Normal Transferrin Saturation Research Articles

Almost Normal Transferrin Saturation in Thalassemic Patients on Deferasirox May Predict Better Outcome

Iron overload is a serious consequence of the repeated blood transfusions required in the treatment of Thalassaemia major patients. Transferrin saturation (TfS) above 75% is a marker of iron overload of any cause. In thalassemia TfS levels can reach 80% to 100% and even higher. Most of the Non Transferrin Bound Iron (NTBI) is chelated by iron chelators. The fraction of NTBI, which in thalassemia is referred to as ‘‘directly chelatable iron”, has also recently been termed labile plasma iron (LPI). Labile iron appears in plasma and interstitial fluids when the transferrin binding capacity is surpassed. Iron chelators are agents designed to remove LPI and iron from the cells. There are now 3 different classes of iron chelators used mainly as monotherapy and one combination regimen with Desferrioxamine (DFO) and Deferiprone (DFP). Deferasirox (DFS) is a new active oral chelator. The mode of action of each chelator is different and the choice is individualized.Aim: To evaluate the effect of 4 different types of chelation therapy on TfS.Methods: Serum iron (Fe, normal range 60–180μg/dl), Total Iron Binding Capacity (TIBC, normal range 155–300μg/dl), TfS (which was calculated by the formula (serum Fex100/TIBC) and serum Ferritin were measured in 100 thalassaemia patients (46M/54F, median age 30,4 ± 8,4 years (range 18–54 years). All patients were regularly transfused (pre-transfusion hemoglobin: 9,7g/dl) and well chelated. Twenty-nine of the above-mentioned 100 patients were on DFS (Group A), 32 on DFP (Group B), 25 on combination therapy (Group C) and 14 on DFO (Group D). Median duration of therapy was 16±7 months for group A and 5,2±2,8, 3,4±2,1 and 29,8±4,3 years for groups B, C and D respectively. T-test was used in statistical analysis to compare means.Results: All patients of groups B, C and D had low levels of TIBC below normal values (40±26, 67±35 and 104±39μg/dl respectively) but patients on DFS (A) had values in the normal range (292±114μg/dl). Patients on DFS had the lowest TfS (103±46%), followed by patients on DFO (251±132%). The DFP group had the highest TfS value 1245±1510%, while the combined therapy group's value was 531±545%. Group's A ferritin was 1617±1363 ng/ml while ferritin of groups B, C and D were 1210±799, 1683±1950 and 1058±514 ng/ml respectively. Although TfS differed significantly between groups (T-test: A vs B, A vs C and A vs D: p=0,0001, p=0,0006 and p= 0,001 respectively) differences in ferritin were not statistically significant.Conclusions: TfS can theoretically be used to predict with whatever type of treatment applied, the NTBI and consequently the LPI. Very high TfS values may mean a great amount of circulating toxic NTBI which is not successfully chelated. In our patients TfS was found lowest in those on DFS. Differences in TIBC and TfS can be explained by the different mode of action of the three chelators used. Further comparative studies are needed to conclude whether DFS therapy, leads to not only the lowest TfS levels but also to the lowest LPI values. In that case it could be considered a more “reasonable” therapy than other chelators

Revaluation of clinical and histological criteria for diagnosis of dysmetabolic iron overload syndrome

To re-evaluate the diagnostic criteria of insulin resistance hepatic iron overload based on clinical, biochemical and histopathological findings. We studied 81 patients with hepatic iron overload not explained by known genetic and acquired causes. The metabolic syndrome (MS) was defined according to ATPIII criteria. Iron overload was assessed by liver biopsy. Liver histology was evaluated by Ishak's score and iron accumulation by Deugnier's score; steatosis was diagnosed when present in >or=5% of hepatocytes. According to transferrin saturation levels, we observed significant differences in the amount of hepatic iron overload and iron distribution, as well as the number of metabolic abnormalities. Using Receiving Operating Curve analysis, we found that the presence of two components of the MS differentiated two groups with a statistically significant different hepatic iron overload (P<0.0001). Patients with >or=2 metabolic alterations and steatosis had lower amount of hepatic iron, lower transferrin saturation and higher sinusoidal iron than patients with <2 MS components and absence of steatosis. In our patients, the presence of >or2 alterations of the MS and hepatic steatosis was associated with a moderate form of iron overload with a prevalent sinusoidal distribution and a normal transferrin saturation, suggesting the existence of a peculiar pathogenetic mechanism of iron accumulation. These patients may have the typical dysmetabolic iron overload syndrome. By contrast, patients with transferrin saturation>or=60% had more severe iron overload, few or no metabolic abnormalities and a hemochromatosis-like pattern of iron overload.

