Haemochromatosis in children: A national retrospective cohort promoted by the A.I.E.O.P. (Associazione Italiana Emato-Oncologia Pediatrica) study group.

Haemochromatosis (HC) encompasses a range of genetic disorders. HFE-HC is by far the most common in adults, while non-HFE types are rare due to mutations of HJV, HAMP, TFR2 and gain-of-function mutations of SLC40A1. HC is often unknown to paediatricians as it is usually asymptomatic in childhood. We report clinical and biochemical data from 24 paediatric cases of HC (10 cases of HFE-, 5 TFR2-, 9 HJV-HC), with a median follow-up of 9.6 years. Unlike in the adult population, non-HFE-HC constitutes 58% (14/24) of the population in our series. Transferrin saturation was significantly higher in TFR2- and HJV-HC compared to HFE-HC, and serum ferritin and LIC were higher in HJV-HC compared to TFR2- and HFE-HC. Most HFE-HC subjects had relatively low ferritin and LIC at the time of diagnosis, so therapy could be postponed for most of them after the age of 18. Our results confirm that HJV-HC is a severe form already in childhood, emphasizing the importance of early diagnosis and treatment to avoid the development of organ damage and reduce morbidity and mortality. Although phlebotomies were tolerated by most patients, oral iron chelators could be a valid option in early-onset HC.

Long-term outcome of a cohort of Italian patients affected with alpha-Mannosidosis.

Alpha-mannosidosis (MIM #248500) is an ultra-rare autosomal recessive lysosomal storage disease with multi-system involvement and a wide phenotypic spectrum. Information on long-term outcomes remains poor. We present the long-term outcomes (median, 19 years) of nine patients with alpha-mannosidosis, three females and six males, followed at a single center. The findings of the nine patients were collected from medical records and reported as mean ± SD or median, and range. The age of onset of the first symptoms ranged from 0-1 to 10 years. The diagnostic delay ranged from 2 to 22 years (median= 11 years). Coarse face, hearing, heart valves, joints, gait, language, dysarthria, psychiatric symptoms, I.Q., MRI, walking disabilities, orthopedic disturbances and surgeries showed a slow worsening over the decades. Our patients showed a slowly worsening progressive outcome over the decades. Psychiatric symptoms were present in 100% of our population and improved with the appropriate pharmacological intervention. This aspect requires attention when following up on these patients. Our description of the long-term evolution of alpha-mannosidosis patients may provide basic knowledge for understanding the effects of specific treatments.

A form of inherited hyperferritinemia associated with bi-allelic pathogenic variants of STAB1.

Hyperferritinemia is a frequent finding in several conditions, both genetic and acquired. We previously studied eleven healthy subjects from eight different families presenting with unexplained hyperferritinemia. Their findings suggested the existence of an autosomal-recessive disorder. We carried out whole-exome sequencing to detect the genetic cause of hyperferritinemia. Immunohistochemistry and flow cytometry assays were performed on liver biopsies and monocyte-macrophages to confirm the pathogenic role of the identified candidate variants. Through a combined approach of whole-exome sequencing and homozygosity mapping, we found bi-allelic STAB1 variants in ten subjects from seven families. STAB1 encodes the multifunctional scavenger receptor stabilin-1. Immunohistochemistry and flow cytometry analyses showed absent or markedly reduced stabilin-1 in liver samples, monocytes, and monocyte-derived macrophages. Our findings show a strong association between otherwise unexplained hyperferritinemia and bi-allelic STAB1 mutations suggesting the existence of another genetic cause of hyperferritinemia without iron overload and an unexpected function of stabilin-1 in ferritin metabolism.

Distribution of Exonic Variants in Glycogen Synthesis and Catabolism Genes in Late Onset Pompe Disease (LOPD).

