal fraction was detected. NF-kappaB p65, IkappaBalpha and IkappaBbeta mRNA levels were increased as was the level of nuclear NF-kappaB p65. From these findings, it was suggested that STZ-induced hyperglycemia caused the upregulation of Mdr1b P-gp expression through the activation of PKCalpha and NF-kappaB.

Ursodeoxycholic acid induces glutathione synthesis through activation of PI3K/Akt pathway in HepG2 cells

Ursodeoxycholic acid (UDCA) is widely recognized as an effective compound in the treatment of chronic hepatitis and is known to modulate the redox state of the liver accompanied by an increase of GSH. In the present study, to access the antioxidative effect of UDCA and to clarify the molecular basis of the action on GSH level, we evaluated its effects in HepG2 cells exposed to excessive iron. UDCA inhibited both a decrease in the GSH level and an increase in the reactive oxygen species caused by excessive iron in the cells. UDCA increased the gene expression of the catalytic- and modifier-units of glutamine-cysteine ligase (GCL), which is a key enzyme in GSH synthesis. We further investigated the effect of UDCA on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and obtained results showing that UDCA-induced increase in the GSH level was prevented by LY294002, a PI3K inhibitor. In addition, Western blot analysis of Akt showed that, while the total Akt level remained unchanged, the phosphorylated Akt level was increased by UDCA, and this increase was also prevented by LY294002. Moreover, UDCA promoted the translocation of a transcription factor, nuclear factor-E2-related factor-2 (Nrf2), into the nucleus, and this action was inhibited by LY294002. From these results, it was indicated that UDCA increased the GSH synthesis through an activation of the PI3K/Akt/Nrf2 pathway. This may be a primary mechanism of antioxidative action of UDCA concerned with its therapeutic effectiveness in chronic hepatitis.

Measurement of serum hepcidin-25 levels as a potential test for diagnosing hemochromatosis and related disorders

BackgroundIron overload syndromes include a wide spectrum of genetic and acquired conditions. Recent studies suggest suppressed hepcidin synthesis in the liver to be the molecular basis of hemochromatosis. However, a liver with acquired iron overload synthesizes an adequate amount of hepcidin. Thus, hepcidin could function as a biochemical marker for differential diagnosis of iron overload syndromes.MethodsWe measured serum iron parameters and hepcidin-25 levels followed by sequencing HFE, HJV, HAMP, TFR2, and SLC40A1 genes in 13 Japanese patients with iron overload syndromes. In addition, we performed direct measurement of serum hepcidin-25 levels using liquid chromatography–tandem mass spectrometry in 3 Japanese patients with aceruloplasminemia and 4 Italians with HFE hemochromatosis.ResultsOne patient with HJV hemochromatosis, 2 with TFR2 hemochromatosis, and 3 with ferroportin disease were found among the 13 Japanese patients. The remaining 7 Japanese patients showed no evidence for genetic basis of iron overload syndrome. As far as the serum hepcidin-25 was concerned, seven patients with hemochromatosis and 3 with aceruloplasminemia showed markedly decreased serum hepcidin-25 levels. In contrast, 3 patients with ferroportin disease and 7 with secondary iron overload syndromes showed serum hepcidin levels parallel to their hyperferritinemia. Patients with iron overload syndromes were divided into 2 phenotypes presenting as low and high hepcidinemia. These were then associated with their genotypes.ConclusionDetermining serum hepcidin-25 levels may aid differential diagnosis of iron overload syndromes prior to genetic analysis.

Identification of a novel mutation in the HAMP gene that causes non-detectable hepcidin molecules in a Japanese male patient with juvenile hemochromatosis.

Hepcidin is an iron-regulatory hepatic peptide hormone encoded by the HAMP gene that downregulates iron export from enterocytes and macrophages into the blood plasma. In this study, we identified a novel mutation in the HAMP gene of a 58-year-old Japanese male patient with hemochromatosis. By direct sequencing of the five hereditary hemochromatosis-related genes, HFE, HAMP, HJV, TFR2, and SLC40A1, the previously unreported p.R75X mutation was identified, and the patient was found to be homozygous for the mutation. No other potentially pathogenic mutations were detected. In an LC-MS/MS analysis, hepcidin molecules were not detected in the patients serum or urine. These results indicate that the p.R75X mutation causes iron overload by impairing the hepcidin system.

