Severe iron overload with a novel aminolevulinate synthase mutation and hepatitis C infection. A case report

A 55 year old man with a history of chronic hepatitis C infection was found to have severe hemochromatosis: hepatic cirrhosis, cardiomyopathy, arrhythmia, hypogonadism, diabetes and bronzed skin color. After 50 phlebotomies, he underwent a combined heart and liver transplant. Genetic analyses identified a novel mutation in the iron responsive element of the ALAS2 gene. No mutations were found in other genes associated with adult or juvenile hemochromatosis including HFE, transferrin receptor-2 (TFR2), ferroportin (SLC40A1), hepcidin (HAMP) and hemojuvelin (HJV). We suggest that the ALAS2 mutation together with chronic hepatitis C infection may have caused the severe iron overload phenotype.     

Hereditary iron overload: update on pathophysiology, diagnosis, and treatment

Hereditary hemochromatosis, a very common genetic defect in the Caucasian population, is characterized by progressive tissue iron overload which leads to irreversible organ damage if it is not treated timely. The elucidation of the molecular pathways of iron transport through cells and its control has led to the understanding of various genetic iron-loading conditions. Four types of inherited iron overload have been recognized: type 1, the most common form with an autosomal recessive inheritance, is associated with mutations in the HFE gene on chromosome 6; type 2 (juvenile hemochromatosis) is an autosomal recessive disorder with causative mutations identified in the HJV gene (subtype A) on chromosome 1 and the HAMP gene (subtype B) on chromosome 19; type 3 has also an autosomal recessive inheritance with mutations in the TfR2 gene on chromosome 3; type 4 is an autosomal dominant condition with heterozygous mutations in the ferroportin 1 gene on chromosome 2. In this review, the genetics, pathophysiology, diagnosis, clinical features, and management of these different types of hereditary hemochromatosis are briefly discussed.     

Juvenile hemochromatosis due to G320V/Q116X compound heterozygosity of hemojuvelin in an Irish patient

Hemojuvelin (HJV) is a recently discovered gene responsible for 1q-linked juvenile hemochromatosis. The majority of mutations characterized in this gene are rare and private, except G320V, identified in patients from different countries. Here, we report the clinical features and the molecular study of a young Irish patient presenting with severe cardiac disease related to iron overload. We sequenced the coding region and the exon-intron boundaries of genes associated with juvenile hemochromatosis, HAMP and HJV encoding hepcidin and hemojuvelin respectively. Two heterozygous HJV mutations were identified: the G320V mutation and the new Q116X mutation that cause a premature stop codon in the protein. This finding increases the number of mutations identified in HJV gene and underlines that the G320V is a recurrent mutation, even in Northern Europe.     

Early onset hereditary hemochromatosis resulting from a novel TFR2 gene nonsense mutation (R105X) in two siblings of north French descent

The molecular basis of hereditary hemochromatosis (HH) is more complex than previously expected. More than 80% of hemochromatosis probands of Northern European descent are homozygous for the C282Y HFE gene mutation. However, five novel non-related-HFE HH forms have now been identified. The transferrin receptor(TFR2)-linked form is inherited in an autosomal recessive pattern and is considered to be an adult-onset syndrome. Until now, it has been associated with five mutations that have only been detected in Japanese and southern European patients. Here, we report the identification of a novel TFR2 nonsense mutation in two related French adolescents. We discuss the phenotype of this sibling pair from precedent biological and clinical findings as well as the expected role of TFR2 in iron homeostasis. Finally, we suggest that iron overload phenotypes associated with mutations in TFR2 may be intermediate between those related to mutations in HFE and those related to mutations in juvenile hemochromatosis genes.     

