The stepwise formation of bridging (mu-) hydrides of diiron dithiolates is discussed with attention on the pathway for protonation and subsequent isomerizations. Our evidence is consistent with protonations occurring at a single Fe center, followed by isomerization to a series of mu-hydrides. Protonation of Fe(2)(edt)(CO)(4)(dppv) (1) gave a single mu-hydride with dppv spanning apical and basal sites, which isomerized at higher temperatures to place the dppv into a dibasal position. Protonation of Fe(2)(pdt)(CO)(4)(dppv) (2) followed an isomerization pathway similar to that for [1H](+), except that a pair of isomeric terminal hydrides were observed initially, resulting from protonation at the Fe(CO)(3) or Fe(CO)(dppv) site. The first observable product from low temperature protonation of the tris-phosphine Fe(2)(edt)(CO)(3)(PMe(3))(dppv) (3) was a single mu-hydride wherein PMe(3) is apical and the dppv ligand spans apical and basal sites. Upon warming, this isomer converted fully but in a stepwise manner to a mixture of three other isomeric hydrides. Protonation of Fe(2)(pdt)(CO)(3)(PMe(3))(dppv) (4) proceeded similarly to the edt analogue 3, however a terminal hydride was observed, albeit only briefly and at very low temperatures (-90 degrees C). Low-temperature protonation of the bis-chelates Fe(2)(xdt)(CO)(2)(dppv)(2) produced exclusively the terminal hydrides [HFe(2)(xdt)(mu-CO)(CO)(dppv)(2)](+) (xdt = edt and pdt), which subsequently isomerized to a pair of mu-hydrides. At room temperature these (dppv)(2) derivatives convert to an equilibrium of two isomers, one C(2)-symmetric and the other C(s)-symmetric. The stability of the terminal hydrides correlates with the (C(2)-isomer)/(C(s)-isomer) equilibrium ratio, which reflects the size of the dithiolate. The isomerization was found to be unaffected by the presence of excess acid, by solvent polarity, and the presence of D(2)O. This isomerization mechanism is proposed to be intramolecular, involving a 120 degrees rotation of the HFeL(3) subunit to an unobserved terminal basal hydride as the rate-determining step. The observed stability of the hydrides was supported by DFT calculations, which also highlight the instability of the basal terminal hydrides. Isomerization of the mu-hydride isomers occurs on alternating FeL(3) via 120 degree rotations without generating D(2)O-exchangeable intermediates.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Iron overload represents a significant risk factor in the development of HCC. Hereditary hemochromatosis (HH) is a genetic iron overload disease characterized by hepatic iron accumulation. The potential link between these two conditions leads to significant curiosity about regulation of iron homeostasis. Importantly, one of the HH genes, HAMP, encodes the master regulator of iron homeostasis, hepcidin, which is expressed by hepatocytes. Recent studies have shown that the remaining HH genes are either upstream regulators (HFE, HFE2 and TFR2) or downstream targets (FPN) of hepcidin. Moreover, the presence of additional signaling pathways in the liver that contribute to regulation of hepcidin expression has been documented. The function of these iron-regulatory proteins is currently being investigated to determine if they play a role in abnormal iron uptake in tumors. This review summarizes these recent studies and briefly discusses new directions in the treatment of iron overload in HCC patients.

Juvenile hemochromatosis (JH) is a severe form of hemochromatosis, which involves rapid iron overload and leads to organ damage, typically before the age of 30. We report a single case of a 25-year-old man suffering from juvenile hemochromatosis, with aggressive clinical manifestations, typically characterized by transaminasemia and progressive erectile dysfunction, due to hypogonadotropic hypogonadism. The clinical case appears interesting, as the patient also had secondary osteoporosis accompanied by increased bone resorption, which prevalently affected trabecular bone. Approximately 6 months after normalization of serum ferritin levels was achieved by frequent phlebotomies, he became eugonadal and bone mineral density of the lumbar spine increased. Our observations suggest that osteoporosis might occur in the state of JH even at a young age, mainly due to the deprivation of sex steroids and the direct tissue toxicity of iron.