(R)-Mevalonate 3-Phosphate Is an Intermediate of the Mevalonate Pathway in Thermoplasma acidophilum

Background: A few enzyme reactions in the mevalonate pathway remain unclear in most archaea. Results: (R)-Mevalonate 3-phosphate is synthesized by Ta1305 protein from Thermoplasma acidophilum and is converted into isopentenyl diphosphate in cell-free extract from the archaeon. Conclusion: The mevalonate pathway of T. acidophilum passes through (R)-mevalonate 3-phosphate rather than (R)-mevalonate 5-phosphate. Significance: A third alternative mevalonate pathway is discovered. The lack of a few conserved enzymes in the classical mevalonate pathway and the widespread existence of isopentenyl phosphate kinase suggest the presence of a partly modified mevalonate pathway in most archaea and in some bacteria. In the pathway, (R)-mevalonate 5-phosphate is thought to be metabolized to isopentenyl diphosphate via isopentenyl phosphate. The long anticipated enzyme that catalyzes the reaction from (R)-mevalonate 5-phosphate to isopentenyl phosphate was recently identified in a Cloroflexi bacterium, Roseiflexus castenholzii, and in a halophilic archaeon, Haloferax volcanii. However, our trial to convert the intermediates of the classical and modified mevalonate pathways into isopentenyl diphosphate using cell-free extract from a thermophilic archaeon Thermoplasma acidophilum implied that the branch point intermediate of these known pathways, i.e. (R)-mevalonate 5-phosphate, is unlikely to be the precursor of isoprenoid. Through the process of characterizing the recombinant homologs of mevalonate pathway-related enzymes from the archaeon, a distant homolog of diphosphomevalonate decarboxylase was found to catalyze the phosphorylation of (R)-mevalonate to yield (R)-mevalonate 3-phosphate. The product could be converted into isopentenyl phosphate, probably through (R)-mevalonate 3,5-bisphosphate, by the action of unidentified T. acidophilum enzymes fractionated by anion-exchange chromatography. These findings demonstrate the presence of a third alternative “Thermoplasma-type” mevalonate pathway, which involves (R)-mevalonate 3-phosphotransferase and probably both (R)-mevalonate 3-phosphate 5-phosphotransferase and (R)-mevalonate 3,5-bisphosphate decarboxylase, in addition to isopentenyl phosphate kinase.

PIEZO1 gene mutation in a Japanese family with hereditary high phosphatidylcholine hemolytic anemia and hemochromatosis-induced diabetes mellitus

Hereditary xerocytosis (HX) or dehydrated hereditary stomatocytosis (DHS) [OMIM 194380], in which PIEZO1 gene mutation has recently been identified, is difficult to diagnose. We report here the discovery of a PIEZO1 gene mutation in a Japanese family (father, daughter, and son) who were previously diagnosed with hereditary high phosphatidylcholine hemolytic anemia (HPCHA). All of the affected family members had non-spherocytic hemolytic anemia associated with severe hemochromatosis-related diabetes mellitus. Although the causative correlation between HPCHA and PIEZO1-gene mutated HX/DHS remains to be clarified, our findings raise an important question as to whether any of the HPCHA cases previously diagnosed in Japan may have in fact been the form of hemolytic anemia known as HX/DHS with PIEZO1 gene mutation.

Clinicopathological study of Japanese patients with genetic iron overload syndromes.

In addition to hemochromatosis, aceruloplasminemia and ferroportin disease may be complicated by iron‐induced multiple organ damage. Therefore, clinicopathological features should be evaluated in a wider range of genetic iron disorders. This study included 16 Japanese patients with genetic iron overload syndromes. The responsible genes were CP in four, HAMP in one, HJV in three, TFR2 in five, and SLC40A1 in three patients. No phenotype dissociation was observed in patients with the CP, TFR2, or HAMP genotypes. Two of the three patients with the HJV genotype displayed classic hemochromatosis instead of the juvenile type. Patients with the SLC40A1 genotype were affected by mild iron overload (ferroportin A) or severe iron overload (ferroportin B). Transferrin saturation was unusually low in aceruloplasminemia patients. All patients, except those with ferroportin disease, displayed low serum hepcidin‐25 levels. Liver pathology showed phenotype‐specific changes; isolated parenchymal iron loading in aceruloplasminemia, periportal fibrosis associated with heavy iron overload in both parenchymal and Kupffer cells of ferroportin B, and parenchyma‐dominant iron‐loading cirrhosis in hemochromatosis. In contrast, diabetes occurred in all phenotypes of aceruloplasminemia, hemochromatosis, and ferroportin disease B. In conclusion, clinicopathological features were partially characterized in Japanese patients with genetic iron overload syndromes.

Hemojuvelin hemochromatosis receiving iron chelation therapy with deferasirox: improvement of liver disease activity, cardiac and hematological function.

