International Journal of Molecular Sciences Article Genetic and Clinical Heterogeneity in Thirteen New Cases with Aceruloplasminemia. Atypical Anemia as a Clue for an Early Diagnosis Marc Vila Cuenca 1,† , Giacomo Marchi 2,† , Anna Barqué 1 , Clara Esteban-Jurado 1 , Alessandro Marchetto 3 , Alejandro Giorgetti 3 , Viorica Chelban 4,5,6 , Henry Houlden 4,5,7 , Nicholas W Wood 4,7 , Chiara Piubelli 8 , Marina Dorigatti Borges 9 , Dulcinéia Martins de Albuquerque 9 , Kleber Yotsumoto Fertrin 9,10 , Ester Jové-Buxeda 11 , Jordi Sanchez-Delgado 12,13 , Neus Baena-Díez 14 , Birute Burnyte 15 , Algirdas Utkus 15 , Fabiana Busti 2 , Gintaras Kaubrys 16 , Eda Suku 3 , Kamil Kowalczyk 17 , Bartosz Karaszewski 17 , John B. Porter 18 , Sally Pollard 19 , Perla Eleftheriou 18 , Patricia Bignell 20 , Domenico Girelli 2, * and Mayka Sanchez 21,22,23, * 1 Iron Metabolism: Regulation and Diseases Group, Josep Carreras Leukaemia Research Institute (IJC), Campus Can Ruti, Badalona, 08916 Barcelona, Spain; m.vila.cuenca@gmail.com (M.V.C.); annabarque@gmail.com (A.B.); darth.clara@gmail.com (C.E.-J.) 2 EuroBloodNet Referral Center for Iron Disorders and Gruppo Interdisciplinare Malattie del Ferro, Internal Medicine Unit, Azienda Ospedaliera Universitaria Integrata di Verona, 37134 Verona, Italy; markallbutone@gmail.com (G.M.); fabiana.busti@gmail.com (F.B.) 3 Department of Biotechnology, University of Verona, 37134 Verona, Italy; alessandromarchetto@outlook.it (A.M.); alejandro.giorgetti@univr.it (A.G.); eda.suku@univr.it (E.S.) 4 National Hospital for Neurology and Neurosurgery, London WC1N 3BG, UK; v.chelban@ucl.ac.uk (V.C.); h.houlden@ucl.ac.uk (H.H.); n.wood@ucl.ac.uk (N.W.W.) 5 Department of Neuromuscular Diseases, Institute of Neurology, University College London, London WC1N 3BG, UK 6 Department of Neurology and Neurosurgery, Institute of Emergency Medicine, Toma Ciorbă 1, Chisinau, MD-2052 Chisinau, Republic of Moldova 7 Neurogenetics Laboratory, The National Hospital for Neurology and Neurosurgery, London WC1N 3BG, UK 8 Centre for Tropical Diseases, Ospedale Sacro Cuore - Don Calabria, 37024 Negrar (VR), Italy; chiara.piubelli@univr.it 9 Hematology and Hemotherapy Center—Hemocentro Campinas, University of Campinas—UNICAMP, Campinas 13083-878, Brazil; ma.borges@yahoo.com.br (M.D.B.); dulmal@unicamp.br (D.M.d.A.); kleber@uw.edu (K.Y.F.) 10 Division of Hematology, Department of Medicine, University of Washington, Seattle, WA 98195, USA 11 Internal Medicine Department, Parc Tauli Hospital Universitari, Institut d’ Investigació i Innovació Parc Tauli I3PT, Universidad Autonoma de Barcelona, 08208 Sabadell, Spain; ejoveb@tauli.cat 12 Hepatology Unit, Digestive Diseases Department, Parc Tauli Hospital Universitari. Institut d’ Investigació i Innovació Parc Tauli I3PT, Universidad Autonoma de Barcelona, 08208 Sabadell, Spain; jsanchezd@tauli.cat 13 Centro de Investigación Biomedica y en red Enfermedades hepáticas y digestivas (CIBERehd), Instituto de Salud Carlos III, 28029 Madrid, Spain 14 Genetic Department, Parc Tauli Hospital Universitari, Institut d’ Investigació i Innovació Parc Tauli I3PT, Universidad Autonoma de Barcelona, 08208 Sabadell, Spain; NBaena@tauli.cat 15 Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, LT-08661 Vilnius, Lithuania; birute.burnyte@gmail.com (B.B.); algirdas.utkus@mf.vu.lt (A.U.) 16 Clinic of Neurology and Neurosurgery, Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, 08661 Vilnius, Lithuania; gintaras.kaubrys@santa.lt 17 Department of Adult Neurology, Medical University of Gdańsk, 80-210 Gdańsk, Poland; kkowalczyk@gumed.edu.pl (K.K.); bartkar@gumed.edu.pl (B.K.) 18 Joint Red Cell Unit, Haematology Department, University College London NHS Foundation Trust, Cancer Services, 250 Euston Road, London NW1 2PG, UK; j.porter@ucl.ac.uk (J.B.P.); perla.eleftheriou@nhs.net (P.E.) 19 Consultant Paediatrician, Bradford Royal Infirmary, Duckworthlane, Bradford BD9 6RJ, UK; sally.pollard1@nhs.net Int. J. Mol. Sci. 2020, 21, 2374; doi:10.3390/ijms21072374 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2374 2 of 14 20 Oxford Regional Genetics Laboratory, Oxford University Hospitals NHS Foundation Trust, The Churchill Hospital, Oxford OX3 7LE, UK; patricia.bignell@ouh.nhs.uk 21 Iron Metabolism: Regulation and Diseases Group, Department of Basic Sciences, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya (UIC); Sant Cugat del Valles, 08017 Barcelona, Spain 22 Program of Program of Predictive and Personalized Medicine of Cancer (PMPPC), Institut d ‘Investigació Germans Trias i Pujol (IGTP), Campus Can Ruti, Badalona, 08916 Barcelona, Spain 23 BloodGenetics S.L., Esplugues de Llobregat, 08950 Barcelona, Spain * Correspondence: domenico.girelli@univr.it (D.G.); msanchezfe@uic.es (M.S.) † These authors contributed equally to this work.  