Transfusion-dependent myelodysplastic (MDS) patients are prone to iron overload. We evaluated 43 transfused MDS patients with T2* magnetic resonance imaging scans. 81% had liver and 16·8% cardiac iron overload. Liver R2* (1000/T2*), but not cardiac R2*, was correlated with number of units transfused (r=0·72, P<0·0001) and ferritin (r=0·53, P<0·0001). The area under the curve of a time-ferritin plot was found to be much greater in patients with cardiac iron loading (median 53·7x10(5) Megaunits vs. 12·2x10(5) Megaunits, P=0·002). HFE, HFE2, HAMP or SLC40A1 genotypes were not predictors of iron overload in these patients.

Elucidation of the molecular pathways of iron transport through cells and its control is leading to an understanding of genetic iron loading conditions. The general phenotype of haemochromatosis is iron accumulation in liver parenchymal cells, a raised serum transferrin saturation and ferritin concentration. Four types have been identified: type 1 is the common form and is an autosomal recessive disorder of low penetrance strongly associated with mutations in the HFE gene on chromosome 6(p21.3); type 2 (juvenile haemochromatosis) is autosomal recessive, of high penetrance with causative mutations identified in the HFE2 gene on chromosome 1 (q21) and the HAMP gene on chromosome 19 (q13); type 3 is also autosomal recessive with mutations in the TfR2 gene on chromosome 3 (7q22); type 4 is an autosomal dominant condition with heterozygous mutations in the ferroportin 1 gene. In type 4, iron accumulates in both parenchymal and reticuloendothelial cells and the transferrin saturation may be normal. There are also inherited neurodegenerative conditions associated with iron accumulation. The current research challenges include understanding the central role of the HAMP gene (hepcidin) in controlling iron absorption and the reasons for the variable penetrance in HFE type 1.

Juvenile hemochromatosis (JH) is a rare autosomal recessive disorder that causes iron overload. In the French Canadian region of Saguenay Lac-Saint-Jean the worldwide largest cohort of JH cases has been identified. Here, we report the mapping of this large cohort of cases to the HFE2 locus on chromosome 1q. A maximum multipoint location score of 7.02 was observed with marker D1S2344. A common ancestral haplotype, showing the presence of a founder effect, was identified. The analysis of recombinants allowed us to confirm the JH candidate region.

The availability of a facile treatment for hemochromatosis renders early diagnosis of iron overload syndromes mandatory, and in many instances genetic testing allows identification of individuals at risk of developing clinical disease before pathologic iron storage occurs. Numerous proteins implicated in iron homeostasis have recently come to light, and defects in the cognate genes are associated with iron storage. Although most adult patients with hereditary iron overload are homozygous for the C282Y mutation of the HFE gene, an increasing number with hereditary iron storage have an HFE genotype not characteristic of the disease. Heterozygosity for mutations in the gene encoding ferroportin 1 (FPN1) is probably the second most common genetic cause of hereditary iron storage in adults; here the primarily affected cell is the macrophage. Rare defects, including mutations in the transferrin receptor 2 (TFR2) gene, have also been identified in pedigrees affected with "non-HFE hemochromatosis." Homozygous mutations in the newly identified genes encoding hemojuvelin (HFE2) and hepcidin (HAMP) cause juvenile hemochromatosis. At the same time, heterozygosity for mutations in these genes can modify the clinical expression of iron storage in patients predisposed to iron storage in adult life. Hemochromatosis might thus be considered as a polygenic disease with strong environmental influences on its clinical expression. As our mechanistic understanding of iron pathophysiology improves, our desire to integrate clinical decision making with the results of laboratory tests and molecular analysis of human genes poses increasing challenges.

BACKGROUND

New genetic forms of hereditary hemochromatosis (HH) or hereditary hyperferritinemia (HF) have been identified over the last few years, and abnormalities of various genes may interact in a single patient. This study aimed to develop a rapid automated method for sequencing the main genes involved.