Juvenile hemochromatosis associated with pathogenic mutations of adult hemochromatosis genes

BACKGROUND AND AIMS: Juvenile hemochromatosis is a severe form of hereditary iron overload that has thus far been linked to pathogenic mutations of the gene coding for hemojuvelin (HJV), on chromosome 1, or, more rarely, that coding for hepcidin ( HAMP ), on chromosome 19. A milder adult-onset form is due to pathogenic mutations of HFE or, rarely, serum transferrin receptor 2. METHODS: We studied a pedigree with siblings affected by both juvenile and adult-onset hereditary hemochromatosis. Affected subjects underwent full clinical evaluation, as well as microsatellite and gene sequencing analysis. RESULTS: Two siblings (male and female, aged 24 and 25 years, respectively) were hospitalized for severe endocrinopathy and cardiomyopathy. At age 18 and 17 years, they had presented with impotence and amenorrhea, respectively, and increased serum iron levels. Hypogonadotropic hypogonadism was confirmed in both, and liver biopsy showed marked hepatic iron accumulation and micronodular cirrhosis. Iron levels were normalized after 24 months (female) and 36 months (male) of weekly phlebotomies. Microsatellite analysis showed no linkage with chromosome 1 and 19, and gene sequencing showed no hemojuvelin or hepcidin gene mutations. Instead, combined mutations of HFE (C282Y/H63D compound heterozygosity) and serum transferrin receptor 2 (Q317X homozygosity) were found. A 21-year-old brother with a milder phenotype resembling classic adult-onset hereditary hemochromatosis carried only the Q317X serum transferrin receptor 2 homozygote mutation. CONCLUSIONS: Juvenile hereditary hemochromatosis is not a distinct monogenic disorder invariably due to hemojuvelin or hepcidin mutations: it may be genetically linked to the adult-onset form of hereditary hemochromatosis.     

Hemojuvelin (HJV) mutations in persons of European, African-American and Asian ancestry with adult onset haemochromatosis

Mutations in the chromosome 1q-linked gene hemojuvelin (HJV) have recently been found to be a cause of juvenile haemochromatosis. We addressed the question of whether hemojuvelin mutations may influence the phenotype of patients with adult-onset haemochromatosis with or without mutations of the HFE gene. We sequenced the complete coding region of 133 subjects with iron overload. To screen a large number of patients, we also developed conditions for analysis by denaturing high-performance liquid chromatography (dHPLC). This diagnostic modality detects many mutations of the HJV gene. One patient with severe iron overload was found to be a compound heterozygote for HJV mutations, one of which had previously been identified in patients with juvenile haemochromatosis (G320V) and the other was novel (C321W). A number of other mutations were identified, but none were clearly associated with increases in the body iron burden. Notable among these was a DNA triplet insert, predicting an insertion of glycine, found in two African-American subjects, one with and one without iron storage disease.     

Competitive regulation of hepcidin mRNA by soluble and cell-associated hemojuvelin

Mutations in a recently identified gene HJV (also called HFE2, or repulsive guidance molecule C, RgmC) are the major cause of juvenile hemochromatosis (JH). The protein product of HJV, hemojuvelin, contains a C-terminal glycosylphosphatidylinositol anchor, suggesting that it can be present in either a soluble or a cell-associated form. Patients with HJV hemochromatosis have low urinary levels of hepcidin, the principal iron-regulatory hormone secreted by the liver. However, neither the specific role of hemojuvelin in maintaining iron homeostasis nor its relationship to hepcidin has been experimentally established. In this study we used hemojuvelin-specific siRNAs to vary hemojuvelin mRNA concentration and showed that cellular hemojuvelin positively regulated hepcidin mRNA expression, independently of the interleukin 6 pathway. We also showed that recombinant soluble hemojuvelin (rs-hemojuvelin) suppressed hepcidin mRNA expression in primary human hepatocytes in a log-linear dose-dependent manner, suggesting binding competition between soluble and cell-associated hemojuvelin. Soluble hemojuvelin was found in human sera at concentrations similar to those required to suppress hepcidin mRNA in vitro. In cells engineered to express hemojuvelin, soluble hemojuvelin release was progressively inhibited by increasing iron concentrations. We propose that soluble and cell-associated hemojuvelin reciprocally regulate hepcidin expression in response to changes in extracellular iron concentration.     