Analysis of SLC40A1 gene at the mRNA level reveals rapidly the causative mutations in patients with hereditary hemochromatosis type IV

Mutations in the SLC40A1 gene result in a dominant genetic disorder [ferroportin disease; hereditary hemochromatosis type (HH) IV], characterized by iron overload with two different clinical manifestations, normal transferrin saturation with macrophage iron accumulation (the most prevalent type) or high transferrin saturation with hepatocyte iron accumulation (classical hemochromatosis phenotype). In previous studies, the mutational analysis of SLC40A1 gene has been performed at the genomic DNA level by PCR amplification and direct sequencing of all coding regions and flanking intron–exon boundaries (usually in 9 PCR reactions). In this study, we analyzed the SLC40A1 gene at the mRNA level, in two RT-PCR reactions, followed by direct sequencing and/or NIRCA (non-isotopic RNase cleavage assay). This protocol turned out to be rapid, sensitive and reliable, facilitating the detection of the SLC40A1 gene mutations in two patients with hyperferritinemia, normal transferrin saturation and iron accumulation predominantly in macrophages and Kupffer cells. The first one displayed the well-described alteration V162Δ and the second a novel mutation (R178G) that was further detected in two relatives in a pedigree analysis. The proposed procedure would facilitate the wide-range molecular analysis of the SLC40A1 gene, contributing to better understanding the pathogenesis of the ferroportin disease.

Bivariate mixture modeling of transferrin saturation and serum ferritin concentration in Asians, African Americans, Hispanics, and whites in the Hemochromatosis and Iron Overload Screening (HEIRS) Study

Bivariate mixture modeling was used to analyze joint population distributions of transferrin saturation (TS) and serum ferritin concentration (SF) measured in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. Four components (C1, C2, C3, and C4) with successively age-adjusted increasing means for TS and SF were identified in data from 26,832 African Americans, 12,620 Asians, 12,264 Hispanics, and 43,254 whites. The largest component, C2, had normal mean TS (21% to 26% for women, 29% to 30% for men) and SF (43-82 microg/L for women, 165-242 microg/L for men), which consisted of component proportions greater than 0.59 for women and greater than 0.68 for men. C3 and C4 had progressively greater mean values for TS and SF with progressively lesser component proportions. C1 had mean TS values less than 16% for women (<20% for men) and SF values less than 28 microg/L for women (<47 microg/L for men). Compared with C2, adjusted odds of iron deficiency were significantly greater in C1 (14.9-47.5 for women, 60.6-3530 for men), adjusted odds of liver disease were significantly greater in C3 and C4 for African-American women and all men, and adjusted odds of any HFE mutation were increased in C3 (1.4-1.8 for women, 1.2-1.9 for men) and in C4 for Hispanic and white women (1.5 and 5.2, respectively) and men (2.8 and 4.7, respectively). Joint mixture modeling identifies a component with lesser SF and TS at risk for iron deficiency and 2 components with greater SF and TS at risk for liver disease or HFE mutations. This approach can identify populations in which hereditary or acquired factors influence metabolism measurement.