Pompe disease (PD) is a monogenic autosomal recessive disorder caused by biallelic pathogenic variants of the GAA gene encoding lysosomal alpha-glucosidase; its loss causes glycogen storage in lysosomes, mainly in the muscular tissue. The genotype-phenotype correlation has been extensively discussed, and caution is recommended when interpreting the clinical significance of any mutation in a single patient. As there is no evidence that environmental factors can modulate the phenotype, the observed clinical variability in PD suggests that genetic variants other than pathogenic GAA mutations influence the mechanisms of muscle damage/repair and the overall clinical picture. Genes encoding proteins involved in glycogen synthesis and catabolism may represent excellent candidates as phenotypic modifiers of PD. The genes analyzed for glycogen synthesis included UGP2, glycogenin (GYG1-muscle, GYG2, and other tissues), glycogen synthase (GYS1-muscle and GYS2-liver), GBE1, EPM2A, NHLRC1, GSK3A, and GSK3B. The only enzyme involved in glycogen catabolism in lysosomes is α-glucosidase, which is encoded by GAA, while two cytoplasmic enzymes, phosphorylase (PYGB-brain, PGL-liver, and PYGM-muscle) and glycogen debranching (AGL) are needed to obtain glucose 1-phosphate or free glucose. Here, we report the potentially relevant variants in genes related to glycogen synthesis and catabolism, identified by whole exome sequencing in a group of 30 patients with late-onset Pompe disease (LOPD). In our exploratory analysis, we observed a reduced number of variants in the genes expressed in muscles versus the genes expressed in other tissues, but we did not find a single variant that strongly affected the phenotype. From our work, it also appears that the current clinical scores used in LOPD do not describe muscle impairment with enough qualitative/quantitative details to correlate it with genes that, even with a slightly reduced function due to genetic variants, impact the phenotype.

Hereditary Hyperferritinemia.

Ferritin is a ubiquitous protein that is present in most tissues as a cytosolic protein. The major and common role of ferritin is to bind Fe2+, oxidize it and sequester it in a safe form in the cell, and to release iron according to cellular needs. Ferritin is also present at a considerably low proportion in normal mammalian sera and is relatively iron poor compared to tissues. Serum ferritin might provide a useful and convenient method of assessing the status of iron storage, and its measurement has become a routine laboratory test. However, many additional factors, including inflammation, infection, metabolic abnormalities, and malignancy-all of which may elevate serum ferritin-complicate interpretation of this value. Despite this long history of clinical use, fundamental aspects of the biology of serum ferritin are still unclear. According to the high number of factors involved in regulation of ferritin synthesis, secretion, and uptake, and in its central role in iron metabolism, hyperferritinemia is a relatively common finding in clinical practice and is found in a large spectrum of conditions, both genetic and acquired, associated or not with iron overload. The diagnostic strategy to reveal the cause of hyperferritinemia includes family and personal medical history, biochemical and genetic tests, and evaluation of liver iron by direct or indirect methods. This review is focused on the forms of inherited hyperferritinemia with or without iron overload presenting with normal transferrin saturation, as well as a step-by-step approach to distinguish these forms to the acquired forms, common and rare, of isolated hyperferritinemia.

Possible Use of Minocycline in Adjunction to Intranasal Esketamine for the Management of Difficult to Treat Depression following Extensive Pharmacogenomic Testing: Two Case Reports.

The advent of intra-nasal esketamine (ESK), one of the first so called fast-acting antidepressant, promises to revolutionize the management of treatment resistant depression (TRD). This NMDA receptor antagonist has proven to be rapidly effective in the short- and medium-term course of the illness, revealing its potential in targeting response in TRD. Although many TRD ESK responders are able to achieve remission, a considerable portion of them undergo a metamorphosis of their depression into different clinical presentations, characterized by instable responses and high recurrence rates that can be considered closer to the concept of Difficult to Treat Depression (DTD) than to TRD. The management of these DTD patients usually requires a further complex multidisciplinary approach and can benefit from the valuable contribution of new personalized medicine tools such as therapeutic drug monitoring and pharmacogenetics. Despite this, these patients usually come with long and complex previous treatments history and, often, advanced and sophisticated ongoing pharmacological schemes that can make the finding of new alternative options to face the current recurrences extremely challenging. In this paper, we describe two DTD patients-already receiving intranasal ESK but showing an instable course-who were clinically stabilized by the association with minocycline, a semisynthetic second-generation tetracycline with known and promising antidepressant properties.

Intravenous iron therapy improves the hypercapnic ventilatory response and sleep disordered breathing in chronic heart failure.

