Molecular analyses of patients with hyperferritinemia and normal serum iron values reveal both L ferritin IRE and 3 new ferroportin (slc11A3) mutations

Unexplained hyperferritinemia is a common clinical finding, even in asymptomatic persons. When early onset bilateral cataracts are also present, the hereditary hyperferritinemia-cataract syndrome (HHCS), because of heterozygous point mutation in the L ferritin iron-responsive element (IRE) sequence, can be suspected. We sequenced the L ferritin exon 1 in 52 DNA samples from patients referred to us for molecular diagnosis of HHCS. We identified 24 samples with a point mutation/deletion in the IRE. For the 28 samples in which no IRE mutation was present, we also genotyped HFE mutations and sequenced both H ferritin and ferroportin genes. We found an increased frequency of His63Asp heterozygotes (12 of 28) but no H ferritin mutations. We identified 3 new ferroportin mutations, producing, respectively, Asp157Gly, Gln182His, and Gly323Val amino acid replacements, suggesting that these patients have dominant type 4 hemochromatosis. This study demonstrates that both L ferritin IRE and ferroportin mutations can account for isolated hyperferritinemia. The presence of cataract does not permit the unambiguous identification of patients with HHCS, although the existence of a family history of cataract was only encountered in these patients. This raises the intriguing possibility that lens ferritin accumulation might be a factor contributing to age-related cataract in the general population. Additional causes of isolated hyperferritinemia remain to be identified.     

A point mutation in the iron-responsive element of the L-ferritin in a family with hereditary hyperferritinemia cataract syndrome.

BACKGROUND: Hereditary hyperferritinemia cataract syndrome is an autosomal dominant condition that is characterized by a high serum ferritin level and bilateral early-onset cataracts in the absence of iron overload. The genetic abnormality is identified as a mutation in the 5' regulatory region of the L-ferritin messenger RNA known as the iron-responsive element (IRE). The IRE controls ferritin synthesis in response to cytoplasmic iron pools by interacting with regulatory proteins called iron responsive proteins. Mutations in the IRE decrease its affinity for iron responsive proteins, leading to the constitutive synthesis of L-ferritin which results in hyperferritinemia and the intracellular accumulation of ferritin in the lens and eventual cataract formation. PATIENTS AND METHODS: A 22-year-old woman who was being investigated for hyperferritinemia was diagnosed with hereditary hyperferritinemia cataract syndrome after an extensive workup, including genetic testing for hemochromatosis and a liver biopsy to rule out iron overload. She developed anemia with phlebotomy treatments and subsequently developed symptomatic cataracts. The pedigree of her family affected with cataracts was consistent with an autosomal dominant transmission pattern. DNA was extracted from peripheral leukocytes of eight family members, four of whom were affected by cataracts. Polymerase chain reaction amplification of the 5' region of the L-ferritin gene was performed and a heterozygous point mutation (G32T) was identified in the bulge region of the IRE. CONCLUSION: The combination of early-onset cataracts and an elevated ferritin level should suggest this genetic syndrome.     

Clinical, biochemical and molecular findings in a series of families with hereditary hyperferritinaemia–cataract syndrome

Hereditary hyperferritinaemia-cataract syndrome (HHCS) is an autosomal dominant disease caused by mutations in the iron responsive element (IRE) of the l-ferritin gene. Despite the elucidation of the genetic basis, the overall clinical spectrum of HHCS has been less well studied as, to date, only individual case reports have been described. Therefore, we studied a total of 62 patients in 14 unrelated families, with nine different mutations. No relevant symptoms other than visual impairment were found to be associated with the syndrome. A marked phenotypic variability was observed, particularly with regard to ocular involvement (i.e. age range at which cataract was diagnosed in 16 subjects with the C39T: 6-40 years). Similarly, serum ferritin levels varied substantially also within subjects sharing the same mutation (i.e. range for the A40G: 700-2412 microg/l). We followed an HHCS newborn in whom well-defined lens opacities were not detectable either at birth or at 1 year. The lens ferritin content was analysed in two subjects who underwent cataract surgery at different ages, with different cataract morphology. Values were similar and about 1500-fold higher than in controls. These observations suggest that: (i) in HHCS the cataract is not necessarily congenital; (ii) in addition to the IRE genotype, other genetic or environmental factors may modulate the phenotype, especially the severity of the cataract.     