Plasma Ghrelin Levels in Various Stages of Development of Iron Deficiency Anemia

Ghrelin stimulates food intake and induces metabolic changes leading to an increase in body weight and body fat mass. Iron-deficiency anemia (IDA) is the most frequently seen cause of nutritional anemia, that is a type of starvation. There is no available study related to levels of ghrelin in IDA. The aim of this study is to show an association with ghrelin levels and iron deficiency and to demonstrate whether changes seen in iron deficiency (ID) are explained by ghrelin, as opposed to whether ghrelin levels correlate with ID. The study group was consisted of children who were admitted in the outpatient clinic of pediatrics. Control group (C) was defined as cases with normal hemoglobin (Hb), serum iron (SI), transferrin saturation (TS), and ferritin (F) (>12 ng/mL) values; group hypoferritinemia (IDec) Hb: N, SI: N, TS: N, F<12 ng/mL; group iron deficiency (IDef), Hb: N, SI: decreased, TS<or=16%, F<12 ng/mL, and group IDA, Hb and SI decreased, TS<or=16%, F<12 ng/mL. The patients were categorized into 4 groups [group 1 (C), n=25, age=82.4+/-16.56 mo, F=40.87+/-6.17 ng/mL; group 2 (IDec), n=30, age=57.5+/-20.71 mo, F=29.95+/-3.77 ng/mL; group 3 (IDef), n=28, age=50.21+/-19.87 mo, F=14.82+/-3.41 ng/mL; group 4 (IDA), n=25, age=31.55+/-13.21 mo, F=11.75+/-4.01 ng/mL]. Mean value of ghrelin was detected to be 396.53+/-85.56 pg/mL, 332.26+/-74.35 pg/mL, 309.66+/-68.62 pg/mL, and 177.66+/-27.81 pg/mL in control, groups IDec, IDef, and IDA, respectively. A statistically significant difference was detected between groups control and IDef (P<0.01), control and IDA, IDec and IDA, IDef and IDA (P<0.001). A significant positive correlation was demonstrated between iron status of the body and levels of ghrelin. Decrease in ghrelin levels in IDA can lead to loss of appetite, desire to eat diverse foods with resultant delay in growth and development.

Lack of clinical manifestation of hereditary haemochromatosis in South African patients with multiple sclerosis

Caucasian South African patients with multiple sclerosis (MS) were screened for the most common hereditary haemochromatosis (HH) mutations, H63D and C282Y, in order to determine the impact of iron overload on clinical outcome of MS. DNA screening for mutations H63D and C282Y in 118 apparently unrelated MS patients did not reveal significant differences in allele frequencies in comparison with a control group from the same population. Of 17 MS patients heterozygous for C282Y, 3 had below normal and none had above normal transferrin saturation levels. One of the index MS patients, and subsequently also her sister who also has MS, tested positive for two copies of mutation C282Y. Determination of iron status revealed high serum ferritin and transferrin saturation levels in both patients. However, the index patient, being unaware of her C282Y status, had received treatment for iron deficiency in the past and her MS symptoms were less severe than those of her sister who has been wheelchair bound for the past 12 years and who did not take iron supplements. Lack of clinical manifestation of HH without any signs of organ damage in the C282Y homozygous MS patients is in accordance with a role of iron dysregulation in the aetiology of MS.

Hiperferritinemia familiar y cataratas congénitas asociadas a mutación del gen HFE. Dos nuevas familias españolas y una nueva mutación (A37T: «Zaragoza»)

Nuclear congenital cataracts associated with hyperferritinemia--hereditary hyperferritinemia cataract syndrome (HHCS)--without clinical or biochemical signs of iron overload have been recently described in several Spanish families. This HHCS is associated with mutations in the gene of ferritin subunit L, located in chromosome 19. We describe 2 new families with HHCS, one of them presenting a new L-ferritin mutation (A37T: -Zaragoza-). Patients and probands were studied according to the Anemia Unit protocol: complete blood count, biochemical profile (diabetes, hepatic and renal), hepatic serologies, iron metabolism (iron, transferrin, ferritin, transferrin saturation, reactive C protein) and mutation HFE gene studies (C282Y, H63D). All of them were sent to the Ophthalmology Service for cataract study. L-ferritin mutational scanning was performed by denaturing high performance liquid chromatography (DHPLC). Samples displaying an altered elution profile, as compared to a wild type control, were directly sequenced for the precise characterization of the L-ferritin mutation. Family A proband was a 54 year-old-female, with cataracts, ferritin level: 942 pg/I, transferrin saturation: 14%, HFE gen study: H63D/H63D; L-ferritin gene study: C33T mutation/-. Her two sons had cataracts, hyperferritinemia (1607, and 1188 pg/I, respectively), normal transferrin saturation (40% and 9%), HFE gene study: H63D/N; and L-ferritin gen study: C33T/-. Family B proband was a 39 year-old-female, with cataract, ferritin level: 636 pg/I, transferrin saturation: 25%, HFE gene study: H63D/N; and L-ferritin gene study: A37T/-. Her two sons, sister, brother and nephew, who were affected with A37T mutation, showed hyperferritinemia (883, 747, 835, 613 and 1396 pg/I) with normal transferrin saturation levels (17%, 34%, 25%, 18% and 24%); but the ferritin levels of those non-affected were normal (35 and 50 pg/I). HHCS is a dominant autosomic condition, with a possible world-wide distribution,which should be included in the differential diagnosis of hyperferritinemia studies. It is important to suspect it in order to avoid wrong treatment.