METHODS

We used a standard 96-well microplate with a single PCR condition in an adaptation of the SCAIP (single-condition amplification with internal primer) method to sequence the HFE (hemochromatosis), HAMP (hepcidin antimicrobial peptide), HFE2/HJV [hemochromatosis type 2 (juvenile)], SLC40A1 (ferroportin), and TFR2 (transferrin receptor 2) genes, and the 5' untranslated region of the FTL (ferritin, light polypeptide) gene. To further simplify the method, we adjusted PCR conditions to avoid the use of an internal primer and applied this single-condition amplification method to 38 selected, unrelated patients. We tailored the genetic investigation according to the clinical picture, with the patients falling into 2 groups. Group 1 consisted of patients with hyperferritinemia and high transferrin saturation (TS) (classic adult and juvenile HH forms, groups 1A and 1B, respectively), and group 2 consisted of patients with hyperferritinemia and low, typical, or slightly increased TS, with or without iron overload (groups 2A and 2B, respectively).

RESULTS

With this strategy we identified single-gene and multigene abnormalities, including 6 previously undescribed abnormalities in HFE (c.794dupA), HFE2 (c.-89-4dupT), and SLC40A1 (c.262A>G, c.533G>A, c.1468G>A, and c.-59_-45del).

CONCLUSIONS

This method is a simple approach for investigating hereditary iron overload or HF and allows rapid evaluation of patients.

Hereditary iron overload includes several disorders characterized by iron accumulation in tissues, organs, or even single cells or subcellular compartments. They are determined by mutations in genes directly involved in hepcidin regulation, cellular iron uptake, management and export, iron transport and storage. Systemic forms are characterized by increased serum ferritin with or without high transferrin saturation, and with or without functional iron deficient anemia. Hemochromatosis includes five different genetic forms all characterized by high transferrin saturation and serum ferritin, but with different penetrance and expression. Mutations in HFE, HFE2, HAMP and TFR2 lead to inadequate or severely reduced hepcidin synthesis that, in turn, induces increased intestinal iron absorption and macrophage iron release leading to tissue iron overload. The severity of hepcidin down-regulation defines the severity of iron overload and clinical complications. Hemochromatosis type 4 is caused by dominant gain-of-function mutations of ferroportin preventing hepcidin-ferroportin binding and leading to hepcidin resistance. Ferroportin disease is due to loss-of-function mutation of SLC40A1 that impairs the iron export efficiency of ferroportin, causes iron retention in reticuloendothelial cell and hyperferritinemia with normal transferrin saturation. Aceruloplasminemia is caused by defective iron release from storage and lead to mild microcytic anemia, low serum iron, and iron retention in several organs including the brain, causing severe neurological manifestations. Atransferrinemia and DMT1 deficiency are characterized by iron deficient erythropoiesis, severe microcytic anemia with high transferrin saturation and parenchymal iron overload due to secondary hepcidin suppression. Diagnosis of the different forms of hereditary iron overload disorders involves a sequential strategy that combines clinical, imaging, biochemical, and genetic data. Management of iron overload relies on two main therapies: blood removal and iron chelators. Specific therapeutic options are indicated in patients with atransferrinemia, DMT1 deficiency and aceruloplasminemia.