Juvenile hemochromatosis caused by a novel combination of hemojuvelin G320V/R176C mutations in a 5-year old girl

During a screening program we identified a 5-year old girl with elevated iron parameters. The child was found to have a combination of a novel R176C mutation together with the G320V mutation in the juvenile hemochromatosis gene (HJV). The girl was also homozygous for the H63D mutation in HFE. The possibility of detecting juvenile hemochromatosis before the onset of clinical manifestations raises questions about the management of such young children in order to prevent iron overload.     

Iron overload and prolonged ingestion of iron supplements: clinical features and mutation analysis of hemochromatosis-associated genes in four cases

We evaluated and treated four white adults (one man, three women) who had iron overload associated with daily ingestion of iron supplements for 7, 15, 35, and 61 years, respectively. We performed HFE mutation analysis to detect C282Y, H63D, and S65C in each patient; in two patients, HFE exons were sequenced. In two patients, direct sequencing was performed to detect coding region mutations of TFR2, HAMP, FPN1, HJV, and ALAS2. Patients 1-4 ingested 153, 547, 1,341, and 4,898 g of inorganic iron as supplements. Patient 1 had hemochromatosis, HFE C282Y homozygosity, and beta-thalassemia minor. Patient 2 had spherocytosis and no HFE coding region mutations. Patient 3 had no anemia, a normal HFE genotype, and no coding region mutations in HAMP, FPN1, HJV, or ALAS2; she was heterozygous for the TFR2 coding region mutation V583I (nt 1,747 G-->A, exon 15). Patient 4 had no anemia and no coding region mutations in HFE, TFR2, HAMP, FPN1, HJV, or ALAS2. Iron removed by phlebotomy was 32.4, 10.4, 15.2, and 4.0 g, respectively. There was a positive correlation of log(10) serum ferritin and the quantity of iron removed by phlebotomy (P = 0.0371). Estimated absorption of iron from supplements in patients 1-4 was 20.9%, 1.9%, 1.1%, and 0.08%. We conclude that the clinical phenotypes and hemochromatosis genotypes of adults who develop iron overload after ingesting iron supplements over long periods are heterogeneous. Therapeutic phlebotomy is feasible and effective, and would prevent complications of iron overload.     

Genetic heterogeneity underlies juvenile hemochromatosis phenotype: analysis of three families of northern Greek origin

Hereditary hemochromatosis is a genetically heterogeneous disease. Common HFE mutations (C282Y and H63D) are related to the majority of hereditary hemochromatosis cases in populations of Northern European ancestry (HFE1). Juvenile hemochromatosis (JH) is a more severe iron overload disorder, usually presenting at the second decade of life. The gene responsible for JH lies on a genetic locus at chromosome 1q. We have performed a genetic linkage study in three families of Northern Greek origin with typical clinical features of JH. In two families results were in accordance with linkage to chromosome 1q. In one family linkage of the disease to the genetic loci at 1q21, 7q22, and 6p22 was excluded. We suggest that more than one gene may underlie the JH phenotype. This genetic type of hemochromatosis may be designated 1q unlinked juvenile hemochromatosis. Family studies are necessary to establish the genetic diagnosis of JH.     