Fatty liver in H63D homozygotes with hyperferritinemia

To study the clinical correlates of the H63D mutation we have analysed the phenotype of H63D homo-zygotes identified through mutation analysis in a referral laboratory. A total of 366 blood samples referred for HFE analysis were screened for C282Y and H63D mutations. Four H63D homozygotes were identified. All had raised serum ferritin but normal transferrin saturation. They were negative for hepatitis B and C and only one patient consumed excess alcohol. In all 4 cases ultrasonography revealed fatty liver. In two patients a liver biopsy was done and showed mild siderosis with an unusual distribution and macrovesicular steatosis. These data confirm the association between fatty liver, hyperferritinaemia and increased hepatic iron, but do not clarify whether siderosis was related to steatosis rather than homozygosity for the H63D mutation. Patients with fatty liver may complicate the interpretation of data in population studies of the expression of H63D homozygosity.

Unexplained Serum Ferritin Elevations in Primary Care Patients with Elevated Serum Ferritin Concentrations and High Normal Transferrin Saturations.

Non-HFE primary iron overload exists in African Americans and other ethnic groups, but the prevalence and spectrum of clinical manifestations are not known. In the HEIRS (Hereditary Hemochromatosis and Iron Overload Screening) Study, participants were considered for further evaluation if the serum ferritin concentration was elevated and the transferrin saturation was more than 45% for women or 50% for men. We hypothesized that these screening criteria would miss a substantial number of African Americans and members of other ethnic groups with increased iron stores. In the process of screening 21,231 predominantly African-American and Hispanic primary care patients at the Howard University field center of the HEIRS Study, we identified 161 non-HFE-C282Y homozygotes ≥ 25 years of age with serum ferritin concentrations above the 97.5 percentile for the population (>700 ng/ml for men and >500 ng/ml for women) but transferrin saturations in the upper part of the normal range (35–50% for men and 30–45% for women). Of the 123 participants we were able to contact, 68 (55%) participated in a clinical evaluation, including 64 African Americans, three Hispanics and one Asian American with a mean ± SD age of 57 ± 13 years. Thirty-eight (56%) were females, 6 (9%) were HFE H63D heterozygotes and 2 (3%) were C282Y heterozygotes. Seven patients (10%) had normal serum ferritin concentration on repeat testing while 42 (62%) had potential reasons for elevated serum ferritin concentration other than a primary increase in body iron including (sequentially) multiple blood transfusions (>10 lifetime; n = 4), abnormal liver function tests (hepatitis C positive or AST >60 IU/L and AST>ALT; n = 17), hemoglobin < 10 g/dL men or 9 g/dL women (n = 1), elevated C-reactive protein with transferrin saturation not elevated (n = 17), and excessive alcohol use (n = 3). Nineteen patients did not have these explanations for increased serum ferritin concentration and were considered to have a possible primary iron-loading process (see Table).One of the patients with unexplained elevated serum ferritin concentration (an African American) had a diagnostic liver biopsy showing 2-3+ hepatocellular iron and heavy iron deposition in Kupffer cells and is on phlebotomy therapy; the others have been advised to have diagnostic liver biopsy or quantitative phlebotomy. We conclude that there are substantial numbers of African Americans with elevated serum ferritin concentration and normal transferrin saturation who have transfusional iron overload or a probable primary increase in body iron stores.Characteristics of 19 Patients with Unexplained Serum Ferritin ElevationsNo. (%) of Women8 (42)Age inyears (mean ± SD)63 ± 14Race (African American:Hispanic:Asian)16:2:1Hemoglobin in g.dL (mean ± SD) Men13.8 ±1.5Hemoglobin in g.dL (mean ± SD) Women12.9 ± 0.8HFE mutations in no. (%) C282Y heterozygotes0 (0)HFE mutations in no. (%) H63D heterozygotes2 (11)Ferritin category in no. (%) < 500 ng/ml7 (37)Ferritin category in no. (%) 500–1000 ng/ml9(57)Ferritin category in no. (%) 1000 ng/ml>3 (16)