The application of molecular genetics to haemochromatosis and experimental mutagenesis in animals has transformed our capacity to investigate the unique physiology of iron homeostasis-a key problem in biology and medicine. The identification of HFE, the principal determinant of adult haemochromatosis (HFE1; OMIM 235200) and TfR2, recently implicated in a rarer form of the inherited disorder (HFE3; OMIM 604250), and the promise of candidate genes for juvenile haemochromatosis (HFE2; OMIM 602390) and neonatal haemochromatosis (OMIM 231100) provide the foundation for important studies into the control mechanism of iron balance in humans. The rare conditions atransferrinaemia (OMIM 209300) and acaeruloplasminaemia (OMIM 604290), each associated with tissue iron overload, have already implicated the iron transport ligand transferrin and the copper transporter caeruloplasmin in the control of iron homeostasis. Gene mapping studies in animal mutants with anaemia due to defects in the uptake or tissue transfer of iron have yielded novel proteins involved in iron transport: DMT1 (brush border transporter of ferrous iron) in the mk/mk mouse, hephaestin (basolateral multi-copper ferroxidase) in the sex-linked anaemic mouse (sla) and ferroportin1 (basolateral iron exporter) in zebrafish weh mutants. The discovery of genes that determine heritable defects of iron absorption and regulation in animals and humans thus holds promise for a complete mechanistic understanding of the molecular pathophysiology of iron metabolism.

Density functional theory has been used to study diiron dithiolates [HFe2(xdt)(PR3)n(CO)5-nX] (n = 0, 2, 4; R = H, Me, Et; X = CH3S(-), PMe3, NHC = 1,3-dimethylimidazol-2-ylidene; xdt = adt, pdt; adt = azadithiolate; pdt = propanedithiolate). These species are related to the [FeFe]-hydrogenases catalyzing the 2H(+) + 2e(-) ↔ H2 reaction. Our study is focused on the reduction step following protonation of the Fe2(SR)2 core. Fe(H)s detected in solution are terminal (t-H) and bridging (μ-H) hydrides. Although unstable versus μ-Hs, synthetic t-Hs feature milder reduction potentials than μ-Hs. Accordingly, attempts were previously made to hinder the isomerization of t-H to μ-H. Herein, we present another strategy: in place of preventing isomerization, μ-H could be made a stronger oxidant than t-H (E°μ-H>> E°t-H). The nature and number of PR3 unusually affect ΔE°t-H-μ-H: 4PEt3 models feature a μ-H with a milder E° than t-H, whereas the 4PMe3 analogues behave oppositely. The correlation ΔE°t-H-μ-H ↔ stereoelectronic features arises from the steric strain induced by bulky Et groups in 4PEt3 derivatives. One-electron reduction alleviates intramolecular repulsions only in μ-H species, which is reflected in the loss of bridging coordination. Conversely, in t-H, the strain is retained because a bridging CO holds together the Fe2 core. That implies that E°μ-H>> E°t-H in 4-PEt3 species but not in 4PMe3 analogues. Also determinant to observe E°μ-H>> E°t-H is the presence of a Fe apical σ-donor because its replacement with a CO yields E°μ-H < E°t-H even in 4PEt3 species. Variants with neutral NHC and PMe3 in place of CH3S(-) still feature E°μ-H>> E°t-H. Replacing pdt with (Hadt)(+) lowers E° but yields E°μ-H < E°t-H, indicating that μ-H activation can occur to the detriment of the overpotential increase. In conclusion, our results indicate that the electron richness of the Fe2 core influences ΔE°t-H-μ-H, provided that (i) the R size of PR3 must be greater than that of Me and (ii) an electron donor must be bound to Fe apically.

Repulsive guidance molecule c (RGMc; gene symbol: Hfe2) plays a critical role in iron metabolism. Inactivating mutations cause juvenile hemochromatosis, a severe iron overload disorder. Understanding mechanisms controlling RGMc biosynthesis has been hampered by minimal information about the RGMc gene. Here we define the structure, examine the evolution, and establish mechanisms of regulation of the mouse RGMc gene. RGMc is a 4-exon gene that undergoes alternative RNA splicing to yield 3 mRNAs with 5' different untranslated regions. Gene transcription is induced during myoblast differentiation, producing all 3 mRNAs. We identify 3 critical promoter elements responsible for transcriptional activation in skeletal muscle, comprising paired E-boxes, a putative Stat and/or Ets element, and a MEF2 site, and muscle transcription factors myogenin and MEF2C stimulate RGMc promoter function in non-muscle cells. As these elements are conserved in RGMc genes from multiple species, our results suggest that RGMc has been a muscle-enriched gene throughout its evolutionary history.

