A newly identified ferritin L-subunit variant results in increased proteasomal subunit degradation, impaired complex assembly, and severe hypoferritinemia.

Ferritin is a hetero-oligomeric nanocage, composed of 24 subunits of two types, FTH1 and FTL. It protects the cell from excess reactive iron, by storing iron in its cavity. FTH1 is essential for the recruitment of iron into the ferritin nanocage and for cellular ferritin trafficking, whereas FTL contributes to nanocage stability and iron nucleation inside the cavity. Here we describe a female patient with a medical history of severe hypoferritinemia without anemia. Following inadequate heavy IV iron supplementation, the patient developed severe iron overload and musculoskeletal manifestations. However, her serum ferritin levels rose only to normal range. Genetic analyses revealed an undescribed homozygous variant of FTL (c.92A > G), which resulted in a Tyr31Cys substitution (FTLY31C ). Analysis of the FTL structure predicted that the Y31C mutation will reduce the variant's stability. Expression of the FTLY31C variant resulted in significantly lower cellular ferritin levels compared with the expression of wild-type FTL (FTLWT ). Proteasomal inhibition significantly increased the initial levels of FTLY31C , but could not protect FTLY31C subunits from successive degradation. Further, variant subunits successfully incorporated into hetero-polymeric nanocages in the presence of sufficient levels of FTH1. However, FTLY31C subunits poorly assembled into nanocages when FTH1 subunit levels were low. These results indicate an increased susceptibility of unassembled monomeric FTLY31C subunits to proteasomal degradation. The decreased cellular assembly of FTLY31C -rich nanocages may explain the low serum ferritin levels in this patient and emphasize the importance of a broader diagnostic approach of hypoferritinemia without anemia, before IV iron supplementation.

A case of inter-assay HbA1c discrepancy due to Hemoglobin G-Copenhagen.

BACKGROUND AND OBJECTIVE: A clinician was intrigued about HbA1c upper 9% (75 mmol/mol) in a 76 year-old women with normal glycemia. Further explorations were performed in order to understand this discordance. METHODS: First HbA1c test was performed on a HLC -723 G11 apparatus (Tosoh Bioscience) and thereafter compared to the HLC-723-G8 (Tosoh Bioscience), the Capillaris 3 Tera (Sebia) and the DCA Vantage point of care testing (POCT) (Siemens) apparatus. In addition, study of Hemoglobin (Hb) fraction and mutation analysis of HBB gene was realized due to the suspicion of an Hb variant. RESULTS: Twice high results of HbA1c (9.3%, 78 mmol/mol and 10%, 86 mmol/mol) on the HLC-723 G11 was not confirmed with other instruments. HbA1c result for the same sample was 5.2% (33 mmol/mol) for the HLC-723 G8, 5.3% (34 mmol/mol) for the Capillaris and 6.2% (44 mmol/mol) for the DCA Vantage POCT. The subject had normal glycemia and none signs of diabetes mellitus. An abnormal Hb fraction was visualized on the graphs for the HLC-723 G11 and Capillaris but not for the HLC-723 G8 analyzer. Study of Hb fraction confirmed the presence of an abnormal Hb fraction that was identifed as an Hb G-Copenhagen through mutation analysis of HBB gene. CONCLUSION: This case evidenced an interference on HbA1c test in presence of Hb G-Copenhagen depending to the analyzer used. This report help to alert of such possibility and to remain that a discordance between HbA1c and glycemia can be due to an Hb variant.

A simple clinical score to promote and enhance ferroportin disease screening.

BACKGROUND & AIMS: Ferroportin disease is a rare genetic iron overload disorder which may be underdiagnosed, with recent data suggesting it occurs at a higher prevalence than suspected. Costs and the lack of defined criteria to prompt genetic testing preclude large-scale molecular screening. Hence, we aimed to develop a readily available scoring system to promote and enhance ferroportin disease screening. METHODS: Our derivation cohort included probands tested for ferroportin disease from 2008 to 2016 in our rare disease network. Data were prospectively recorded. Univariate and multivariate logistic regression were used to determine significant criteria, and odds ratios were used to build a weighted score. A cut-off value was defined using a ROC curve with a predefined aim of 90% sensitivity. An independent cohort was used for cross validation. RESULTS: Our derivation cohort included 1,306 patients. Mean age was 55±14 years, ferritin 1,351±1,357 µg/L, and liver iron concentration (LIC) 166±77 µmol/g. Pathogenic variants (n = 32) were identified in 71 patients. In multivariate analysis: female sex, younger age, higher ferritin, higher LIC and the absence of hypertension or diabetes were significantly associated with the diagnosis of ferroportin disease (AUROC in whole derivation cohort 0.83 [0.78-0.88]). The weighted score was based on sex, age, the presence of hypertension or diabetes, ferritin level and LIC. An AUROC of 0.83 (0.77-0.88) was obtained in the derivation cohort without missing values. Using 9.5 as a cut-off, sensitivity was 93.6 (91.7-98.3) %, specificity 49.5 (45.5-53.6) %, positive likelihood ratio 1.8 (1.6-2.0) and negative likelihood ratio 0.17 (0.04-0.37). CONCLUSION: We describe a readily available score with simple criteria and good diagnostic performance that could be used to screen patients for ferroportin disease in routine clinical practice. LAY SUMMARY: Increased iron burden associated with metabolic syndrome is a very common condition. Ferroportin disease is a dominant genetic iron overload disorder whose prevalence is higher than initially thought. They can be difficult to distinguish from each other, but the limited availability of genetic testing and the lack of definitive guidelines prevent adequate screening. We herein describe a simple and definitive clinical score to help clinicians decide whether to perform genetic testing.