Received: 12 March 2020; Accepted: 26 March 2020; Published: 30 March 2020  Abstract: Aceruloplasminemia is a rare autosomal recessive genetic disease characterized by mild microcytic anemia, diabetes, retinopathy, liver disease, and progressive neurological symptoms due to iron accumulation in pancreas, retina, liver, and brain. The disease is caused by mutations in the Ceruloplasmin (CP) gene that produce a strong reduction or absence of ceruloplasmin ferroxidase activity, leading to an impairment of iron metabolism. Most patients described so far are from Japan. Prompt diagnosis and therapy are crucial to prevent neurological complications since, once established, they are usually irreversible. Here, we describe the largest series of non-Japanese patients with aceruloplasminemia published so far, including 13 individuals from 11 families carrying 13 mutations in the CP gene (7 missense, 3 frameshifts, and 3 splicing mutations), 10 of which are novel. All missense mutations were studied by computational modeling. Clinical manifestations were heterogeneous, but anemia, often but not necessarily microcytic, was frequently the earliest one. This study confirms the clinical and genetic heterogeneity of aceruloplasminemia, a disease expected to be increasingly diagnosed in the Next-Generation Sequencing (NGS) era. Unexplained anemia with low transferrin saturation and high ferritin levels without inflammation should prompt the suspicion of aceruloplasminemia, which can be easily confirmed by low serum ceruloplasmin levels. Collaborative joint efforts are needed to better understand the pathophysiology of this potentially disabling disease. Keywords: aceruloplasminemia; ceruloplasmin; iron metabolism; neurodegenerative disease; anemia; ferritin 1. Introduction Aceruloplasminemia (ACP) (OMIM#604290, ORPHA48818) is an adult-onset rare autosomal recessive disorder due to mutations in the CP gene (3q24-q25) encoding ceruloplasmin (CP), a copper-containing ferroxidase involved in maintaining iron homeostasis [1,2]. The pathological basis of ACP includes mild microcytic anemia and iron overload in several tissues, which can lead to diabetes mellitus, liver disease, progressive neurodegeneration, and retinopathy [1]. ACP was first described in 1987 in a 52-year-old Japanese female suffering from retinal degeneration, diabetes mellitus, and blepharospasm [3]. Usually, the onset of clinical manifestations is around the fourth or fifth decade of life, while biochemical signs of mild microcytic anemia with low transferrin saturation (TSAT) and paradoxically high ferritin may be evident since childhood [2]. Its prevalence is estimated at about one per two million offspring in Japanese non-consanguineous marriages, while epidemiologic data in the non-Japanese population are substantially missing [1]. Diagnosis is usually based on the evidence of very low or undetectable levels of serum CP and clinical, biochemical, or radiologic signs of iron overload in target organs. Current treatments are mainly based on iron-chelating agents, which are quite effective in reducing liver iron accumulation and may prevent further brain iron deposition but are poorly effective in patients with already established neurologic damage [4–6]. Administration of fresh-frozen human plasma [7], vitamin E, and oral Diagnosis is usually based on the evidence of very low or undetectable levels of serum CP and clinical, biochemical, or radiologic signs of iron overload in target organs. Current treatments are mainly based on iron‐chelating agents, which are quite effective in reducing liver iron accumulation and Int. J. may prevent Mol. Sci. 2020, 21,further 2374 brain iron deposition but are poorly effective in patients with already 3 of 14 established neurologic damage [4–6]. Administration of fresh‐frozen human plasma [7], vitamin E, and oral administration of zinc [8] have also been reported. The administration of a CP enzyme administration replacement of zincshowed therapy [8] have also beenresults promising reported. The administration in CP‐knockout mice [9]. of a CP enzyme replacement therapy showed promising results in CP-knockout mice [9]. Understanding the pathophysiology of ACP is of interest also in other more common Understandingdisorders, neurodegenerative the pathophysiology such as Alzheimer’s of ACPand is of interest also Parkinson’s in other diseases, more common characterized by an neurodegenerative disorders, such as Alzheimer’s and Parkinson’s alteration of brain iron homeostasis [10]. CP is a single polypeptide chain of 1046 amino acids diseases, characterized bythat an alteration of brain iron homeostasis [10]. CP is a single polypeptide can bind up to six atoms of copper. CP has six structural domains with a three‐copper catalytic chain of 1046 amino acids that can bind upwhich center, to sixisatoms crucial of for copper. CP has six its oxidative structural function [11] and domains protein with a three-copper stability [12]. Two catalytic isoformscenter, of CP which are produced by alternative splicing in exons 19 and 20: a soluble formof and is crucial for its oxidative function [11] and protein stability [12]. Two isoforms CP are a produced by alternative splicing glycosylphosphatidylinositol in exons 19 andmembrane (GPI)‐anchored 20: a solubleform form[13]. and aThe glycosylphosphatidylinositol soluble form is almost (GPI)-anchored exclusively synthesizedmembrane byform [13]. The while hepatocytes, solublethe form is almost exclusively GPI‐anchored membrane synthesized isoform isbyexpressed hepatocytes, by while