Juvenile hemochromatosis (JH) is an autosomal recessive condition that leads to significant morbidity due to early onset systemic iron overload. The majority of families with JH link to chromosome 1q and were recently found to have mutations in the HFE2 gene encoding hemojuvelin; however, several JH families have been reported to have mutations in the HAMP gene encoding hepcidin. Here, we report a multiply consanguineous family with a father and daughter showing iron overload consistent with JH. Sequence analysis of HAMP revealed homozygosity for amino acid substitution C78T due to a c.233G>> A mutation. This mutation disrupts one of eight highly conserved cysteines that are believed to be critical for the function of the active enzyme. This finding adds support to the importance of the role of these conserved cysteines in the activity of hepcidin.

The prevalence of HFE-related hereditary hemochromatosis (HH) among European populations has been well studied. There are no prevalence data for atypical forms of HH caused by mutations in HFE2, HAMP, TFR2, or SLC40A1. The purpose of this study was to estimate the population prevalence of these non-HFE forms of HH.

A list of HH pathogenic variants in publically available next-generation sequence (NGS) databases was compiled and allele frequencies were determined.

Of 161 variants previously associated with HH, 43 were represented among the NGS data sets; an additional 40 unreported functional variants also were identified. The predicted prevalence of HFE HH and the p.Cys282Tyr mutation closely matched previous estimates from similar populations. Of the non-HFE forms of iron overload, TFR2-, HFE2-, and HAMP-related forms are predicted to be rare, with pathogenic allele frequencies in the range of 0.00007 to 0.0005. Significantly, SLC40A1 variants that have been previously associated with autosomal-dominant ferroportin disease were identified in several populations (pathogenic allele frequency 0.0004), being most prevalent among Africans.

We have, for the first time, estimated the population prevalence of non-HFE HH. This methodology could be applied to estimate the population prevalence of a wide variety of genetic disorders.Genet Med 18 6, 618-626.

Hereditary hemochromatosis (HH) is a group of genetic iron overload disorders that manifest with various symptoms, including hepatic dysfunction, diabetes, and cardiomyopathy. Classic HH type 1, which is common in Caucasians, is caused by bi-allelic mutations of HFE. Severe types of HH are caused by either bi-allelic mutations of HFE2 that encodes hemojuvelin (type 2A) or HAMP that encodes hepcidin (type 2B). HH type 3, which is of intermediate severity, is caused by bi-allelic mutations of TFR2 that encodes transferrin receptor 2. Mutations of SLC40A1 that encodes ferroportin, the only cellular iron exporter, causes either HH type 4A (loss-of-function mutations) or HH type 4B (gain-of-function mutations). Studies on these gene products uncovered a part of the mechanisms of the systemic iron regulation; HFE, hemojuvelin, and TFR2 are involved in iron sensing and stimulating hepcidin expression, and hepcidin downregulates the expression of ferroportin of the target cells. Phlebotomy is the standard treatment for HH, and early initiation of the treatment is essential for preventing irreversible organ damage. However, because of the rarity and difficulty in making the genetic diagnosis, a large proportion of patients with non-HFE HH might have been undiagnosed; therefore, awareness of this disorder is important.

Hereditary hemochromatosis (HH) is a genetically heterogeneous disease. The HFE gene resides on chromosome 6 and its mutations account for the majority of HH cases in populations of northern European ancestry. Recently, two new types of hemochromatosis have been identified: Juvenile hemochromatosis (JH or HFE2), which maps to chromosome 1q21, and an adult form defined as HFE 3, which results from mutations of the TFR 2 gene, located at 7q22. We have performed a linkage study in five unrelated families of Greek origin with non-HFE hemochromatosis. Linkage at the chromosome 1q21 JH locus was detected in affected members with the use of polymorphic markers. Comparison of haplotypes between Greek and Italian JH patients revealed the presence of a common haplotype. However, the fact that many other haplotypes carrying the JH defect were observed in the two populations indicates that the respective mutations may have occurred in different genetic backgrounds. We suggest that hemochromatosis patients without HFE mutations should be evaluated for other possible types of hemochromatosis since hemochromatosis type 3 (HFE3) has a clinical appearance similar to HFE 1, and JH may have a late onset in some cases.