AIMS: Intravenous iron therapy can improve symptoms in patients with heart failure, anaemia and iron deficiency. The mechanisms underlying such an improvement might involve chemoreflex sensing and nocturnal breathing patterns. METHODS AND RESULTS: Patients with heart failure, reduced left ventricular ejection fraction, anaemia (haemoglobin <13 g/dl in men; <12 g/dl in women) and iron deficiency (ferritin <100 or 100-299 µg/L with transferrin saturation <20%) were 2:1 randomized to patient-tailored intravenous ferric carboxymaltose dose or placebo. Chemoreflex sensitivity cardiorespiratory sleep study, symptom assessment and cardiopulmonary exercise test were performed before and 2 weeks after the last treatment dose. Fifty-eight patients (38 active arm/20 placebo arm) completed the study. Intravenous iron was associated with less severe symptoms, higher haemoglobin (12.5 ± 1.4 vs. 11.7 ± 1.0 mg/dl, p < 0.05) and improved haematinic parameters. Ferric carboxymaltose improved the central hypercapnic ventilatory response (-25.8%, p < 0.05 vs. placebo), without changes in peripheral chemosensitivity. In particular, the central hypercapnic ventilatory responses passed from 4.6 ± 6.5 to 2.9 ± 2.9 L/min/mmHg after ferric carboxymaltose and from 4.4 ± 4.6 to 4.6 ± 3.9 L/min/mmHg after placebo (ptreatment*condition  = 0.046). In patients presenting with sleep-related breathing disorder, apnoea-hypopnoea index was reduced with active treatment as compared to placebo (12 ± 11 vs. 19 ± 13 events/h, p < 0.05). After ferric carboxymaltose, but not after placebo, both peak oxygen uptake (VO2 ) increased (Δ1.1 ± 2.0 ml/kg/min, p < 0.05) and VO2 /workload slope was steeper (Δ0.67 ± 1.7 L/min/W, p < 0.01). CONCLUSIONS: Intravenous ferric carboxymaltose improves the hypercapnic ventilatory response and sleep-related breathing disorders in patients with heart failure, anaemia and iron deficiency. These newly described findings, along with improved oxygen delivery to exercising muscles, likely contribute to the favourable effects of ferric carboxymaltose in anaemic patients with heart failure.

Rhabdomyolysis after Intravenous Ferric Gluconate: A Case Report.

Patients with severe iron deficiency, malabsorption or intolerance to oral iron are frequently treated with intravenous iron replacement. We report the case of a 42-year-old woman with non-erosive oligoarticular arthritis with antiparietal cell antibodies and iron deficiency anemia secondary to menorrhagia and unresponsive to oral iron preparations. She was treated with an intravenous infusion of ferric gluconate. After the first infusion of 125 mg (in 250 mL saline), she developed transient pain in her knee and wrist joints. When the dose was subsequently halved, the patient showed no adverse symptoms in the next four infusions and had normalized hemoglobin levels and iron indices. However, after a subsequent 125 mg ferric gluconate infusion she developed severe leg pain, muscular and joint stiffness, and functional impairment of her hands, right foot, and ankle. Laboratory tests showed a progressive increase in creatine kinase, transaminase, and C-reactive protein that normalized several days after the infusion. Although rhabdomyolysis is not reported among endovenous iron-induced adverse events, our findings suggest that intravenous iron infusions might have increased free iron generation promoting oxidative joint and muscular injury, which would explain the joint pain and stiffness, and rhabdomyolysis. Greater attention should be paid to the more frequent cases of myalgia occurring after iron infusion, which may underlie a rhabdomyolytic event requiring clinical observation. LEARNING POINTS: Rhabdomyolysis can occur not only after intramuscular injection of iron dextran but also after infusion of ferric gluconate.The generation of increased free iron following iron administration can promote oxidative joint and muscular injury.Leg cramps and myalgia, which are relatively common adverse events after iron infusions, might represent undiagnosed events of mild to moderate rhabdomyolysis requiring further investigation.

Transfusional Approach in Multi-Ethnic Sickle Cell Patients: Real-World Practice Data From a Multicenter Survey in Italy.

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder characterized by recurrent acute vaso-occlusive crises (VOCs and anemia). Gold standard treatments are hydroxycarbamide (HC) and/or different red blood cell (RBC) transfusion regimens to limit disease progression. Here, we report a retrospective study on 1,579 SCD patients (median age 23 years; 802 males/777 females), referring to 34 comprehensive Italian centers for hemoglobinopathies. Although we observed a similar proportion of Caucasian (47.9%) and African (48.7%) patients, Italian SCD patients clustered into two distinct overall groups: children of African descent and adults of Caucasian descent. We found a subset of SCD patients requiring more intensive therapy with a combination of HC plus chronic transfusion regimen, due to partial failure of HC treatment alone in preventing or reducing sickle cell-related acute manifestations. Notably, we observed a higher use of acute transfusion approaches for SCD patients of African descent when compared to Caucasian subjects. This might be related to (i) age of starting HC treatment; (ii) patients' low social status; (iii) patients' limited access to family practitioners; or (iv) discrimination. In our cohort, alloimmunization was documented in 135 patients (8.5%) and was more common in Caucasians (10.3%) than in Africans (6.6%). Alloimmunization was similar in male and female and more frequent in adults than in children. Our study reinforces the importance of donor-recipient exact matching for ABO, Rhesus, and Kell antigen systems for RBC compatibility as a winning strategy to avoid or limit alloimmunization events that negatively impact the clinical management of SCD-related severe complications. Clinical Trial Registration: ClinicalTrials.gov, identifier: NCT03397017.