GGCX-related congenital combined vitamin K-dependent clotting factors deficiency-1: Description of a fetus with chondrodysplasia punctata.

Congenital combined vitamin K-dependent clotting factors deficiency (VKCFD) is a rare autosomal recessive disease resulting in hemorrhagic symptoms usually associated with developmental disorders and bone abnormalities. Pathogenic variants in two genes encoding enzymes of the vitamin K cycle, GGCX and VKORC1, can lead to this disorder. We present the case of a male fetus with a brachytelephalangic chondrodysplasia punctata (CDP), absence of nasal bone, growth restriction, and bilateral ventriculomegaly at 18 weeks of gestation. Pathological examination showed a Binder phenotype, hypoplastic distal phalanges, stippled epiphyses, and brain abnormalities suggestive of a brain hemorrhage. Two GGCX pathogenic variants inherited respectively from the mother and the father were identified. To our knowledge, this is the first prenatal description of VKCFD. Even if it remains a rare etiology, which is mostly described in children or adult patients, VKCFD should be considered in fetuses with CDP.

Hepcidin: immunoanalytic characteristics.

Hepcidin has progressively become essential in clinical practice for the diagnosis and follow-up of a large spectrum of diseases. Anyway, its own biochemical and structural characteristics have complicated and delayed the acquisition of a standardized quantifying tool of the peptide.

Inherited or acquired modifiers of iron status may dramatically affect the phenotype in dehydrated hereditary stomatocytosis.

Severe iron overload is frequent in dehydrated hereditary stomatocytosis (DHSt) despite well-compensated hemolysis and no or little transfusion requirement. We investigated 4 patients with proven DHSt, in whom the degree of hemolysis was closely related to iron status. Genetic modifiers increasing iron stores (HFE:pCys282Tyr, HAMP:c-153C>T mutations) were accompanied with high liver iron concentrations and increased hemolysis, whereas therapeutic phlebotomies alleviated the hemolytic phenotype. There were no manifestations of hemolysis in one patient with low iron stores. Hemolysis reappeared when iron supplementation was given. The search for genetic or acquired modifiers of iron status and the modulation of iron stores may help in the management of these patients.

Added value of hepcidin quantification for the diagnosis and follow-up of anemia-related diseases.

Iron homeostasis is based on a strict control of both intestinal iron absorption and iron recycling through reticulo-endothelial system. Hepcidin controls the iron fluxes in order to maintain sufficient iron levels for erythropoietic activities, hemoproteins synthesis or enzymes function, but also to limit its toxic accumulation throughout the body. Hepcidin expression is regulated by various stimuli: inflammation and iron stimulate the production of the peptide, while anemia, erythropoiesis and hypoxia repress its production. Regulation of hepcidin expression is not so simple in complex pathological situations such as hemolytic anemia, cancer or chronic inflammation. Serum hepcidin quantification in association with the diagnostic tests currently available is quite promising for the diagnosis or the follow-up of anemia in those conditions. This study is part of the working group « Clinical interests of hepcidin quantification ¼ of the Société française de biologie clinique.

The role of genetic factors in patients with hepatocellular carcinoma and iron overload - a prospective series of 234 patients.

BACKGROUND & AIMS: Iron overload (IO) in HFE-related hereditary haemochromatosis is associated with increased risk of liver cancer. This study aimed to investigate the role of other genes involved in hereditary IO among patients with hepatocellular carcinoma (HCC). METHODS: Patients with HCC diagnosed in our institution were included in this prospective study. Those with ferritin levels ≥300 µg/L (males) or ≥200 µg/L (females) and/or transferrin saturation ≥50% (males) or ≥45% (females) had liver iron concentration (LIC) evaluated by MRI. HFE C282Y and H63D mutations were screened. Genetic analyses of genes involved in hereditary IO (HFE, HJV/HFE2, HAMP, TFR2, SLC40A1, GNPAT) were performed in patients with increased LIC. RESULTS: A total of 234 patients were included; 215 (92%) had common acquired risk factors of HCC (mainly alcoholism or chronic viral hepatitis). 119 patients had abnormal iron parameters. Twelve (5.1%) were C282Y homozygotes, three were compound C282Y/H63D heterozygotes. LIC was measured by MRI in 100 patients. Thirteen patients with a LIC>70 µmol/g were enrolled in further genetic analyses: two unrelated patients bore the HAMP:c.-153C>T mutation at the heterozygous state, which is associated with increased risk of IO and severe haemochromatosis. Specific haplotypes of SLC40A1 were also studied. CONCLUSIONS: Additional genetic risk factors of IO were found in 18 patients (7.7%) among a large series of 234 HCC patients. Screening for IO and the associated at-risk genotypes in patients who have developed HCC, is useful for both determining etiologic diagnosis and enabling family screening and possibly primary prevention in relatives.