Hemochromatosis type 2 (HFE2) or juvenile hemochromatosis (JH) is a rare recessive disorder that causes iron overload, characterized by early onset and severe clinical course. The JH locus maps to chromosome 1q, in a 4-cM region encompassing markers D1S442 and D1S2347. Recently a gene named ZIRTL has been characterized and mapped to 1q21. This gene belongs to a family of divalent metal ion-transporting genes that encode for proteins involved in transport of different metals, including iron. Thus, the ZIRTL gene represents a positional and functional candidate for JH. Here we further restrict the candidate region through segregation analysis of two new polymorphic markers and haplotype analysis in JH families. Furthermore, we exclude ZIRTL as a JH candidate gene showing that it maps outside the critical interval and that its genomic sequence is normal in three patients.

We characterized HFE C282Y homozygotes aged 25-29 years in the HEmochromatosis and IRon Overload Screening (HEIRS) Study using health questionnaire responses, transferrin saturation (TfSat), serum ferritin (SF), and HFE genotyping. In eight homozygotes, we used denaturing high-performance liquid chromatography and sequencing to search for HFE2 (= HJV), TFR2, HAMP, SLC40A1 (= FPN1), and FTL mutations. Sixteen of 4,008 White or Hispanic participants aged 25-29 years had C282Y homozygosity (15 White, 1 Hispanic); 15 were previously undiagnosed. Eleven had elevated TfSat; nine had elevated SF. None reported iron overload-associated abnormalities. No deleterious non-HFE mutations were detected. The prevalence of C282Y homozygosity in White or Hispanic HEIRS Study participants aged 25-29 years did not differ significantly from the prevalence of C282Y homozygosity in older White or Hispanic HEIRS Study participants. The prevalences of reports of iron overload-associated abnormalities were not significantly different in these 16 C282Y homozygotes and in HFE wt/wt control participants aged 25-29 years who did not report having hemochromatosis or iron overload. We conclude that C282Y homozygotes aged 25-29 years diagnosed by screening infrequently report having iron overload-associated abnormalities, although some have elevated SF. Screening using an elevated TfSat criterion would fail to detect some C282Y homozygotes aged 25-29 years.

Mutations in the TMPRSS6 gene are associated with severe iron-refractory iron deficiency anemia resulting from an overexpression of hepcidin, the key regulator of iron homeostasis. The matriptase (MT)-2 protein (encoded by the TMPRSS6 gene) regulates hepcidin expression by cleaving hemojuvelin [HJV/hemochromatosis type 2 (HFE2)], a bone morphogenetic protein (BMP) coreceptor in the hepcidin regulatory pathway. We investigated the functional consequences of five clinically associated TMPRSS6 variants and the role of MT-2 protein domains by generating epitope-tagged mutant and domain-swapped MT-2-MT-1 (encoded by the ST14 gene) chimeric constructs and expressing them in HepG2/C3A cells. We developed a novel cell culture immunofluorescence assay to assess the effect of MT-2 on cell surface HJV expression levels, compatible with HJV cleavage. The TMPRSS6 variants Y141C, I212T, G442R, and C510S were retained intracellularly and were unable to inhibit BMP6 induction of hepcidin. The R271Q variant, although it has been associated with iron-refractory iron deficiency anemia, appears to remain functional. Analysis of the chimeric constructs showed that replacement of sperm protein, enterokinase, and agrin (SEA), low-density-lipoprotein receptor class A (LDLRA), and protease (PROT) domains from MT-2 with those from MT-1 resulted in limited cell surface localization, while the complement C1r/C1s, Uegf, Bmp1 (CUB) domain chimera retained localization at the cell surface. The SEA domain chimera was able to reduce cell surface HJV expression, while the CUB, LDLRA, and PROT domain chimeras were not. These studies suggest that the SEA and LDLRA domains of MT-2 are important for trafficking to the cell surface and that the CUB, LDLRA, and PROT domains are required for cleavage of HJV.