the GPI-anchored membrane isoform is expressed by a number a number of cells, including brain astrocytes glial cells, hepatocytes, macrophages, pancreatic, of cells, including brain astrocytes and glial cells, retinal hepatocytes, epithelial macrophages, cells [14]. This membrane pancreatic, and retinal isoform plays epithelial a key role cells in [14]. Thisiron cellular membrane egress isoform plays a key role in cellular iron egress cooperating with cooperating with ferroportin, the ubiquitous and unique transmembrane protein able to export iron ferroportin, the ubiquitous and unique transmembrane protein able to export iron from the cells [15,16]. from the cells [15,16]. CP, with its copper‐mediated oxidation of ferrous iron (Fe2+) to ferric iron CP, with its copper-mediated oxidation (Fe of ferrous 3+), ensures iron (Fe2+ ) to the appropriate ferric iron binding (Fe3+ ), ensures of extracellular iron thetoappropriate transferrin binding [1]. Since ofmacrophages extracellular iron are to transferrin [1]. Since macrophages are key cells in iron recycling, key cells in iron recycling, a macrophage iron overload would be expected in ACP. Conversely, a macrophage iron overload would iron be expected inisACP. accumulation Conversely, usually observed iron accumulation is usually in hepatocytes/liver (Figureobserved 1) ratherinthan hepatocytes/liver (Figure 1) in macrophages/spleen rather than in macrophages/spleen in affected patients, suggesting in affected patients, suggesting that additional pathophysiologic roles of CP are probably involved that additional pathophysiologic roles of [17]. CP are probably Specifically, it hasinvolved [17]. Specifically, been reported it has been CP that GPI‐anchored reported can alsothat modulate GPI-anchored the CP can also interaction modulateferroportin between the interaction and between hepcidinferroportin [18–20], theand hepcidin master [18–20], regulator of the masteriron systemic regulator of systemic homeostasis [21]. Furthermore, even if soluble CP is not able to pass the blood–brain barrier, it is also present in itthe iron homeostasis [21]. Furthermore, even if soluble CP is not able to pass the blood–brain barrier, is also present brain through in the brain through secretion secretion by epithelial by of cells epithelial cells ofplexus the choroid the choroid plexus into the into the cerebrospinal cerebrospinal fluid [22], fluidthe and [22], andofthe role role this of thisform soluble soluble formbrain in the in theisbrain is still unclear. still unclear. Overall, Overall, it emerges it emerges that CPthatisCP is part part of a of a complex regulatory system involved in maintaining iron balance, complex regulatory system involved in maintaining iron balance, but further research is needed to but further research is needed to better elucidate better elucidate ACP ACP pathophysiology, pathophysiology, hopefullyhopefully helping helping to to develop develop more moreefficient efficienttreatments. treatments. Figure 1. 1. Liver Liverhistology histologyofof family family 7 proband 7 proband II.2, pearls II.2, pearls stain scale stain (20×, (20×,bar = 100 scale barµm). = 100 μm).analysis Biopsy Biopsy analysis showed liver showed preserved preserved liver with architecture architecture marked iron with markediniron deposition deposition hepatocytes, in hepatocytes, predominating at the predominating at the biliary pole of the biliary pole of the cells. cells. To address this need, in our international collaborative study, we describe the largest series of non-Japanese patients with ACP published so far, including 13 individuals from 11 families carrying 13 mutations in the CP gene, 10 of which are novel. Int. J. Mol. Sci. 2020, 21, 2374 4 of 14 2. Patients and Methods 2.1. Patients We got written informed consent from patients and relatives. Project: Uncovering new molecular and pathophysiological networks in iron metabolism (SAF2015-70412-R) approved on 10 July 2015 by the Ethics Research Committee from the Hospital Germans Trias i Pujol, Badalona, Spain. Protocol n.36792 from Project n.1460 was approved by the Ethics Research Committee from Hospital of Verona, Italy. Obtained informed written consent for molecular studies was obtained from all of the patients in accordance with the Declaration of Helsinki 1975, as revised in 2008 for The Diagnostic Lab Oxford Medical Genetics Laboratories from Oxford University Hospitals NHS Foundation Trust, United Kingdom. A detailed description of the biochemical, clinical, and genetic data of the 13 affected members in the 11 studied families with ACP is provided in the Supplementary Methods. 