Down-regulation of hepcidin in porphyria cutanea tarda

Hepatic siderosis is common in patients with porphyria cutanea tarda (PCT). Mutations in the hereditary hemochromatosis (hh) gene (HFE) explain the siderosis in approximately 20% patients, suggesting that the remaining occurrences result from additional genetic and environmental factors. Two genes known to modify iron loading in hh are hepcidin (HAMP) and hemojuvelin (HJV). To determine if mutations in or expression of these genes influenced iron overload in PCT, we compared sequences of HAMP and HJV in 96 patients with PCT and 88 HFE C282Y homozygotes with marked hepatic iron overload. We also compared hepatic expression of these and other iron-related genes in a group of patients with PCT and hh. Two intronic polymorphisms in HJV were associated with elevated serum ferritin in HFE C282Y homozygotes. No exonic polymorphisms were identified. Sequencing of HAMP revealed exonic polymorphisms in 2 patients with PCT: heterozygosity for a G-->A transition (G71D substitution) in one and heterozygosity for an A-->G transition (K83R substitution) in the other. Hepatic HAMP expression in patients with PCT was significantly reduced, regardless of HFE genotype, when compared with patients with hh but without PCT with comparable iron overload. These data indicate that the hepatic siderosis associated with PCT likely results from dysregulated HAMP.     

Prevalence of the G320V mutation of the HJV gene, associated with juvenile hemochromatosis, in Greece

Mutations of the HJV gene, which maps on chromosome 1q21, underlie most cases of juvenile hemochromatosis. We evaluated the frequency of the most common mutation (G320V) of the HJV gene in the Greek population, since 50% of cases of hereditary hemochromatosis in Greece carry mutations of the HJV gene.     

Hemochromatosis and severe iron overload associated with compound heterozygosity for TFR2 R455Q and two novel mutations TFR2 R396X and G792R

We report three mutations of transferrin receptor 2 (TFR2)--R396X (exon 9; nt 1186C-->T), R455Q (exon 10; nt 1364G-->A) and G792R (exon 18; nt 2374G-->A)--in a man of Scottish descent with hemochromatosis and severe iron overload. He was also heterozygous for the common HFE H63D polymorphism. The patient did not have coding region mutations in HAMP, FPN1, HJV or ALAS2. We conclude that this patient represents another example of hemochromatosis due to mutations of the gene encoding transferrin receptor 2.     

Genetic background of primary iron overload syndromes in Japan

The different prevalences of iron overload syndromes between Caucasians and Asians may be accounted for by the differences in genetic background. The major mutation of hemochromatosis in Celtic ancestry, C282Y of HFE, was reported in a Japanese patient. Five patients of 3 families with the hepatic transferrin receptor gene (TFR2)-linked hemochromatosis were found in different areas of Japan, suggesting that TFR2 is a major gene in Japanese people. Three patients with mutations in the hemojuvelin gene, HJV, showed also middle-age-onset hemochromatosis. A heterozygous mutation in the H ferritin gene, FTH1, was found in a family of 3 affected patients. Another autosomal dominant SLC40A1-linked hyperferritinemia (ferroportin disease) was found in 3 patients of 2 families. Two patients with hemochromatosis were free from any mutations in the genes investigated. In conclusion, the genetic backgrounds of Japanese patients with primary iron overload syndromes were partially clarified, showing some phenotype-genotype correlations.     

Inherited HFE-unrelated hemochromatosis in Italian families

Hemochromatosis (HH) is usually caused by the homozygous state for C282Y mutation in the HFE gene. A minority of iron loaded patients have no mutations in this gene. An infrequent subset shows an early-onset aggressive disorder, denoted juvenile hemochromatosis (JH), which has no linkage to 6p. In this report we describe six patients from three unrelated Italian families, four men and two women, aged 21 to 44 with the typical hemochromatosis phenotype, who are homozygous for the wild type allele at the HFE gene. In two families the disorder is unlinked to 6p; in one family some features of the juvenile form are seen, but linkage to 6p is not excluded. Our results point to genetic forms of hemochromatosis not associated with HFE and raise the problem of whether non-HFE hemochromatosis in Italy is related to the "juvenile" form. They also emphasize the importance of phenotypic as well as genetic diagnosis of HH.     

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 patient's serum or urine. These results indicate that the p.R75X mutation causes iron overload by impairing the hepcidin system.     