Relations entre surcharges en fer et insulinorésistance

There is increasing evidence that moderately elevated body iron stores, below levels commonly found in genetic hemochromatosis, may be associated with adverse health outcomes. Genetic hemochromatosis, characterized by transferrin saturation (TS) greater than 45%, is most often linked to homozygosity of the HFE C282Y allele. The phenotype is also modulated by mutations of more recently discovered genes (including ferroportin, hemojuvelin, hepcidin, and transferrin receptor) and environmental factors (including alcohol, viruses, diet, blood loss). Iron overload without hemochromatosis is characterized by high levels of serum ferritin and normal TS, as seen in dysmetabolic hepatosiderosis. Elevated serum ferritin levels predict incident type 2 diabetes in prospective studies and have been associated with hypertension, dyslipidemia, glucose tolerance disturbances, central adiposity, and metabolic syndrome. High ferritin levels are not synonymous with iron overload and may in some cases be a simple marker of insulin resistance.

Microcytic anemia, erythropoietic protoporphyria, and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2

AbstractIron-regulatory proteins (IRP14144) 1 and 2 posttranscriptionally regulate expression of transferrin receptor (TfR), ferritin, and other iron metabolism proteins. Mice with targeted deletion of IRP2 overexpress ferritin and express abnormally low TfR levels in multiple tissues. Despite this misregulation, there are no apparent pathologic consequences in tissues such as the liver and kidney. However, in the central nervous system, evidence of abnormal iron metabolism in IRP2-/- mice precedes the development of adult-onset progressive neurodegeneration, characterized by widespread axonal degeneration and neuronal loss. Here, we report that ablation of IRP2 results in iron-limited erythropoiesis. TfR expression in erythroid precursors of IRP2-/- mice is reduced, and bone marrow iron stores are absent, even though transferrin saturation levels are normal. Marked overexpression of 5-aminolevulinic acid synthase 2 (Alas2) results from loss of IRP-dependent translational repression, and markedly increased levels of free protoporphyrin IX and zinc protoporphyrin are generated in IRP2-/- erythroid cells. IRP2-/- mice represent a new paradigm of genetic microcytic anemia. We postulate that IRP2 mutations or deletions may be a cause of refractory microcytic anemia and bone marrow iron depletion in patients with normal transferrin saturations, elevated serum ferritins, elevated red cell protoporphyrin IX levels, and adult-onset neurodegeneration.

Resistance to hepcidin is conferred by hemochromatosis-associated mutations of ferroportin

AbstractFerroportin (FP141414148) mediates iron export from cells; FPN mutations are associated with the iron overloading disorder hemochromatosis. Previously, we found that the A77D, V162del, and G490D mutations inhibited FPN activity, but that other disease-associated FPN variants retained full iron export capability. The peptide hormone hepcidin inhibits FPN as part of a homeostatic negative feedback loop. We measured surface expression and function of wild-type FPN and fully active FPN mutants in the presence of hepcidin. We found that the Y64N and C326Y mutants of FPN are completely resistant to hepcidin inhibition and that N144D and N144H are partially resistant. Hemochromatosis-associated FPN mutations, therefore, either reduce iron export ability or produce an FPN variant that is insensitive to hepcidin. The former mutation type is associated with Kupffer-cell iron deposition and normal transferrin saturation in vivo, whereas patients with the latter category of FPN mutation have high transferrin saturation and tend to deposit iron throughout the liver parenchyma. FPN-linked hemochromatosis may have a variable pathogenesis depending on the causative FPN mutant.

Comparison of the Unsaturated Iron-Binding Capacity with Transferrin Saturation as a Screening Test to Detect C282Y Homozygotes for Hemochromatosis in 101 168 Participants in the Hemochromatosis and Iron Overload Screening (HEIRS) Study