Iron overload in HFE C282Y heterozygotes at first genetic testing: a strategy for identifying rare HFE variants.

BACKGROUND: Heterozygotes for the p.Cys282Tyr (C282Y) mutation of the HFE gene do not usually express a hemochromatosis phenotype. Apart from the compound heterozygous state for C282Y and the widespread p.His63Asp (H63D) variant allele, other rare HFE mutations can be found in trans on chromosome 6. DESIGN AND METHODS: We performed molecular investigation of the genes implicated in hereditary hemochromatosis in six patients who presented with iron overload but were simple heterozygotes for the HFE C282Y mutation at first genetic testing. Functional impairment of new variants was deduced from computational methods including molecular modeling studies. RESULTS: We identified four rare HFE mutant alleles, three of which have not been previously described. One mutation is a 13-nucleotide deletion in exon 6 (c.1022_1034del13, p.His341_Ala345 > LeufsX119), which is predicted to lead to an elongated and unstable protein. The second one is a substitution of the last nucleotide of exon 2 (c.340G > A, p.Glu114Lys) which modifies the relative solvent accessibility in a loop interface. The third mutation, p.Arg67Cys, also lies in exon 2 and introduces a destabilization of the secondary structure within a loop of the α1 domain. We also found the previously reported c.548T > C (p.Leu183Pro) missense mutation in exon 3. No other known iron genes were mutated. We present an algorithm at the clinical and genetic levels for identifying patients deserving further investigation. Conclusions Our results suggest that additional mutations in HFE may have a clinical impact in C282Y carriers. In conjunction with results from previously described cases we conclude that an elevated transferrin saturation level and elevated hepatic iron index should indicate the utility of searching for further HFE mutations in C282Y heterozygotes prior to other iron gene studies.

The Southern French registry of genetic hemochromatosis: a tool for determining clinical prevalence of the disorder and genotype penetrance.

BACKGROUND: Despite great progress in understanding the mechanisms underlying genetic hemochromatosis, data on the prevalence and the penetrance of the disorder are conflicting. DESIGN AND METHODS: A registry of patients with genetic hemochromatosis was established in the South of France and a regional health network was developed to allow the inclusion of all the diagnosed patients. C282Y homozygous patients classified in stages 2 (biological iron overload), 3 and 4 (clinical manifestations of iron overload, stage 4 being the more severe) according to the classification of the French National Authority for Health were included in the registry over a 6-year period. RESULTS: A total of 352 symptomatic C282Y homozygotes were identified, resulting in a total prevalence of 1.83 per 10,000 (95% CI: 1.63 to 2.02) in subjects over 20 years and 2.40 per 10,000 (95% CI, 2.15 to 2.65) among subjects of European descent. Among Europeans, the total calculated penetrance was 15.8% in stage 2 or higher, 12.1% in stage 3 or 4 and 2.9% in stage 4. The penetrance was slightly higher in males (18.7%) than in females (13.2%). It was 19.9% for individuals over 40 years of age (24.1% and 16.3% in males and females, respectively) with a maximum of 31% in subjects between 50 and 54 years old. Among 249 patients with complete records, 24% were in stage 2, the majority (58%) were in stage 3, and 18% in stage 4. There was a higher proportion of males, and excessive alcohol intake was more prevalent in stage 4 than in stages 2 and 3 combined. CONCLUSIONS: A French Mediterranean regional hemochromatosis registry with strict inclusion criteria is a useful tool for characterizing the history of this disease, particularly for the most severely affected patients, as defined by the disease severity classification. The total prevalence of symptomatic C282Y homozygotes in the region was found to be low. However, clinical penetrance (stages 3 and 4) was not negligible.

Global sequencing approach for characterizing the molecular background of hereditary iron disorders.

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

A novel mutation of the beta-globin gene promoter (-102 C>A) and pitfalls in family screening.

We describe a family with beta-thalassemia in which several pitfalls of genetic diagnoses were present. These include coherent family phenotypes with discrepancies in molecular findings because of nonpaternity, and a false beta-globin gene homozygous genotype due to a large deletion in the second locus. These findings underline the difficulties of family genetic studies and the need for tight relationship between professionals involved in laboratory studies and those in-charge of the clinical follow-up and genetic counselling. In this family, we also report a new silent beta-thalassemia mutation, -102 (C>A), in the distal CACCC box of the beta-globin gene promoter.

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

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