Three patients with middle-age-onset hemochromatosis caused by novel mutations in the hemojuvelin gene

Hemochromatosis is a genetically heterogeneous condition. Mutations in the recently described hemojuvelin gene were found in patients with juvenile hemochromatosis, who usually manifest clinical signs of iron overload, including cardiomyopathy and hypogonadism, in their teens and early 20s. In this report, we describe three Japanese patients who showed typical clinical and hepatic histological damage compatible with hemochromatosis at around 50 years of age. Genetic analyses showed that all three patients carried mutations in the hemojuvelin gene. The first patient was homozygous for a novel mutation (745G > C [D249H]), and the second and third patients from the same family were homozygous for another novel mutation (934C > T [Q312X]). No mutations in their HFE, hepcidin, transferrin receptor 2, or ferroportin genes were found. One patient had chronic infection with Helicobacter pylori. The age at initial presentation of hemojuvelin-hemochromatosis occurs over a wider range than previously described.     

C282Y and H63D mutations in the HFE gene have no effect on iron overload disorders in Japan

OBJECTIVE: The gene responsible for hereditary hemochromatosis close to the human leukocyte antigen A locus was previously identified and designated as HFE. This study was performed to evaluate the clinical significance of two mutations, C282Y and H63D of HFE, in Japanese patients with hepatic iron overload. PATIENTS AND METHODS: We examined C282Y and H63D in 11 patients with primary hemochromatosis, 94 patients with chronic hepatitis C, 54 patients with miscellaneous liver diseases, and 151 healthy volunteers. The HFE gene region of DNA samples extracted from peripheral leukocytes was amplified by polymerase chain reaction. Restriction enzyme analysis was performed using SnaBI for C282Y and BclI for H63D. Direct sequence analysis was then performed when products suggested the presence of a mutation. RESULTS: All the subjects studied were free from C282Y. None of the patients with hemochromatosis had H63D. One patient with chronic hepatitis C was homozygous, and 4 patients were heterozygous for H63D. Two patients with alcoholic liver disease were heterozygous for H63D. The prevalence of chromosomes with H63D was 6/188 (3.2%) in patients with chronic hepatitis C, 2/108 (1.9%) in patients with miscellaneous liver diseases, and 8/302 (2.6%) in healthy volunteers. These differences were not significant. CONCLUSION: Our results suggested that neither C282Y nor H63D in HFE affect Japanese patients with hemochromatosis or chronic hepatitis C.     

Transferrin receptor-2 (TFR2) mutation Y250X in Alabama Caucasian and African American subjects with and without primary iron overload

Most cases of hemochromatosis are associated with mutations of the HFE gene on Ch6p. In southern Italy and central Alabama, the percentages of patients with hemochromatosis who have "atypical" HFE genotypes (defined as lack of C282Y homozygosity, C282Y/H63D compound heterozygosity, or H63D homozygosity) are relatively great. A mutation of the transferrin receptor-2 gene (TFR2; exon 6, nt 750 C --> G, replaces TAC with stop signal TAG; Y250X) on Ch7q22 was recently identified in two Sicilian families with HFE mutation-negative hemochromatosis. We wanted to estimate the frequency of this mutation in persons from central Alabama. We evaluated Caucasian hemochromatosis probands with atypical HFE genotypes and African Americans with primary iron overload. We also studied control Caucasians, including persons of southern Italian/Sicilian heritage, and control African Americans. Analysis of genomic DNA was performed using a PCR-sequence-specific priming assay and positive control specimens from Sicilian hemochromatosis subjects heterozygous and homozygous for Y250X. Among Alabama subjects, this allele was not detected in 113 Caucasians, including 21 hemochromatosis probands with atypical HFE genotypes and 92 normal control subjects (including 27 of southern Italian/Sicilian descent). In African Americans, Y250X was not detected in 20 index cases with primary iron overload or in 274 unrelated control subjects. We conclude that Y250X is uncommon in Caucasians with hemochromatosis associated with atypical HFE genotypes, in African Americans with primary iron overload, and in the general Caucasian and African American population subgroups in central Alabama.