2.2. DNA Extraction, PCR Amplification, and DNA Sequencing Genetic studies were performed with minor differences for all the pedigrees. Genomic DNA was extracted from peripheral blood using the FlexiGene DNA kit (Qiagen) or Wizard Genomic Purification kit (Promega, Madison, WI) or phenol-chloroform extraction (QIAsymphony DNA extraction kits, Qiagen) according to manufacturer’s instructions. Ceruloplasmin gene regions (exonic, intron-exon boundaries, and untranslated regions) were sequenced using the Sanger method or Next-Generation Sequencing (NGS) methods. For Sanger methods, the CP gene was amplified using 50 ng of genomic DNA. Primer sequences and PCR conditions are available upon request. For patients in families 9 and 10, apart from sequencing the regions of the CP gene in which most mutations have been described [19], the whole CP pseudogene (CPP) was also sequenced. The resulting amplification products were verified on a 2% ethidium bromide agarose gel. The purified PCR products were sequenced using the conventional Sanger method. Sequencing results were analyzed using Mutation Surveyor software (SoftGenetics LLC, PA, USA) or Chromas software (Technelysium Pty Ltd., Australia). For NGS methods, a patient in family 2 was analyzed using the targeted NGS gene panel (v14) for iron-related anemias that included the following 5 genes: CP, TF, TMPRSS6, SLC11A2, STEAP3. For patients in families 7 and 8, NGS sequencing was performed by targeted capture and sequencing of CP, along with the five canonical “hemochromatosis” genes (HFE, HFE2, HAMP, TFR2, and SLC40A1), as previously described [23]. Patients in families 4, 11, and 12 were analyzed using a targeted NGS gene panel including the following genes: HFE, HJV, HAMP, TFR2, SLC40A1, ALAS2, CP, HEPH, TF, FTH1, FTL, SLC11A2, TMPRSS6, BMP4, BMP6, and SMAD4. Briefly, the capture of key genomic regions of interest was conducted starting from 225 ng of gDNA using a custom design HaloPlexTM Target Enrichment 1–500 kb Kit (Agilent Technologies, Santa Clara, CA) or TruSeq® Custom Amplicon version 1.5 kits (Illumina, CA) according to manufacturer instructions. Library quality was determined using the Agilent High Sensitivity DNA kit on the Agilent 2100 bioanalyzer or Agilent 4200 TapeStation System. Libraries were sequenced with the NextSeq 500 High Output Reagent Cartridge v2 (300 cycles) or MiSeq reagent kit v2 (300 cycles) (Illumina, San Diego, CA) on an Illumina NextSeq 500 or a MiSeq sequencer (Illumina, San Diego, CA), generating 150-bp paired-end reads. Samples were aligned with the reference human genome GRCh37/hg19 and data analysis was performed as previously described [24] or using the BasespaceTruSeq® Amplicon App version 1.1 or own developed algorithms. Variant interpretation followed the American College of Medical Genetics and Genomics (ACMG) guidelines [25]. Mutations were confirmed by conventional Sanger sequencing. Genetic variants are reported following official Human Genome Variation Sequence (HGVS) nomenclature and refer to NM_000096.3 for the Homo sapiens CP transcript variant, and NP_000087.1 for Homo sapiens ceruloplasmin precursor protein. Reported mutations in this study have been submitted Int. J. Mol. Sci. 2020, 21, 2374 5 of 14 to the Leiden Open Variation Database (http://www.lovd.nl) or to ClinVar (http://www.ncbi.nlm.nih. gov/clinvar) [26,27]. 2.3. Bioinformatics and Computational Analysis In silico predictions of missense variants, pathogenicity was performed using SIFT and Polyphen-2 bioinformatics tools [28,29]. Ceruloplasmin sequences from different species were retrieved from the PFAM database [30] and aligned using the MUSCLE program [31] for multiple alignments. Conservation analysis and alignment visualization were performed by Jalview software (version 2, www.jalview.org) [32]. The structural analysis of the missense variants was made based on the available human CP crystallographic structure [33]. Human Ceruloplasmin crystallographic structure was downloaded from the PDB server (PDB code: 4ENZ, crystallographic resolution: 2.6 Å; (http://www.rcsb.org/pdb/ home/home.do). The Consurf server was used to map conservation features on the structure [34]. The prediction of the putative effects of the variants in the structure/function of the protein was performed by visual inspection using the Chimera program [35]. The wild-type residues and the modeled mutants were included in the IronGenes database, publicly accessible from: http: //molsim.sci.univr.it/marchetto/php/gene_detail.php?geneId=CP#tabellaInit: from the first page, the results can be accessed graphically by selecting the ‘Variants Structures and Models’ button, and then directly exploring the different variants. To analyze the plausible splicing effects of the CP c.1864+5G>A intronic variant, we used the bioinformatic NNSplice algorithm based on neural networks (http://www.fruitfly.org/seq_tools/splice. html [36]) and the Human Splicing Finder software [37] based on position-dependent logic. 