To the Editor: As hemojuvelin hemochromatosis is a severe subtype of hemochromatosis, in affected persons, intensive iron removal is essential to prevent the irreversible multiorgan damage (1). Although phlebotomy is the standard treatment, compliance with this therapy can vary. The once-daily, oral iron chelator deferasirox (DFX) (Exjade; Novartis Pharma AG, Basel, Switzerland) is an option to phlebotomy treatment for patients with hemochromatosis and iron overload, although the safety and efficacy of DFX remain unproven for this option (2, 3). Herein, we report the case of a 29-yr-old Japanese man with hemojuvelin hemochromatosis who was treated with DFX. He was diagnosed with hemochromatosis at the age of 13. He was treated with phlebotomy, but discontinued treatment at the age of 20. After 9 yrs, he visited our hospital because of symptomatic heart failure. He had no history of excessive iron ingestion, alcohol consumption, or transfusion. He was not married and did not have children. One of his brothers had been diagnosed with type 1 diabetes mellitus at the age of 31. His mother was healthy and his father suffered from hepatic dysfunction and arrhythmia; however, iron deposition was not evident. Consanguinity was not known to be present in this kinship. There was no sign of hepatosplenomegaly. White blood cells (WBC) were 2.8 · 10 ⁄L, red blood cells were 3.76 · 10 ⁄L, reticulocytes were 26.3 · 10 ⁄L, and platelets were 99.0 · 10 ⁄L. Aspartate transaminase (AST) level was 83 U ⁄L, and alanine transaminase (ALT) level was 80 U ⁄L. Serum ferritin was 6624 ng ⁄mL, serum iron was 306 lg ⁄dL, and the total iron binding capacity was 324 lg ⁄dL. Bone marrow aspiration showed the nucleated cell count to be 20 000 ⁄ lL. Iron deposition was observed in macrophages, and no dysplastic blood cells were observed. Cardiac and liver iron overload was evident on magnetic resonance imaging, and the left ventricular ejection fraction (EF) was 24% by echocardiography. The genes analyzed in this patient included HFE, HJV, human antimicrobial peptide (HAMP), transferrin receptor 2 (TFR2), and SLC40A1 (4–6). The protocol was approved by the ethics committees in both Showa University Hospital and Aichi Gakuin University. The patient provided written informed consent for genetic testing, measurement of all biochemical markers, and treatment with DFX according to the Declaration of Helsinki. Molecular analysis revealed a homozygous genotype 515_6insC mutation in exon 3 of HJV, without detectable mutations of HFE, HAMP, TFR2, and SLC40A1 genes. The novel mutation predicts a stop signal at codon 196 (D172fsX196). The patient was treated daily with DFX 10 mg ⁄kg. Serum liver enzyme levels and ferritin decreased rapidly. The symptoms of the cardiac failure disappeared, and the EF improved up to 40%. At 28 weeks, the platelet increased to the normal range, and reticulocyte and WBCs increased further to 34.9 · 10 and 4.9 · 10 ⁄L, respectively (Fig. 1). The DFX dose of 10 mg ⁄kg ⁄day reduced the serum levels of ferritin and transaminases. It is a lower dose than that generally recommended for patients with transfusion-induced iron overload (2, 3, 7). He continued iron chelation therapy and did not experience any adverse events. This suggests that a small dose of DFX is effective to remove iron deposits in several organs, although iron accumulation in the tissues was not directly evaluated. During DFX treatment, we observed a substantial increase in WBCs, reticulocytes, and platelet counts, although the levels were below normal range at diagnosis. The mechanisms by which iron chelation may lead to hematologic improvement are unclear. He did not receive any specific therapeutics or blood transfusion during this period. Decreased splenic consumption of blood cells was not a plausible explanation for these increases, because splenomegaly was not evident throughout the clinical course. In some patients with myelodysplastic syndrome, iron chelation therapy appears to stimulate hematopoiesis, leading to a reduction or even complete weaning from the need for transfusion (8, 9). Iron overload itself exhibits a suppressive effect on erythroid progenitors and seems to increase transfusion requirement (10). Iron chelators promote iron release from storage sites, facilitating its usage for hematopoietic tissues. Iron excess is associated with increased oxidative stress. It would appear that the level of oxidative stress is directly responsible for organ dysfunction, including bone doi:10.1111/j.1600-0609.2011.01693.x European Journal of Haematology 87 (467–469)

A male patient with ferroportin disease B and a female patient with iron overload similar to ferroportin disease B

Reticuloendothelial iron overload is associated with secondary hemochromatosis including repeated transfusions and iron over-supplementation. Ferroportin disease B is a severe subtype of hereditary iron overload syndrome with an activated reticuloendothelial system. The iron exporter ferroportin may be insensitive to hepcidin 25 in this subtype. However, the interactions between the hepcidin–ferroportin system and modifiers of reticuloendothelial iron overload have not yet been elucidated. We describe two patients with iron overload conditions that were compatible with ferroportin disease B, but their genetic backgrounds and habitual states differed. Both patients had diabetes, periportal fibrosis with severe iron deposits in their hepatocytes and Kupffer cells, and adequate levels of circulating hepcidin 25. However, the first patient was heterozygous for a mutation in the FP gene and free from the acquired factors of iron overload, while the second patient was a heavy drinker with a heterozygous mutation in the TFR2 gene and no mutations in the FP gene. The first patient was the second reported case of ferroportin disease B in Japan. Our study on these 2 patients suggests that liver fibrosis associated with compound iron overload of reticuloendothelial cells and hepatocytes may occur via multi-etiological backgrounds.