Thalassaemia is paradoxically associated with a reduced risk of in-hospital complications and mortality in COVID-19: Data from an international registry.

Although numerous patient-specific co-factors have been shown to be associated with worse outcomes in COVID-19, the prognostic value of thalassaemic syndromes in COVID-19 patients remains poorly understood. We studied the outcomes of 137 COVID-19 patients with a history of transfusion-dependent thalassaemia (TDT) and transfusion independent thalassaemia (TIT) extracted from a large international cohort and compared them with the outcomes from a matched cohort of COVID-19 patients with no history of thalassaemia. The mean age of thalassaemia patients included in our study was 41 ± 16 years (48.9% male). Almost 81% of these patients suffered from TDT requiring blood transfusions on a regular basis. 38.7% of patients were blood group O. Cardiac iron overload was documented in 6.8% of study patients, whereas liver iron overload was documented in 35% of study patients. 40% of thalassaemia patients had a history of splenectomy. 27.7% of study patients required hospitalization due to COVID-19 infection. Amongst the hospitalized patients, one patient died (0.7%) and one patient required intubation. Continuous positive airway pressure (CPAP) was required in almost 5% of study patients. After adjustment for age-, sex- and other known risk factors (cardiac disease, kidney disease and pulmonary disease), the rate of in-hospital complications (supplemental oxygen use, admission to an intensive care unit for CPAP therapy or intubation) and all-cause mortality was significantly lower in the thalassaemia group compared to the matched cohort with no history of thalassaemia. Amongst thalassaemia patients in general, the TIT group exhibited a higher rate of hospitalization compared to the TDT group (p = 0.001). In addition, the rate of complications such as acute kidney injury and need for supplemental oxygen was significantly higher in the TIT group compared to the TDT group. In the multivariable logistic regression analysis, age and history of heart or kidney disease were all found to be independent risk factors for increased in-hospital, all-cause mortality, whereas the presence of thalassaemia (either TDT or TIT) was found to be independently associated with reduced all-cause mortality. The presence of thalassaemia in COVID-19 patients was independently associated with lower in-hospital, all-cause mortality and few in-hospital complications in our study. The pathophysiology of this is unclear and needs to be studied in vitro and in animal models.

Iron-mediated tissue damage in acquired ineffective erythropoiesis disease: It's more a matter of burden or more of exposure to toxic iron form?

Iron is essential in cellular life, however, when in excess, it favors the production of reactive oxygen species (ROS) that, when overwhelm the physiological cellular antioxidant system, produce an oxidative stress state leading to cellular damages and organ failure. What is not yet completely clear is whether the damage is related more to the amount of iron or to the duration of exposure to ROS. Various cellular pathways are sensitive to the detrimental action of ROS in a non-dose-dependent manner. In addition, different organs have a different capacity to respond to iron-mediated toxicity, suggesting that the toxicity thresholds are disease-specific and patient-dependent. The aim of this article is to review the recent understanding of the concept of exposure to free iron-mediated damage, comprehending the need to design protocols in which reducing organ exposure to ROS is the primary objective in order to prevent or delay the development of organ damage.

New Mutations in HFE2 and TFR2 Genes Causing Non HFE-Related Hereditary Hemochromatosis.

Hereditary hemochromatosis (HH) is an iron metabolism disease clinically characterized by excessive iron deposition in parenchymal organs such as liver, heart, pancreas, and joints. It is caused by mutations in at least five different genes. HFE hemochromatosis is the most common type of hemochromatosis, while non-HFE related hemochromatosis are rare cases. Here, we describe six new patients of non-HFE related HH from five different families. Two families (Family 1 and 2) have novel nonsense mutations in the HFE2 gene have novel nonsense mutations (p.Arg63Ter and Asp36ThrfsTer96). Three families have mutations in the TFR2 gene, one case has one previously unreported mutation (Family A-p.Asp680Tyr) and two cases have known pathogenic mutations (Family B and D-p.Trp781Ter and p.Gln672Ter respectively). Clinical, biochemical, and genetic data are discussed in all these cases. These rare cases of non-HFE related hereditary hemochromatosis highlight the importance of an earlier molecular diagnosis in a specialized center to prevent serious clinical complications.