3. Results 3.1. Clinical and Biochemical Profile In this international collaborative study, we describe 11 new ACP families with 13 affected individuals (six men and seven women). Table 1 summarizes the biochemical, clinical, and genetic data of the patients. Pedigrees are shown in Figure 2. Family origins were spread worldwide, but none of the patients had Japanese ancestry (Table 1). Age at diagnosis ranged from 16 to 75 years (median 40 years), with an average diagnostic delay (i.e., time from clinical onset to the molecular diagnosis) of 10 years or more. Int. J. Mol. Sci. 2020, 21, 2374 6 of 14 Table 1. Biochemical, clinical, and genetic data of the 13 affected members in the 11 studied families with ACP. Family 1 Family 2 Family 3 Family 4 Family 4 Family 5 Family 6 Family 7 Family 8 Family 8 Family 9 Family 10 Family 11 Normal Characteristic II.3 II.3 II.3 II.2 (proband) II.3 II.1 II.2 II.3 II.3 (proband) II.2 II.2 II.1 II.1 Values Country of Origin Lithuania Spain India India India Poland Italy Italy Brazil Brazil Brazil India Pakistan Sex F F M M M F M M F F F F M Age at Dx (years) 75 33 40 66 61 40 46 62 37 29 46 25 16 Hb (g/dl) 11.1 10.9 12.1 11.6 10.7 11.1 12.5 12.2 11.7 12.3 9.4 9.2 13.4 12–16 MCV (fl) 82,0 85.1 77 66.8 81 88.0 84 70.6 75.2 71.4 64.5 71.5 69 79–99 Retyculocytes (%) 0.5 0.8 n/a 0.9 1.2 n/a 0.58 1 1.77 1.1 1.77 0.51 n.a. 1.1–2.7 RDW (%) 16.8 15.2 14.3 17.3 15.5 15.5 15.4 16.1 16 17.2 18 17.1 18 11.3–14.5 Serum iron (µg/dl) 82.8 15 28 19 39.1 81.0 33 215 23 23 22 9.5 33.5 37–170 Ferritin (ng/ml) 12159 355.4 1077 1112 3845 1143 2100 3650 791 732 1060 757 1065 10–290 Transferrin 12.4 4.3 10 5 12 39 9 88 9.24 9.83 8.2 4 8.8 20–55 Saturation (%) CP (mg/dl) < 0.02 < 2.0 < 0.03 < 0.03 < 0.03 0.12 undetectable 0.12 11 9 <2 n.a. < 0.02 17–65 ALT (U/l) 55 12 32 24 36 19 37 113 26 179 37 24 87 14–36 AST (U/l) 43 17 n.a n.a 24 16 20 80 12 82 29 n/a n/a 8–40 Low level of Cp. Low level of Cp. overexpressed HFE Hair loss, mild No Cu urine Low serum Cu and Iron deposition at Iron deposition at Iron deposition at Hemochromatosis, Low CP in executive excretion low urine Cu. brain MRI, brain MRI, brain MRI, Symptoms supposed Clue to ACP unexplained Iron deposition at Familial investigation of dysfunction on Hepatocellular Hepatic iron Unexplained Unexplained Unexplained (including tremor) additional low CP level diagnosis hyperferritinemia brain MRI investigation iron-refractory formal siderosis. Iron overload. Iron hyperferritinemia, hyperferritinemia, hyperferritinemia, and very low CP non-HFE anaemia neurocognitive deposition in basal deposit in basal low CP low CP low CP mutation(s) tested assessment ganglia. ganglia. with NGS Moderate dementia with prevalent Unspecific Neurological Head and postural frontal features, symptoms: Fatigue; symptoms: Neurological tremor of upper April 2015 acute cerebellar ataxia, abdominal progressive symptoms: and lower limbs, Choreiform psychotic episode oromandibular discomfort and cognitive decline, Concentration and Clinical progressive(over 5 slight dysmetria, Iron overload. Mild movement disorder, Mild anaemia, with catatonia, hair dystonia, torsion of chronic anaemia. diabetes, mild intellectual ability Asymptomatic. presentation years) cognitive ataxia, proximal Liver iron overload anaemia was mild anaemia, DM-2, asthenia, loss, mild executive Concentration/memory the trunk, severe Liver bradykinesia with decline. Familial (symptoms and decline, diabetes, weakness of lower and mild anaemia consistent also with DM-2, mild movement dysfunction on lapses chorea-athetosis ultrasonografy mild finger nose Unexplained investigation signs) tremor left hand extremities, b-thalassemia trait asymptomatic disorder formal with choreiform showed hepatic ataxia and anaemia, DM. Unexplained horizontal retina pigmentation neurocognitive movements. Mild lesions that justified dysdiadochokinesia anaemia nystagmus, and assessment type-2 DM. Retinal a MRI, that showed .Unexplained tunnel vision. degeneration. Mild iron overload anaemia anaemi Anaemia Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes No Neurological Yes No Yes Yes Yes Yes No No Yes No Yes Yes Yes symptoms Liver Iron overload Yes Yes n.a. Yes Yes No Yes n.a. n.a. Yes n.a Yes Yes Diabetes Yes No Yes Yes Yes No No No Yes No Yes No No Yes but not typical Retinopathy Yes No No No No Not evaluated for No Yes Not evaluated Not evaluated No Yes aceruloplasminemia MRI SWI with marked SWI increased susceptibility susceptibility Iron overload in Iron overload in Iron overload in predominantly FLAIR and T2 Iron overload in Iron overload in Brain MRI (sites of Iron overload in involving the lentiform caudate, thalami, basal choroid plexus, Iron overload in involving the hypointenseties in thalami, basal thalami, basal iron accumulation, lenticular, dentate cerebelum, basal dorsal lateral no iron overload no iron overload ganglia, red nuclei, bilateral dentate n.a. putamen lateral putamen, the putamen and ganglia, and ganglia, dentate, in brief) and thalamus ganglia, thalami, thalami and dentate, cerebellum nuclei thalamic and red nucleus, substantia nigra cerebellum and cerebellum red and dentate dentate nuclei and brain cortex basal ganglia striaum, thalamic, nuclei. pulvinar, cerebellar dentate nucleus Biopsy: small depositions of LIC 340 µM/g = HII Iron overload in Liver MRI or hepatocellular HII = 9.58, severe Feriscan LIC: 9.4 LIC by Ferriscan: yellow-brown LIC by Ferriscan LIC by Ferriscan: n.a. 7.9 (severe iron n.a. not performed liver and pancreas not performed HII < 1.9 (biopsy) siderosis grade III iron overload mg/g dw 6.3 mg/g dw pigment (stain for 9.0 mg/g/dw 5.3 mg/g dw overload) (qualitative) ferrum - negative) - probably lipofuscin Diagnostic delay 20 years 1.5 years 5 years 2 years 4 years 13 years 3 years 30 years 1 year Not applicable 32 years 3 years Not known Int. J. Mol. Sci. 2020, 21, 2374 7 of 14 Table 1. Cont. Family 1 Family 2 Family 3 Family 4 Family 