Hereditary hyperferritinaemia-cataract syndrome: a challenging diagnosis for the hepatogastroenterologist

Hereditary hyperferritinaemia-cataract syndrome (HHCS) is a relatively rare disorder with an autosomal dominant trait. It can be caused by various mutations within the iron responsive element (IRE) of the L-ferritin gene. These mutations result in an increased translation of L-ferritin mRNA and consequently the accumulation of L-ferritin in different fluids and tissues. HHCS patients present with an isolated hyperferritinaemia in the absence of any sign of iron overload. Early onset bilateral cataract, probably due to accumulation of ferritin crystals in the lens, is the only presenting clinical manifestation. Internists, especially gastrohepatologists, should be aware of this syndrome and differentiate it from haemochromatosis which is much more frequent, in order to avoid unnecessary imaging procedures, liver biopsies and an eventual venesection therapy, which will only lead to microcytic anaemia. In the present paper we report the first cases with HHCS diagnosed in Belgium. At diagnosis, the seven known affected members of three different families had ferritin levels between 603 and 3432 microg/l (normal < 150 microg/l), and this in combination with normal iron and transferrin values. All of them were known with early-onset bilateral cataract and our postulated diagnosis of HHCS was confirmed after genetic sequencing of the L-ferritin gene, which showed a C39U point mutation in the first family, and an A40G point mutation in the IRE loop segment in the two other families. The other investigated members of the three families had normal ferritin values, no history of early-onset cataract and genetic analyses could not reveal a mutation in the IRE of their L-ferritin gene. In current clinical practice, gastroenterologists should remember that elevated ferritin levels in the absence of documented iron overload is not haemochromatosis.     

Hereditary hyperferritinemia cataract syndrome in three unrelated families of western Greek origin caused by the C39 > G mutation of L-ferritin IRE

Hereditary hyperferritinemia-cataract syndrome (HHCS) is a well-characterized autosomal dominant disease caused by mutations in the iron responsive element (IRE) of ferritin L-chain (FTL) mRNA. Mutations in the IRE result in reduced binding of the trans-acting iron regulatory proteins (IRPs) and hence in upregulation of ferritin L-chain synthesis. The disease is characterized by increased L-ferritin in serum and tissues and early onset of bilateral cataracts. Iron metabolism is normal, and there is no tissue iron overload. At least 25 nucleotide substitutions and deletions in the L-ferritin IRE have been described in families with HHCS, originating from diverse European, Australian and North American populations. We studied the molecular pathogenesis of HHCS in three unrelated kinderships of western Greek origin, with 19 affected members. We identified a relatively rare C39G mutation located in the hexanucleotide loop of L-ferritin IRE. Computational analysis of mRNA folding of mutant FTL IRE predicted that the C39 > G mutation leads to a rearrangement of base pairing in this critical region, which is likely to modify the IRP binding affinity. All subjects with HHCS were heterozygotes for the same C39G mutation. Clinical and laboratory phenotypes were described. Moreover, there was evidence of an association between this FTL IRE stem-loop mutation and very high ferritin levels. Our findings broaden the list of populations where HHCS has been described.     

Mutation spectrum in Australian pedigrees with hereditary hyperferritinaemia-cataract syndrome reveals novel and de novo mutations

Hereditary hyperferritinaemia-cataract syndrome (HHCS) (OMIM #600886) is a rare autosomal dominant condition identified by high serum ferritin levels with normal iron saturation and distinctive bilateral cataract. It may be misdiagnosed as haemochromatosis and such patients become anaemic as a result of inappropriate venesection. The elevated serum ferritin is due to a mutation in the iron-responsive element (IRE) of the l-ferritin gene, resulting in excessive l-ferritin production. We report the identification of three Australian pedigrees; one with a previously described mutation at position 40, a pedigree with a novel mutation at position 39 and an individual with a de novo mutation at position 32 of the l-ferritin IRE.     

