Predicting C282Y homozygote genotype for hemochromatosis using serum ferritin and transferrin saturation values from 44,809 participants of the HEIRS Study

INTRODUCTION:

The simultaneous interpretation of serum ferritin level and transferrin saturation has been used as a clinical guide to diagnose genetic hemochromatosis. The Hemochromatosis and Iron Overload Screening (HEIRS) Study screened 101,168 North American participants for serum ferritin level and transferrin saturation, and C282Y genotyping for the HFE gene.

METHODS:

Logistic regression involving a subsample of Caucasians (n=44,809) was used to predict individual probabilities of HFE C282Y homozygosity using serum ferritin and transferrin saturation values. Men (n=17,323) and women (n=27,486) were analyzed separately. Regression equations were evaluated using area under the curve from ROC analysis and variance explained by Nagelkerke’s pseudo R-squared. An Android smartphone App and website application were developed to provide physicians with easy access to predicting C282Y homozygosity of the HFE gene.

RESULTS:

The logistic equation had an area under the ROC curve of 0.91 for men and 0.89 for women. The pseudo R-squared was 0.44 for men and 0.34 for women. An example analysis was a Caucasian man with a transferrin saturation of 50% and a ferritin level of 500 µg/L, who had a 1.3% (95% CI 1.1% to 8.8%) probability of being a C282Y homozygote.

CONCLUSIONS:

A large primary care-based sample of 44,809 participants contributed to the development of a new computer/smartphone tool that predicts the probability of being a C282Y homozygote of the HFE gene from serum ferritin and transferrin saturation values.     

Dietary iron intake and serum ferritin concentration in 213 patients homozygous for the HFEC282Y hemochromatosis mutation

BACKGROUND:

HFE^C282Y homozygotes have an increased risk for developing increased iron stores and related disorders. It is controversial whether dietary iron restrictions should be recommended to such individuals.

OBJECTIVE:

To determine whether dietary iron content influences iron stores in HFE^C282Y homozygotes as assessed by serum ferritin concentration.

DESIGN:

Serum ferritin concentration was measured and a dietary iron questionnaire was completed as part of the evaluation of 213 HFE^C282Y homozygotes who were identified through screening of >100,000 primary care patients at five HEmochromatosis and IRon Overload Screening (HEIRS) Study Field Centers in the United States and Canada.

RESULTS:

No significant relationships between serum ferritin concentration and dietary heme iron content, dietary nonheme iron content or reports of supplemental iron use were found.

CONCLUSION:

These results do not support recommending dietary heme or nonheme iron restrictions for HFEC^282Y homozygotes diagnosed through screening in North America.     

HFE mutations in Caucasian participants of the Hemochromatosis and Iron Overload Screening study with serum ferritin level <1000 μg/L

BACKGROUND:

Many patients referred for an elevated serum ferritin level <1000 μg/L are advised that they likely have iron overload and hemochromatosis.

AIMS:

To determine the prevalence of HFE mutations in the hemochromatosis gene for 11 serum ferritin concentration intervals from 200 μg/L to 1000 μg/L in Caucasian participants in a primary care, population-based study.

METHODS:

The Hemochromatosis and Iron Overload Screening study screened 99,711 participants for serum ferritin levels, transferrin saturation and genetic testing for the C282Y and H63D mutations of the HFE gene. This analysis was confined to 17,160 male and 27,465 female Caucasian participants because the HFE C282Y mutation is rare in other races. Post-test likelihood was calculated for prediction of C282Y homozygosity from a ferritin interval. A subgroup analysis was performed in participants with both an elevated serum ferritin level and transferrin saturation.

RESULTS:

There were 3359 male and 2416 female participants with an elevated serum ferritin level (200 μg/L to 1000 μg/L for women, 300 μg/L to 1000 μg/L for men). There were 69 male (2.1%) and 87 female (3.6%) C282Y homozygotes, and the probability of being a homozygote increased as the ferritin level increased. Post-test likelihood values were 0.3% to 16% in men and 0.3% to 30.4% in women.

CONCLUSIONS:

Iron loading HFE mutations are unlikely to be the most common cause of an elevated serum ferritin level in patients with mild hyperferritinemia. Patients should be advised that there are many causes of an elevated serum ferritin level including iron overload.     

Screening for iron overload: Lessons from the HEmochromatosis and IRon Overload Screening (HEIRS) Study

BACKGROUND:

The HEmochromatosis and IRon Overload Screening (HEIRS) Study provided data on a racially, ethnically and geographically diverse cohort of participants in North America screened from primary care populations.