4 Family 5 Family 6 Family 7 Family 8 Family 8 Family 9 Family 10 Family 11 Normal Characteristic II.3 II.3 II.3 II.2 (proband) II.3 II.1 II.2 II.3 II.3 (proband) II.2 II.2 II.1 II.1 Values Deferasirox and Started on FFP Deferasirox from desferoxamine both OctaplasLG every 2 Deferiprone started Desferoxamine, Iron chelation or 10/2014 to 11/2015; Deferiprone started suspended for Desferoxamine and No Iron chelation therapy. weeks on diagnosis. on diagnosis (25 Zinc Deferiprone combination with Deferiprone Deferiprone other therapy Deferiprone from 2011 renal insufficiency, “micro”-phlebotomies Later added mg/kg/d) deferiprone 11/2015; Vitamin E. actually on Deferiprone deferiprone Iatrogenic iron overload due to oral iron supplementation for microcytic White nails, 3-4 alcoholic Beta-Thal trait; anaemia, Acquired hypertension, units/day, arterial fully penetrant Hypothyroidism, Hypothyroidism, hypothyroid, leukopenia, previous CVA, low 2013: DM, 2011: hypertension, HFE-related HH Hypercholesterolemia, lower limb venous glaucoma, kidney amenorhoea Bilateral lattice Other clinical data Bilateral cataracts Psoriasis None Hypertension Vitamin B12, hearing loss overweight, central (C282Y macroalbuminuria thrombosis, stones, chronic (thought to be due degeneration of bilateral cataracts serous homozygous) in the migraine diarrhea to polycystic fundi-risk of retinal chorioretinopathy 3rd decade of life ovaries), microcytic detachment. Vit D anaemia, depletion hypothyroid, borderline oral glucose tolerance test. c.[1783_1787delGATAA(;) c.[2050_2051delAC]; c.[2684G>C(;) c.[2756T>C]; c.[1679G>T]; Genetics CP gene c.[1012T>A(;) 2520_2523delAACA] [2050_2051delAC] c.[1864 + 5G>A]; c.[1864 + 5G>A]; c.[1864 + c.[389A>C]; [=] 1602T>G] p. c.[2879-1G>T]; c.[2879-1G>T]; [2756T>C] [1679G>T] c.[1713+1delG]; NM_000096.3; 2972T>C] p. p.(Asp595Tyrfs*2; p.(Thr684Alafs*6); [1864 + 5G>A] [1864 + 5G>A] 5G>A];[1864+5G>A] p.(His130Pro); (=) (Gly895Ala); [2879-1G>T] [2879-1G>T] p.(Leu919Pro); p.(Cys560Phe); [1713+1delG] NP_000087.1 (Cys338Ser);(Ile991Thr) Thr841Argfs*52) (Thr684Alafs*6) (Cys534Trp) (Leu919Pro) (Cys560Phe) Reference values are indicated in the last column. Sex: M: male, F: female; ACP: Aceruloplasminemia; CP: ceruloplasmin; MRI: magnetic resonance imaging; SWI Susceptibility weighted imaging; HII hypoxic-ischemic injury; LIC: liver iron content; Hb: Hemoglobin; MCV: mean corpuscular volume; RDW: red cell distribution width; DM-2: Diabetes mellitus type 2; NGS: next-generation sequencing; n.a.: not available. Int. Int. J. Mol. Sci. 2018, J. Mol. 19, x21, Sci. 2020, FOR PEER REVIEW 2374 11 of 17 8 of 14 Figure 2. Pedigree trees from 11 studied families affected by ACP. Squares indicate males and circles Figure 2. Pedigree trees from 11 studied families affected by ACP. Squares indicate males and females. Index cases are indicated with an arrow. Filled symbols indicate affected members, half-filled circles females. Index cases are indicated with an arrow. Filled symbols indicate affected members, black symbols denote unaffected carriers, and barred symbols indicate deceased subjects. “?”, indicates half‐filled black symbols denote unaffected carriers, and barred symbols indicate deceased subjects. individuals that were not possible to test because of refusal of testing or non-locatable person. Mutations “?”, indicates individuals that were not possible to test because of refusal of testing or non‐locatable are named according to the Human Genome Variation Sequence (HGVS) nomenclatures. person. Mutations are named according to the Human Genome Variation Sequence (HGVS) nomenclatures. In our cohort, 11/13 (84.6%) patients had anemia (mild in 9/11), and 8/13 had microcytosis (61,5%). In all patients, serum ferritin values were high (geometrical mean 1409 ng/mL, CI95% 809.5-2452.5), In our cohort, 11/13 (84.6%) patients had anemia (mild in 9/11), and 8/13 had microcytosis and ceruloplasmin levels were very low or even undetectable. TSAT was decreased in 11/13 patients (61,5%). In all patients, serum ferritin values were high (geometrical mean 1409 ng/mL, CI95% (84.6%), and serum iron was decreased in 10/13 patients (76.9%). Neurological symptoms were present 809.5‐2452.5), and ceruloplasmin levels were very low or even undetectable. TSAT was decreased in in 75% of patients (data available from 12/13 patients) and ranged from severe complex neurological 11/13 patients (84.6%), and serum iron was decreased in 10/13 patients (76.9%). Neurological and psychiatric syndromes to mild cognitive dysfunctions. Brain iron overload at MRI was proven in symptoms were present in 75% of patients (data available from 12/13 patients) and ranged from ten (83.3%) of such patients. Eight out of nine patients (88.9%) had liver iron overload assessed by severe complex neurological and psychiatric syndromes to mild cognitive dysfunctions. Brain iron either liver biopsy (Figure 1) or MRI. Diabetes mellitus and retinopathy were present in 46.2% and 40% overload at MRI was proven in ten (83.3%) of such patients. Eight out of nine patients (88.9%) had of cases, respectively. All patients are alive since our last contact with them. liver iron overload assessed by either