Two Dutch families with hereditary hyperferritinaemia-cataract syndrome and heterozygosity for an HFE-related haemochromatosis gene mutation

Hereditary haemochromatosis is an autosomal recessive disorder, leading to progressive iron overload, which is very common among the Caucasian population. In the vast majority of the cases, the hereditary iron overload is caused by mutations in the HFE gene. Most prominently this is the homozygous Cys282Tyr mutation. We report two Dutch families in which both propositi were found to be heterozygous for Cys282Tyr in the work-up of hyperferritinaemia. Frequent phlebotomies had no effect on the ferritin level, but led to microcytic anaemia. Finally, the family history with bilateral cataracts was the clue for the correct diagnosis. Hereditary hyperferritinaemia-cataract syndrome (HHCS) is an autosomal dominant disease characterised by elevated serum ferritin levels and bilateral cataracts in the absence of iron overload. Several point mutations and deletions within the iron-responsive element (IRE) in the 5' noncoding region of the L-ferritin gene have been found in HHCS families. In the first Dutch family a G to C transition at position 32 was found and a G to A mutation at the same location was found in the second Dutch family. In individuals with an isolated hyperferritinaemia (normal transferrin saturation), the presence of early onset (familial) cataract should raise the possibility of HHCS, even when Cys282Tyr heterozygosity is found.     

Recurrent mutations in the iron regulatory element of L-ferritin in hereditary hyperferritinemia-cataract syndrome.

BACKGROUND AND OBJECTIVE: Hereditary hyperferritinemia-cataract syndrome (HHCS) is an autosomal dominant disorder characterized by bilateral cataracts and increased serum and tissue L-ferritin, in the absence of iron overload. The deregulation of ferritin production is caused by heterogeneous mutations in the iron regulatory element (IRE) of L-ferritin that interfere with the binding of iron regulatory proteins. DESIGN AND METHODS: We have identified several patients from three unrelated Italian families with HHCS. Iron parameters were assessed by standard methods. The IRE element of L-ferritin was amplified by PCR using appropriate primers and directly sequenced. RESULTS: Ferritin levels ranged from 918 microg/L to 2490 microg/L in the patients studied. In one family bilateral cataracts were diagnosed early in life, whereas in the others cataracts were diagnosed around 40-50 years. The female proband of family 3 presented with a severe iron deficiency anemia, which was unrecognized because of the increased ferritin values. Sequencing of the IRE element of L-ferritin in the probands of the three families identified three different nucleotide substitutions (+32 GAE A, +40 AAE G and +39 CT) in the IRE of L-ferritin. These mutations have already been reported in unrelated subjects of different ethnic origins. INTERPRETATION AND CONCLUSIONS: Our findings are consistent with recurrent mutations associated with HHCS and underline the importance of this syndrome in the differential diagnosis of unexplained hyperferritinemia. In addition, the findings highlight the role played by transferrin saturation in the diagnosis of iron deficiency in these patients.     

Pathogenesis of hyperferritinemia cataract syndrome

Hereditary hyperferritinemia-cataract syndrome (HHCS) is an autosomal dominant disorder characterized by bilateral cataracts and increased serum L-ferritin, in the absence of iron overload. Under physiological conditions, ferritin synthesis is finely regulated at the translational level by iron availability. This regulation is achieved by the high-affinity interaction between cytoplasmic mRNA-binding proteins (iron regulatory proteins, IRPs), and mRNA stem-loop structures, known as iron responsive elements (IREs), located in the untranslated regions (UTRs) of the mRNAs. A single IRE is located on the 5' UTR of a series of genes involved in iron metabolism, like L-ferritin, and the binding IRE-IRPs represses these genes translation. The deregulation of ferritin production responsible of HHCS is caused by heterogeneous mutations in the iron regulatory element (IRE) of L-ferritin that interfere with the binding of iron regulatory proteins, disrupting the negative control of L-ferritin synthesis and causing the constitutive up-regulation of ferritin L-chains. The HHCS families originate from different countries of Europe and North America, suggesting that HHCS may be distributed widely throughout the world and not sporadic, whereas its prevalence remains to be established. The lens seems to be particularly sensitive to the increased amount of L-ferritin and the alteration of the proteic equilibrium in this tissue can be responsible of the cataract. In spite of the elucidation of the genetic basis, the genotype phenotype correlation is not clear. Recently, a study based on the thermo-denaturation profile and dissociation constant of the IRE-IRP complex performed for several mutated IREs has provided evidence for a possible correlation between heterogeneous IRE mutations and serum ferritin levels. On the other hand, the in vivo relevance of these conclusions has not been determined completely. A clinical variability among subjects sharing the same mutation, whether they belonged to the same family or not, has also been demonstrated. These findings suggest that, besides the L-ferritin IRE genotype, additional factors are likely to modulate the lens involvement and the rate of progression to severe cataract in HHCS patients.     