METHODS:

A total of 101,168 participants were screened by testing for HFE C282Y and H63D mutations, and measuring serum ferritin concentration and transferrin saturation. In the present review, lessons from the HEIRS Study are highlighted in the context of the principles of screening for a medical disease as previously outlined by the World Health Organization.

RESULTS:

Genetic testing is well accepted, with minimal risk of discrimination. Transferrin saturation has high biological variability and relatively low sensitivity to detect HFE C282Y homozygotes, which limits its role as a screening test. Symptoms attributable to HFE C282Y homozygosity are no more common in individuals identified by population screening than in control subjects.

CONCLUSIONS:

Generalized population screening in a primary care population as performed in the HEIRS Study is not recommended. There may be a role for focused screening in Caucasian men, with some debate regarding genotyping followed by phenotyping, or phenotyping followed by genotyping.     

Liver iron concentration in dysmetabolic hyperferritinemia: Results from a prospective cohort of 276 patients

Introduction and objectivesWe aimed to study the liver iron concentration in patients referred for hyperferritinemia to six hospitals in the Basque Country and to determine if there were differences between patients with or without metabolic syndrome.Patients and methodsMetabolic syndrome was defined by accepted criteria. Liver iron concentration was determined by magnetic resonance imaging.ResultsWe obtained the data needed to diagnose metabolic syndrome in 276 patients; a total of 135 patients (49%), 115/240 men (48%), and 20/36 women (55.6%) presented metabolic syndrome. In all 276 patients, an MRI for the determination of liver iron concentration (mean ± SD) was performed. The mean liver iron concentration was 30.83 ± 19.38 for women with metabolic syndrome, 38.84 ± 25.50 for men with metabolic syndrome, and 37.66 ± 24.79 (CI 95%; 33.44–41.88) for the whole metabolic syndrome group. In 141 patients (51%), metabolic syndrome was not diagnosed: 125/240 were men (52%) and 16/36 were women (44.4%). The mean liver iron concentration was 34.88 ± 16.18 for women without metabolic syndrome, 44.48 ± 38.16 for men without metabolic syndrome, and 43.39 ± 36.43 (CI 95%, 37.32–49.46) for the whole non-metabolic syndrome group. Comparison of the mean liver iron concentration from both groups (metabolic syndrome vs non-metabolic syndrome) revealed no significant differences (p = 0.12).ConclusionsPatients with hyperferritinemia and metabolic syndrome presented a mildly increased mean liver iron concentration that was not significantly different to that of patients with hyperferritinemia and non-metabolic syndrome.     

The global burden of iron overload

There have been major developments in the field of iron metabolism in the past decade following the identification of the HFE gene and the mutation responsible for the C282Y substitution in the HFE protein. While HFE-associated hemochromatosis occurs predominantly in people of northern European extraction, other less-common mutations can lead to the same clinical syndrome and these may occur in other populations in the Asian-Pacific region. The most common of these is the mutation that leads to changes in the ferroportin molecule, the protein responsible for the transport of iron across the basolateral membrane of the enterocyte and from macrophages. Recent research has unraveled the molecular processes of iron transport and regulation of how these are disturbed in hemochromatosis and other iron-loading disorders. At the same time, at least one new oral iron chelating agent has been developed that shows promise in the therapy of hemochromatosis as well as thalassemia and other secondary causes of iron overload. It is pertinent therefore to examine the developments in the global field of iron overload that have provided insights into the pathogenesis, disease penetrance, comorbid factors, and management.     

Haemochromatosis revisited

Haemochromatosis is a genetic disease caused by hepcidin deficiency, responsible for an increase in intestinal iron absorption. Haemochromatosis is associated with homozygosity for the HFE p.Cys282Tyr mutation. However, rare cases of haemochromatosis (non-HFE haemochromatosis) can also be caused by pathogenic variants in other genes (such as HJV, HAMP, TFR2 and SLC40A1). A working group of the International Society for the Study of Iron in Biology and Medicine (BIOIRON Society) has concluded that the classification based in different molecular subtypes is difficult to be adopted in clinical practice and has proposed a new classification approaching clinical questions and molecular complexity. The aim of the present review is to provide an update on classification, pathophysiology and therapeutic recommendations.     

Men with hyperferritinemia and diabetes in the Mediterranean area do not have a higher iron overload than those without diabetes

Aim

To assess the role of iron overload in type 2 diabetic men with hyperferritinemia.

Methods

150 men were recruited from a genetic screening programme for hereditary hemocromatosis (HH) and were tested for type 2 diabetes, other components of the metabolic syndrome, beta cell function (BCF), insulin sensitivity, high-sensitivity C-reactive protein and iron overload.