The diagnosis of hemochromatosis was previously based on a combination of clinical and laboratory assessments that included history and physical examination, increased transferrin saturation (TS) and serum ferritin, liver biopsy, the amount of iron removed by phlebotomy, and pedigree studies identifying other family members with iron overload (1). Since the discovery of the hemochromatosis gene (HFE) in 1996 (2), most studies from referral centers have shown that >90% of typical hemochromatosis patients are homozygous for the C282Y mutation of the HFE gene (3). Before the availability of DNA-based testing, it was assumed that most hemochromatosis patients have increased TS. However, recent population screening studies incorporating HFE genotyping have now shown that many C282Y homozygotes will have a normal TS and may never develop clinical signs and symptoms related to iron overload (4)(5)(6)(7)(8). TS has been recommended in many studies as the most clinically useful screening test for hemochromatosis because it is widely available and may be increased even in young adults with a genetic predisposition to hemochromatosis. Another potential advantage over DNA-based testing as an initial screening test is that TS may detect many types of iron overload other than those associated with HFE mutations. In addition, screening for iron overload instead of performing DNA-based testing may reduce the risks of potential genetic discrimination that some authors suggest is associated with identification of a C282Y homozygote with normal serum iron tests (9)(10)(11). The TS is a 2-step assay in which serum iron is the numerator and the denominator is either total iron-binding capacity (TIBC), [serum iron + unsaturated iron-bonding capacity (UIBC)] or an adjusted serum transferrin. The UIBC is a 1-step automated colorimetric assay that has been reported to have similar or better operating characteristics than TS for …

The Evaluation of Hyperferritinemia: An Updated Strategy Based on Advances in Detecting Genetic Abnormalities

The number of new genes implicated in iron metabolism has dramatically increased during the last few years. Alterations of these genes may cause hyperferritinemia associated or not with iron overload. Correct assignment of the specific disorder of iron metabolism requires the identification of the causative gene mutation. Here, we propose a rational strategy that allows targeting the gene(s) to be screened for a diagnostic purpose. This strategy relies on the age of onset of the disease, the type of clinical symptoms, the biochemical profile (elevated or normal serum transferrin saturation (TfSat)), the presence or not of visceral iron excess, and the mode of inheritance (autosomal recessive or dominant). Then, two main entities can be differentiated: genetic (adult or juvenile) hemochromatosis characterized by elevated TfSat, and hereditary hyperferritinemias where TfSat is normal (or only slightly modified). Adult genetic hemochromatosis (GH) is related mainly to mutations of the HFE gene, and exceptionally to mutations of the TFR2 gene. Juvenile GH is a rare condition related principally to mutations of the HJV gene coding for hemojuvelin, and rarely to mutations of the HAMP gene coding for hepcidin. Hereditary hyperferritinemias are linked to mutations of three genes: the L-ferritin gene responsible for the hereditary hyperferritinemia cataract syndrome (without iron overload), the ferroportin gene leading to a dominant form of iron overload, and the ceruloplasmin (CP) gene corresponding to an iron overload syndrome with neurological symptoms. The proposed strategic approach may change with the identification of other genes involved in iron metabolism.

Transferrin Saturation, Dietary Iron Intake, and Risk of Cancer

Transferrin saturation of more than 60% has been identified as a cancer risk factor. It is unclear whether dietary iron intake increases the risk of cancer among individuals with transferrin saturation of less than 60%. The purpose of this study was to examine the association of dietary iron intake and the risk of cancer among adults with increased transferrin saturation. Analysis of a cohort study, the National Health and Nutrition Examination Survey I Epidemiologic Follow-Up Study, was performed. US adults (aged 25 to 74 years at baseline) were followed up from baseline in 1971-1974 to 1992 (N = 6,309). A total of 7.3% of the US population had a serum transferrin saturation of more than 45% at baseline. Intake of dietary iron was essentially uncorrelated with transferrin saturation (r = 0.04). Compared with individuals who had normal serum transferrin saturation and low dietary iron intake, individuals whose serum transferrin saturation was more than 45% and who had high dietary iron intake also had an increased adjusted relative risk of cancer (2.24; 95% confidence interval [CI], 1.02-4.89). Increased risk was not found for individuals with a transferrin saturation of more than 45% but a normal dietary iron intake (hazard ratio, 1.02; 95% CI, 0.69-1.49). Transferrin saturation levels could be set as low as 41%, and the individuals with high transferrin saturation and high dietary iron intake would still have an increased adjusted relative risk of cancer (hazard ratio, 2.00; 95% CI, 1.04-3.82). Among persons with increased transferrin saturation, a daily intake of dietary iron more than 18 mg is associated with an increased risk of cancer. Future research might focus on the benefits of dietary changes in those individuals with increased serum transferrin saturation.