H ferritin knockout mice: a model of hyperferritinemia in the absence of iron overload

Ferritin, the iron-storing molecule, is made by the assembly of various proportions of 2 different H and L subunits into a 24-mer protein shell. These heteropolymers have distinct physicochemical properties, owing to the ferroxidase activity of the H subunit, which is necessary for iron uptake by the ferritin molecule, and the ability of the L subunit to facilitate iron core formation inside the protein shell. It has previously been shown that H ferritin is indispensable for normal development, since inactivation of the H ferritin gene by homologous recombination in mice is lethal at an early stage during embryonic development. Here the phenotypic analysis of the mice heterozygous for the H ferritin gene (Fth(+/-) mice) is reported, and differences in gene regulation between the 2 subunits are shown. The heterozygous Fth(+/-) mice were healthy and fertile and did not present any apparent abnormalities. Although they had iron-overloaded spleens at the adult stage, this is identical to what is observed in normal Fth(+/+) mice. However, these heterozygous mice had slightly elevated tissue L ferritin content and 7- to 10-fold more L ferritin in the serum than normal mice, but their serum iron remained unchanged. H ferritin synthesis from the remaining allele was not up-regulated. This probably results from subtle changes in the intracellular labile iron pool, which would stimulate L ferritin but not H ferritin synthesis. These results raise the possibility that reduced H ferritin expression might be responsible for unexplained human cases of hyperferritinemia in the absence of iron overload where the hereditary hyperferritinemia-cataract syndrome has been excluded. (Blood. 2001;98:525-532)     

A novel deletion of the L-ferritin iron-responsive element responsible for severe hereditary hyperferritinaemia-cataract syndrome

In the last few years, mutations that cause disease through increased efficiency of mRNA translation have been discovered. Hereditary hyperferritinaemia-cataract syndrome (HHCS) arises from various point mutations or deletions within the iron-responsive element (IRE) in the 5'-UTR of the L-ferritin mRNA. Each unique mutation confers a characteristic degree of hyperferritinaemia and severity of cataract in affected individuals. We report a novel six-nucleotide deletion identified in an Italian family presenting with elevated serum ferritin and early onset bilateral cataract. This deletion involves a sequence with a TCT repetition and may have occurred through a mechanism of slippage mispairing. Because of the above repetition, the observed mutation can be interpreted as deletion 22-27, 23-28, 24-29 or 25-30. Structural modelling predicted an IRE stem modification that is expected to markedly reduce the binding to iron-regulatory proteins. A double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method easily detected the above deletion.     

Hyperferritinaemia not always a sign of iron overload

The level of serum ferritin normally parallels the concentration of storage iron within the body. In the absence of chronic diseases elevated serum ferritin levels will lead to the diagnosis congenital haemochromatosis. However, there are genetic disorders with high ferritin levels without any sign of iron overload. A case history of a patient suffering from cataract at young age and high ferritin levels is described. Because his mother and his three sons also had cataract at young age and high ferritin levels the diagnosis hereditary hyperferritinaemia-cataract syndrome (HHCS) was made. The diagnosis was confirmed by detection of one of the mutations responsible for the syndrome.     