liver biopsy (Figure 1) or MRI. Diabetes mellitus and retinopathy 3.2. Genetic were present in 46.2% and 40% of cases, respectively. All patients are alive since our Spectrum last contact with them. Using a combination of Sanger sequencing and NGS, we identified 13 unique mutations, 10 of 3.2.which Genetic are novel (see details in supplementary data). Spectrum Three novel frameshift mutations were detected in family 1 and family 2 (Figure 2, Table 1). Patient Using (II.3) from a combination of Sangerheterozygous family 1 is compound sequencing and NGS, for two we identified frameshift 13 unique variants, mutations, 10 of the c.1783_1787delGATAA which andare novel (see details inlocated c.2520_2523delAACA supplementary in exon 10data). and 14 of the CP gene, respectively. Both mutations lead Three novel frameshift mutations to the creation of aberrant C-terminal were CP detected in family protein ends 1 and family and premature stop2 codons (Figureat2, position Table 1).596 Patient and 892, respectively p.(Asp595Tyrfs*2 and Thr841Argfs*52). Proband (II.3) from family 2 has athe (II.3) from family 1 is compound heterozygous for two frameshift variants, novel c.1783_1787delGATAA and c.2520_2523delAACA located in exon 10 and 14 of homozygous deletion at position 2050 to 2051 of the CP gene leading to a premature stop codon at the CP gene, respectively. position 689 Both mutations lead of ceruloplasmin to the protein creation of aberrant C‐terminal CP protein ends and p.(Thr684Alafs*6). premature stop codons In families 3 andat4,position 596 and sequencing 892, respectively analysis p.(Asp595Tyrfs*2 revealed in all probands and inand oneThr841Argfs*52). relative (brother of Proband patient II.1 from family 4) a novel homozygous intronic mutation at position +5 to (II.3) from family 2 has a novel homozygous deletion at position 2050 2051 of theexon downstream CP 10 gene leading to a premature stop codon at position 689 of ceruloplasmin protein p.(Thr684Alafs*6). Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 12 of 17 Int. J. Mol. Sci. 2020, 3 In families 21,and 23744, sequencing analysis revealed in all probands and in one relative (brother 9 of 14 of patient II.1 from family 4) a novel homozygous intronic mutation at position +5 downstream exon 10 of the CP gene (Figure 2 and Table 1). All individuals presenting this mutation are from India. of the CP gene (Figure 2 and Table 1). All individuals presenting this mutation are from India. The The NNSplice algorithm program and the Human Splicing Finder splicing program predicted that NNSplice algorithm program and the Human Splicing Finder splicing program predicted that the the substitution of a G by an A at position +5 will abolish the wild‐type donor splicing site of intron substitution of a G by an A at position +5 will abolish the wild-type donor splicing site of intron 10, 10, interfering with CP splicing. interfering with CP splicing. In family 5, a new missense mutation was detected in heterozygous state p.(His130Pro). His130 is In family 5, a new missense mutation was detected in heterozygous state p.(His130Pro). His130 engaged in a hydrogen bond with Tyr155, and its substitution with a proline causes the loss of this is engaged in a hydrogen bond with Tyr155, and its substitution with a proline causes the loss interaction (http://molsim.sci.univr.it/marchetto/php/gene_detail.php?geneId=CP#tabellaInit). Although of this interaction (http://molsim.sci.univr.it/marchetto/php/gene_detail.php?geneId=CP#tabellaInit). this histidine is not well conserved in our multiple sequence alignment (Figure S1), in this position, there Although this histidine is not well conserved in our multiple sequence alignment (Figure S1), in this is always a polar or charged residue present, indicating that a substitution with a hydrophobic residue position, there is always a polar or charged residue present, indicating that a substitution with a could cause protein misfolding. In this patient, we cannot exclude the existence of an additional hydrophobic residue could cause protein misfolding. In this patient, we cannot exclude the existence mutation in non‐studied regions (i.e., deep intronic or promoter regions). Interestingly, additional ACP of an additional mutation in non-studied regions (i.e., deep intronic or promoter regions). Interestingly, cases with only one mutation and a second wild‐type allele have also been previously described in the additional ACP cases with only one mutation and a second wild-type allele have also been previously literature (see Table S1). described in the literature (see Table S1). In family 6, the proband (II.2) is compound heterozygous for two CP missense mutations, In family 6, the proband (II.2) is compound heterozygous for two CP missense mutations, p.(Cys338Ser) and p.(Ile991Thr). These two variants have been recently categorized as pathogenic p.(Cys338Ser) and p.