There are few descriptions of young adults with self-reported hemochromatosis or iron overload (H/IO). We analyzed initial screening data in 7,343 HEmochromatosis and IRon Overload Screening (HEIRS) Study participants ages 25-29 years, including race/ethnicity and health information; transferrin saturation (TS) and ferritin (SF) measurements; and HFE C282Y and H63D genotypes. We used denaturing high-pressure liquid chromatography and sequencing to detect mutations in HJV, TFR2, HAMP, SLC40A1, and FTL. Fifty-one participants reported previous H/IO; 23 (45%) reported medical conditions associated with H/IO. Prevalences of reports of arthritis, diabetes, liver disease or liver cancer, heart failure, fertility problems or impotence, and blood relatives with H/IO were significantly greater in participants with previous H/IO reports than in those without. Only 7.8% of the 51 participants with previous H/IO reports had elevated TS; 13.7% had elevated SF. Only one participant had C282Y homozygosity. Three participants aged 25-29 years were heterozygous for potentially deleterious mutations in HFE2, TFR2, and HAMP promoter, respectively. Prevalences of self-reported conditions, screening iron phenotypes, and C282Y homozygosity were similar in 1,165 participants aged 30 years or greater who reported previous H/IO. We conclude that persons who report previous H/IO diagnoses in screening programs are unlikely to have H/IO phenotypes or genotypes. Previous H/IO reports in some participants could be explained by treatment that induced iron depletion before initial screening, misdiagnosis, or participant misunderstanding of their physician or the initial screening questionnaire.

OBJECTIVE

To determine the molecular basis of a mild hemochromatosis phenotype in a man of Scottish-Irish descent.

METHODS

We sequenced genomic DNA to detect mutations of HFE, SLC40A1, TFR2, HAMP, and HFE2. RNA isolated from blood mononuclear cells was used to make cDNA. RT-PCR was performed to amplify ferroportin from cDNA, and amplified products were visualized by electrophoresis and sequenced.

RESULTS

The proband was heterozygous for the novel mutation c.1402G->>A (predicted G468S) in exon 7 of the ferroportin gene (SLC40A1). Located in the last nucleotide before the splice junction, this mutation results in aberrant splicing to a cryptic upstream splice site located at nt 990 within the same exon. This causes truncation of ferroportin after glycine 330 and the addition of 4 irrelevant amino acids before terminating. The truncated ferroportin protein, missing its C-terminal 241 amino acids, would lack all structural motifs beyond transmembrane region 7. The patient was also heterozygous for the common HFE H63D polymorphism, but did not have coding region mutations in TFR2, HAMP, or HFE2.

CONCLUSIONS

We conclude that this patient represents a unique example of hemochromatosis due to a single base-pair mutation of SLC40A1 that results in aberrant splicing and truncation of ferroportin.

1q21.1 duplication is a rare copy number variant with multiple congenital malformations, including developmental delay, autism spectrum disorder, dysmorphic features and congenital heart anomalies. The present study described a Chinese female patient (age, four years and eight months) with multiple malformations, including congenital heart defect, mental impairment and developmental delay. The parents and the monozygotic twin sister of the patient, however, were physically and psychologically normal. High‑resolution genome‑wide single nucleotide polymorphism array revealed a 1.6‑Mb duplication in chromosome region 1q21.1. This chromosome region contained HFE2, a critical gene involved in hereditary hemochromatosis. However, the parents and monozygotic twin sister of the patient did not carry this genomic lesion. To the best of our knowledge, the present study was the first to report on a 1q21.1 duplication patient in mainland China.