Molecular Diagnosis of the First Ferroportin Mutation (C326Y) in the Far East Causing a Dominant Form of Inherited Iron Overload.

Genetic hemochromatosis (HH) is a common inherited disorder in populations of European origin in which different types of genetic hemochromatosis (type 1–4) have been characterized. Most hemochromatosis-type 1 patients are homozygotes or compound heterozygotes for two HFE mutations C282Y and H63D. Studies of several non-HFE iron overload families led to identification of mutations in hemojuvelin and hepcidin (juvenile form-HFE2A and B), transferrin receptor 2 (HFE3) and ferroportin (HFE4) as a cause of different forms of hemochromatosis. In the Far East, inherited hemochromatosis has rarely been reported and may have been misdiagnosed due to the high prevalence of secondary iron loading from hemoglobin disorders. This report describes, for the first time, non-HFE iron overload in patients from Southeast Asia. The affected Thai family presented with a distinctive clinical phenotype including macrocytosis and elevated transferrin saturation (>95%), increased non-transferrin bound iron (NTBI) as well as raised serum ferritin and marked hepatic hemochromatosis. Our patients tolerated therapeutic phlebotomy well. DNAs from peripheral blood leukocytes were firstly analyzed for three common HFE mutations (C282Y, H63D and IVS5+1 G→A). Subsequently, we screened all coding sequences, promoters and exon/intron boundaries of the HFE, HAMP, TfR2, HJV and SLC40A1 genes using denaturing high performance liquid chromatography (DHPLC). The entire coding region and splice sites of these genes were amplified and directly sequenced. We identified a novel mutation (C326Y) in ferroportin (SLC40A1, IREG-1, MTP-1), a membrane iron transport protein due to a G→A substitution at nucleotide 1281 in exon 7. This mutation was confirmed by restriction fragment length polymorphism (RFLP) analysis using Sfa NI. Six hundred Thai and two hundred Vietnamese chromosomes were analyzed for the C326Y mutation by RFLP analysis and it was not detected in any of the healthy controls studied. This result suggested that the G→A substitution is not a common polymorphism and is likely to be the causative mutation for the phenotype in this family. Previous reported mutations of ferroportin, including A77D and V162del, which lead to type IV hemochromatosis, were characterized by increased serum ferritin despite normal transferrin saturation, in contrast to our patients' phenotype. These autosomal dominant mutants are postulated to lead to disease due to loss of iron exporting function. Preliminary in vivo assay using transient transfection of wild-type and ferroportin mutants in HeLa or 293T cells revealed, as expected, a loss of function and diminished surface membrane localisation in A77D and V162del mutants. Surprisingly, the C326Y mutant was indistinguishable from wt ferroportin in both iron status of the cell and protein localization suggesting different pathophysiology leading to iron overload in our patients.

The mortality risk of elevated serum transferrin saturation and consumption of dietary iron.

Recent data shows an increased mortality risk associated with elevated transferrin saturation. Because ingestion of dietary iron may contribute to iron overload in persons with elevated transferrin saturation, we investigated the relationship between elevated transferrin saturation, ingestion of dietary iron and red meat, and mortality. This 12-year cohort study used data from the second National Health and Nutrition Examination Survey 1976-1980 (NHANES II) and the NHANES II Mortality Study 1992. Population estimates were based on 9,229 persons aged 35 to 70 years at baseline. A Cox proportional hazards analysis was performed based on levels of transferrin saturation, intake of dietary iron, and intake of red meat. The analysis was conducted while controlling for demographics, severity of illness, body mass index, and smoking status. Unadjusted analyses indicated that those who had a high transferrin saturation and reported high dietary iron or red meat consumption had an increased mortality risk. The adjusted survival analysis indicated that persons with elevated transferrin saturation who reported high dietary iron intake had a hazard ratio for death of 2.90 (95% confidence interval [CI], 1.39-6.04) compared with those with normal transferrin saturation levels and reported low dietary iron intake. Persons who had a high transferrin saturation and reported high red meat consumption also had an increased hazard ratio for death (2.26; 95% CI, 1.45-3.52) compared with those who had normal transferrin saturation and reported low red meat consumption. Ingestion of large quantities of dietary iron and red meat in persons with high transferrin saturation is associated with an increase in mortality. Simple dietary restrictions may reduce the mortality risk associated with high transferrin saturation.