Results

Fifty-one men had type 2 diabetes. They were older (p = 0.017) and 99 had lower BCF (p < 0.001) than non-diabetic men. None of the iron overload indexes was associated with diabetes.

Conclusions

Our findings dispute a role of iron overload in the pathogenesis of type 2 diabetes.     

Dysmetabolic hyperferritinemia is associated with normal transferrin saturation,mild hepatic iron overload,and elevated hepcidin

Hyperferritinemia is common in individuals with the metabolic syndrome (dysmetabolic hyperferritinemia), but its pathophysiology and the degree to which it reflects tissue iron overload remains unclear. We conducted a cross-sectional study evaluating ten cases with dysmetabolic hyperferritinemia for liver iron overload and compared their serum iron indices and urine hepcidin levels to healthy controls. Seven out of ten cases had mild hepatic iron overload by magnetic resonance imaging (MRI) (median, 75 μmol/g dry weight). Cases had higher serum ferritin than controls (median, 672 μg/L vs. 105 μg/L, p < 0.001), but the median transferrin saturation was not significantly different (38% vs. 36%, p = 0.5). Urinary hepcidin was elevated in dysmetabolic hyperferritinemia (median; 1,584 ng/mg of creatinine vs. 799 ng/mg of creatinine, p = 0.05). Dysmetabolic hyperferritinemia is characterized by hyperferritinemia with normal transferrin saturation, elevated hepcidin levels, and mild liver iron overload in a subset of patients.     

Multiple Hepatic Abscesses Due to Yersinia enterocolitica Infection Secondary to Primary Haemochromatosis

A case of hepatic abscesses due to Yersinia enterocolitica in an immunocompetent male is presented. Reexamination after 3 months showed that the patient had primary haemochromatosis. Treatment with repeated phlebotomies was instituted. Two years after the patient was first admitted to hospital, 17.2 g iron had been removed and all haematological and biochemical parameters had returned to normal. Genetic analysis of the patients' two sons showed that one was positive for the chromosome defect found in primary haemochromatosis; further investigation is under progress. A study of the literature showed that prior to this case only 45 cases of hepatic abscess secondary to Yersinia enterocolitica have been registered. Of the 45 reported cases, 64% had underlying haemochromatosis and 29% had diabetes mellitus. The overall mortality was 31%. Mortality before 1987 was 60% (n = 20) and since 1987 it has been 8% (n = 25).     

Familial iron overload with possible autosomal dominant inheritance

A 96 member Melanesian kindred with 31 cases of iron overload is reported. Liver biopsies from 19 of these patients showed features similar to those of genetic haemochromatosis in Caucasians, but in contrast to the previous reported HLA-linked autosomal recessive pattern of inheritance for haemochromatosis, this family shows a pattern that is most consistent with autosomal dominant inheritance. This is suggested by involvement of three and possibly four consecutive generations, with a high frequency of transmission from parents to children and equal gender distribution. Linkage and segregation analysis supported dominant inheritance, with no demonstrable HLA linkage.     

A Japanese family with ferroportin disease caused by a novel mutation of SLC40A1 gene: hyperferritinemia associated with a relatively low transferrin saturation of iron

Ferroportin disease, autosomal-dominant reticuloendothelial iron overload, may be more prevalent than hemochromatosis in Japan. Hyperferritinemia of 822 ng/ml with 24.8% transferrin saturation of iron was incidentally noted in a 43-year-old man. His iron overload was selective in Kupffer cells of the liver. Subsequently, his father was found to have asymptomatic hyperferritinemia of 2,283 ng/ml with 62.1% saturation. These affected subjects were heterozygous for 1467A>C (R489S) in SLC40A1, and without other mutations of the hemochromatosis genes. Here, we report a Japanese family with ferroportin disease, characterized by hyperferritinemia with relatively low transferrin saturations of iron.     

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.     