Identification of Novel Mutations by Targeted NGS Panel in Patients with Hyperferritinemia.

BACKGROUND: Several inherited diseases cause hyperferritinemia with or without iron overload. Differential diagnosis is complex and requires an extensive work-up. Currently, a clinical-guided approach to genetic tests is performed based on gene-by-gene sequencing. Although reasonable, this approach is expensive and time-consuming and Next Generation Sequencing (NGS) technology may provide cheaper and quicker large-scale DNA sequencing. METHODS: We analysed 36 patients with non-HFE-related hyperferritinemia. Liver iron concentration was measured in 33 by magnetic resonance. A panel of 25 iron related genes was designed using SureDesign software. Custom libraries were generated and then sequenced using Ion Torrent PGM. RESULTS: We identified six novel mutations in SLC40A1, three novel and one known mutation in TFR2, one known mutation and a de-novo deletion in HJV, and a novel mutation in HAMP in ten patients. In silico analyses supported the pathogenic role of the mutations. CONCLUSIONS: Our results support the use of an NGS-based panel in selected patients with hyperferritinemia in a tertiary center for iron metabolism disorders. However, 26 out of 36 patients did not show genetic variants that can individually explain hyperferritinemia and/or iron overload suggesting the existence of other genetic defects or gene-gene and gene-environment interactions needing further studies.

Prolonged exposure to welding fumes as a novel cause of systemic iron overload.

BACKGROUND & AIMS: Inhalation of welding fume may cause pulmonary disease known as welder's lung. At our centre we came across a number of welders with systemic iron overload and prolonged occupational history and we aimed at characterizing this novel clinical form of iron overload. METHODS: After exclusion of other known causes of iron overload, 20 welders were fully evaluated for working history, hepatic, metabolic and iron status. MRI iron assessment was performed in 19 patients and liver biopsy in 12. We included 40 HFE-HH patients and 24 healthy controls for comparison. RESULTS: 75% of patients showed lung HRCT alterations; 90% had s-FERR > 1000 ng/mL and 60% had TSAT > 45%. Liver iron overload was mild in 8 and moderate-severe in 12. The median iron removed was 7.8 g. Welders showed significantly lower TSAT and higher SIS and SIS/TIS ratio than HFE-HH patients. Serum hepcidin was significantly higher in welders than in HFE-HH patients and healthy controls. At liver biopsy, 50% showed liver fibrosis that was mild in four, and moderate-severe in two. Liver staging correlated with liver iron overload. CONCLUSIONS: Welders with prolonged fume exposure can develop severe liver iron overload. The mechanism of liver iron accumulation is quite different to that of HFE-HH suggesting that reticuloendothelial cells may be the initial site of deposition. We recommend routine measurement of serum iron indices in welders to provide adequate diagnosis and therapy, and the inclusion of prolonged welding fume exposure in the list of acquired causes of hyperferritinemia and iron overload.

HIF1A: A Putative Modifier of Hemochromatosis.

HFE-related hereditary hemochromatosis (HH) is characterized by marked phenotypic heterogeneity. Homozygosity for p.C282Y is a low penetrance genotype suggesting that the HFE-HH is a multifactorial disease resulting from a complex interaction involving a major gene defect, genetic background and environmental factors. We performed a targeted NGS-based gene panel to identify new candidate modifiers by using an extreme phenotype sampling study based on serum ferritin and iron removed/age ratio. We found an increased prevalence of the HIF1A p.Phe582Ser and p.Ala588Thr variants in patients with a severe iron and clinical phenotype. Accordingly, Huh-7 cells transfected with both variants showed significantly lower HAMP promoter activity by luciferase assay. The qRT-PCR assays showed a downregulation of hepcidin and an upregulation of the HIF1A target genes (VEGF, HMOX, FUR, TMPRSS6) in cells transfected with the HIF1A-P582S vector. We identified mutations in other genes (e.g., Serpina1) that might have some relevance in single cases in aggravating or mitigating disease manifestation. In conclusion, the present study identified HIF1A as a possible modifier of the HFE-HH phenotype cooperating with the genetic defect in downregulating hepcidin synthesis. In addition, this study highlights that an NGS-based approach could broaden our knowledge and help in characterizing the genetic complexity of HFE-HH patients with a severe phenotype expression.

Inherited iron overload disorders.

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.