Autosomal dominant iron overload due to a novel mutation of ferroportin1 associated with parenchymal iron loading and cirrhosis

We report the identification of a novel mutation in ferroportin1 in an Australian family with autosomal dominant iron overload. The phenotype of iron overload in one member of this family is associated with high serum ferritin concentration and elevated transferrin saturation. The pattern of iron overload in the liver shows accumulation predominantly in parenchymal cells with some Kupffer cell iron loading. Although some cases of type 4 haemochromatosis have been associated with the development of liver fibrosis this is the first report of a patient with fully established cirrhosis at a relatively young age (32 years). The coexistence of sarcoidosis in this patient may contribute to the more severe phenotype. This report highlights the phenotypic variability that can occur in type 4 haemochromatosis. Some patients have predominant reticuloendothelial iron loading and normal transferrin saturation whereas others have predominant parenchymal iron loading and elevated transferrin saturation. The reasons for this variability remain to be determined. Interestingly this is the third mutation to affect asparagine 144, reinforcing the important role for this amino acid in the function of ferroportin1.

Natural history of the C282Y homozygote for the hemochromatosis gene ( HFE) with a normal serum ferritin level

Background & Aims : The discovery of a genetic test for hereditary hemochromatosis has identified many individuals who are homozygous for the C282Y mutation of the HFE gene with a normal transferrin saturation and serum ferritin level. The long-term prognosis, rate of iron accumulation, and surveillance guidelines for these individuals are unknown. Methods : To determine the degree of iron accumulation over time, an updated serum ferritin level was obtained in patients initially identified as homozygous for the C282Y mutation with a normal serum ferritin level. Results : Twenty-two asymptomatic untreated C282Y homozygotes with a normal serum ferritin level were identified, 10 through population screening, 9 through pedigree analysis, and 3 through a general medical work-up. There were 18 women (4 postmenopausal) and 4 men with a median age of 46 years, range 28–76 years. The median follow-up interval was 4 years, range 2–23 years. The serum ferritin levels of 20 of 22 patients remained below the upper limit of normal during the follow-up period. A decline in serum ferritin level was observed in 13 of 22 patients. Three patients had an increase in serum ferritin level of greater than 50%, with only 1 male patient exceeding the upper limit of normal by having a serum ferritin level increase from 295 to 344 μg/L during a 3-year period. Conclusions : In C282Y homozygotes with a normal ferritin level at the time of diagnosis, 20 of 22 patients failed to show any significant increase in serum ferritin level during a median follow-up of 4 years. This has clinical and economic implications for follow-up and surveillance of this selected population.

Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene☆

The product of the SLC40A1 gene, ferroportin 1, is a main iron export protein. Pathogenic mutations in ferroportin 1 lead to an autosomal dominant hereditary iron overload syndrome characterized by high serum ferritin concentration, normal transferrin saturation, iron accumulation predominantly in macrophages, and marginal anemia. Iron overload occurs in both the African and the African-American populations, but a possible genetic basis has not been established. We analyzed the ferroportin 1 gene in 19 unrelated patients from southern Africa ( N = 15) and the United States ( N = 4) presenting with primary iron overload. We found a new c. 744 C→T (Q248H) mutation in the SLC40A1 gene in 4 of these patients (3 Africans and 1 African-American). Among 22 first degree family members, 10 of whom were Q248H heterozygotes, the mutation was associated with a trend to higher serum ferritin to amino aspartate transferase ratios (means of 14.8 versus 4.3 μg/U; P = 0.1) and lower hemoglobin concentrations (means of 11.8 versus 13.2 g/dL; P = 0.1). The ratio corrects serum ferritin concentration for alcohol-induced hepatocellular damage. We also found heterozygosity for the Q248H mutation in 7 of 51 (14%) southern African community control participants selected because they had a serum ferritin concentration below 400 μg/L and in 5 of 100 (5%) anonymous African-Americans, but we did not find the change in 300 Caucasians with normal iron status and 25 Caucasians with non- HFE iron overload. The hemoglobin concentration was significantly lower in the African community controls with the Q248H mutation than in those without it. We conclude that the Q248H mutation is a common polymorphism in the ferroportin 1 gene in African populations that may be associated with mild anemia and a tendency to iron loading.