Serum transferrin receptors: Distribution and diagnostic performance in pre-school children

Soluble transferrin receptors have gained interest in the field of diagnosing anemias. Reference ranges differ according to the method used for the quantification of sTfR. We aim to explore the distributional properties and diagnostic performance of sTfR in pre-school healthy children as well as in children with β-thalassemia carriers, iron deficiency with normal hematological phenotype (ID) and iron deficiency anemia (IDA). Circulating sTfR as well as biochemical and hematological indices were determined in 521 pre-school children and four groups (normal children, β-thalassemia traits, ID and IDA) were formed. Diagnostic performance and distribution of sTfR according to age and in relation to several parameters were evaluated in every group. Three hundred eighty one children (261 normal, 60 β-thalassemia traits, 44 ID and 16 IDA) aged 1–6 years were included. We found that distribution of sTfR differed significantly among the four groups (Kruskal Wallis p < 0.001) with children in the normal group exhibiting lower concentrations compared to all other. A negative correlation between sTfR and age occurred in the normal (β = − 0.12, p < 0.001) and the ID groups (β = − 0.13, p = 0.035). In the β-thal and IDA groups sTfR is correlated to HbA₂ (β = 0.34, p = 0.001) and ferritin (Spearman's rho = − 0.6, p = 0.014) respectively. An area under the curve equal to 0.63 was achieved by sTfR in distinguishing between normal and ID children. Sensitivity and specificity were 70.5% and 50% respectively at a cut-off of 2.5 mg/l. Levels of sTfR are negatively correlated to age in pre-school children while dyserythropoietic procedures like β-thal, ID, and IDA significantly affect them. These findings indicated that the accuracy of sTfR in diagnosing ID from normal children is limited. Standardization will allow the use of formulas that combine sTfR and ferritin which are of greater diagnostic value than sTfR alone.     

Non-HFE haemochromatosis

Non-HFF hereditary haemochromatosis (HH) refers to a genetically heterogeneous group of iron overload disorders that are unlinked to mutations in the HFF gene. The four main types of non-HFE HH are caused by mutations in the hemojuvelin, hepcidin,transferrin receptor 2 and ferroportin genes. Juvenile haemochromatosis is an autosomal recessive disorder and can be caused by mutations in either hemojuvelin or hepcidin. An adult onset form of HH similar to HFE-HH is caused by homozygosity for mutations in transferrin receptor 2. The autosomal dominant iron overload disorder ferroportin disease is caused by mutations in the iron exporter ferroportin. The clinical characteristics and molecular basis of the various types of non-HFE haemochromatosis are reviewed. The study of these disorders and the molecules involved has been invaluable in improving our understanding of the mechanisms involved in the regulation of iron metabolism.     

Analyses for binding of the transferrin family of proteins to the transferrin receptor 2

Transferrin receptor 2 alpha (TfR2 alpha), the major product of the TfR2 gene, is the second receptor for transferrin (Tf), which can mediate cellular iron uptake in vitro. Homozygous mutations of TfR2 cause haemochromatosis, suggesting that TfR2 alpha may not be a simple iron transporter, but a regulator of iron by identifying iron-Tf. In this study, we analysed the ligand specificity of TfR2 alpha using human transferrin receptor 1 (TfR1) and TfR2 alpha-stably transfected and expressing cells and flow-cytometric techniques. We showed that human TfR2 alpha interacted with both human and bovine Tf, whereas human TfR1 interacted only with human Tf. Neither human TfR1 nor TfR2 alpha interacted with either lactoferrin or melanotransferrin. In addition, by creating point mutations in human TfR2 alpha, the RGD sequence in the extracellular domain of TfR2 alpha was shown to be crucial for Tf-binding. Furthermore, we demonstrated that mutated TfR2 alpha (Y250X), which has been reported in patients with hereditary haemochromatosis, also lost its ability to interact with both human and bovine Tf. Although human TfR1 and TfR2 alpha share an essential structure (RGD) for ligand-binding, they have clearly different ligand specificities, which may be related to the differences in their roles in iron metabolism.     

Estimating Iron Overload in Patients with Suspected Liver Disease and Elevated Serum Ferritin

BackgroundIron status evaluation in patients with suspected liver disease and elevated serum ferritin is often challenging because hyperferritinemia does not always indicate iron overload. A reliable approach to estimate iron overload without exposing the patient to unnecessary investigations would help the clinician to identify patients who may take advantage of iron-removal therapy.MethodsWe analyzed all liver biopsies, including measurement of hepatic iron concentration, performed at the University Hospital Zurich from 1997 to 2010 to identify clinical and laboratory predictors of iron overload in patients with elevated serum ferritin (n = 147).ResultsHyperferritinemia was predictive of iron overload only in patients with a high level of serum ferritin (>2000 μg/L). In patients with moderate hyperferritinemia, liver transaminases inversely correlated with hepatic iron concentration. A combination of both parameters expressed as ferritin/aspartate transaminase ratio was highly predictive of tissue iron overload (sensitivity 83.3%, specificity 78.6%). Receiver operating characteristic analysis resulted in an area under the curve of 0.83.ConclusionsWe established a simple and reliable method to correctly estimate iron overload in patients with suspected liver disease and elevated serum ferritin.