Hyperferritinaemia in the absence of iron overload

Background—Serum ferritin is normally a marker ofiron overload. Ferritin genes are sited at chromosomes 19 and 11. Regulation of ferritin synthesis involves an interaction between aniron regulatory protein (IRP) and part of the ferritin mRNA designated the iron regulatory element (IRE). A disorder of ferritin synthesis resulting in hyperferritinaemia in the absence of iron overload hasbeen described recently.
Patients and methods—Hyperferriti- naemia in theabsence of iron overload was detected in a patient who was investigatedfor possible haemochromatosis. Serum iron, transferrin saturation, andferritin concentration were studied in 11 members of this patient'sfamily from three generations. Eight members had DNA samples analysedby direct cycle sequencing of the 5' untranslated region of the Lferritin gene.
Results—Six of the family members studied hadserum ferritin concentrations greater than 900 µg/l. However, serumiron and transferrin saturation were normal in these subjects who allhad evidence of cataracts. Three affected family members who hadgenetic studies of the L ferritin gene on chromosome 19 had an A to G point mutation which was not found in unaffected members.
Conclusions—There was complete concordance betweena mutated IRE, cataracts, and hyperferritinaemia in three generationsof this family. This family study confirms the finding that hereditary hyperferritinaemia in the absence of iron overload is an autosomal dominant inherited disorder.

     

Hepcidin levels in hereditary hyperferritinemia: Insights into the iron-sensing mechanism in hepatocytes

AIM:To study the role of hepcidin in hereditary hyperferritinemia cataract syndrome(HHCS). METHODS:Six patients from two families with HHCS, confirmed by genetic analysis showing A to G mutation at position+40 in the L-ferritin gene,were recruited to undergo serum hepcidin and prohepcidin measurements using radioimmunoassay and enzyme linked immunoassay,respectively,and measurements were compared with levels in serum from 25 healthy volunteers(14 females),mean age 36±11.9 years.RESULTS:The serum hepcidin an...     

Hereditary hyperferritinaemia/ cataract syndrome

In mammalian cells, cellular iron homeostasis is maintained by the co-ordinated regulation of transferrin receptor and ferritin synthesis that occurs at the translational level. This regulation is mediated by iron-responsive elements (IREs) that are found within the untranslated regions (UTRs) of mRNA and by cytoplasmic mRNA-binding proteins, known as iron regulatory proteins (IRPs). When cellular iron is scarce, IRPs are available for binding the 5' IRE of ferritin mRNA, initiation of translation is prevented and ferritin synthesis is inhibited. By contrast, the presence of abundant intracellular iron prevents binding of the IRPs to the 5' IRE and allows efficient mRNA translation to proceed. Hereditary hyperferritinaemia/cataract syndrome (HHCS) arises as a result of various point mutations or deletions within a protein binding sequence in the 5'-UTR of the L-ferritin mRNA, which results in increased efficiency of L-ferritin translation. Each unique mutation confers a characteristic degree of hyperferritinaemia and severity of cataract in affected individuals. This exemplifies a new paradigm in which mutations in mRNA cis-acting elements may be responsible for phenotypic variability in disease states.     

Two novel mutations in the SLC40A1 and HFE genes implicated in iron overload in a Spanish man

The most common form of hemochromatosis is caused by mutations in the HFE gene. Rare forms of the disease are caused by mutations in other genes. We present a patient with hyperferritinemia and iron overload, and facial flushing. Magnetic resonance imaging was performed to measure hepatic iron overload, and a molecular study of the genes involved in iron metabolism was undertaken. The iron overload was similar to that observed in HFE hemochromatosis, and the patient was double heterozygous for two novel mutations, c.-20G>A and c.718A>G (p.K240E), in the HFE and ferroportin (FPN1 or SLC40A1) genes, respectively. Hyperferritinemia and facial flushing improved after phlebotomy. Two of the patient's children were also studied, and the daughter was heterozygous for the mutation in the SLC40A1 gene, although she did not have hyperferritinemia. The patient presented a mild iron overload phenotype probably because of the two novel mutations in the HFE and SLC40A1 genes.     

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.