(Ile991Thr). These two variants have been recently categorized2+as pathogenic mutations in ACP cases [38]. Indeed, Cys338 forms the binding cavity for one of the Cu ions (Figure 3 mutations in ACP cases [38]. Indeed, Cys338 forms the binding cavity for one of the Cu2+ ions (Figure 3 and http://molsim.sci.univr.it/marchetto/php/gene_detail.php?geneId=CP#tabellaInit), and thus, the lack and http://molsim.sci.univr.it/marchetto/php/gene_detail.php?geneId=CP#tabellaInit), and thus, the of a cysteine residue in that position could dramatically affect protein function. lack of a cysteine residue in that position could dramatically affect protein function. Figure 3. A. Detail of the structural analysis of selected missense mutations mapped on the structure Figure of human3. A. Detail of the structural ceruloplasmin (PDB code: analysis 4ENZ). ofThe selected missense reference and mutations the mutated mapped onare residue theindicated structure of human ceruloplasmin (PDB code: 4ENZ). The reference and the mutated residue within the insights. The six Plastocyanin-like domains, described in the Uniprot Database (https: are indicated //www.uniprot.org/uniprot/P00450) within the insights. The six Plastocyanin‐like I to VI are coloreddomains, in cyan, described in the orange, green, Uniprot yellow, Database pink, and blue, (https://www.uniprot.org/uniprot/P00450) respectively. Copper domains are colored in I to VI are grey. Thesecolored three in cyan, orange, variants, as well green, yellow, as those pink, detailed in and blue, respectively. Copper domains are colored in grey. These three variants, as well the main text, can be retrieved from the IronGenes database (http://molsim.sci.univr.it/marchetto/php/ as those detailed in the main text, can gene_detail.php?geneId=CP#tabellaInit) be retrieved developed from of Verona. at the University the IronGenes database (http://molsim.sci.univr.it/marchetto/php/gene_detail.php?geneId=CP#tabellaInit) developed at the In family 7, University the patient is compound heterozygous for the missense mutation p.(Gly895Ala) of Verona. (rs139633388, previously reported as p.(Gly876Ala) due to alternate nomenclature) and a novel missense variant, p.(Cys534Trp). The Gly895 is an evolutionary highly conserved (Figure S1) amino acid, and Int. J. Mol. Sci. 2020, 21, 2374 10 of 14 in silico modeling analysis indicated that its substitution by alanine could cause local perturbations due to size differences. Cys534 residue is involved in a disulfide bridge with Cys560 (Figure 2 and http://molsim.sci.univr.it/marchetto/php/gene_detail.php?geneId=CP#tabellaInit), and both cysteines are highly conserved (Figure S1); therefore, the p.(Cys534Trp) variant is predicted to have a high impact on protein stability. In family 8, the proband and her elder sister carries a new splicing mutation (c.2879-1 G>T) in homozygosity in the donor site of intron 16 (variant rs386134141 and ClinVar ID 431112 reported by us). This mutation is predicted to lead to the loss of the splicing donor site located upstream of exon 17. A pathogenic mutation in the same position but with the change G>A (c. 2879-1 G>A, reported as nt2878-1G/A) was previously described as causative of ACP (see supplementary Table S1 and supplementary Reference 4). In family 9, the proband is homozygous for a new missense mutation p.(Leu919Pro) in exon 16 (variant rs1135401784 and ClinVar ID 431113 reported by us). According to our computational model, Leu919 is involved in a network of hydrophobic interactions with Leu921 and Leu808 (http://molsim.sci.univr.it/marchetto/php/gene_detail.php?geneId=CP#tabellaInit) and is conserved in 86% of the sequences (Figure S1). The substitution by a proline would probably lead to perturbations in this network, altering the stability of the protein. In family 10, the proband (II.1) is homozygous for a new missense mutation p.(Cys560Phe) in exon 9. Interestingly, Cys560 is 100% conserved (Figure S1) and is engaged in a disulfide bridge with Cys534; therefore, its substitution by Phe would have a high impact on the protein stability and/or structure (Figure 3 and http://molsim.sci.univr.it/marchetto/php/gene_detail.php?geneId=CP#tabellaInit). The effect, breaking of a disulfide bridge, is the same as the one encountered for the mutant Cys534 in family 7, pinpointing to a critical functional region of the protein. In family 11, the proband (II.1) is homozygous for a new splicing mutation in intron 9, c.1713+1delG, which alters the donor splice site of exon 9, which is predicted to result in the skipping of exon 9. His cousin has the same phenotype and his sister is a carrier of this splicing mutation. In Table S1, we provide an updated overview of all ACP cases reported in the literature and, if available, the corresponding CP mutations. Including our cohort, 107 patients with ACP from 83 families with a worldwide distribution were identified. Most reported cases are of Japanese origin, followed by Italian cases. In total, there are 71 different reported mutations causing ACP, distributed along the CP gene. All types of mutations have been reported (13 splicing mutations, 28 missense, 8 nonsense, 19 frameshift, and 2 big deletions), including 10 novel mutations that we reported in this study (3 splicing, 4 missense, and 3 frameshift mutations) (Figure S2). Most CP mutations are restricted to a particular family. 4. Discussion To the best of our knowledge, this study represents the largest series of non-Japanese ACP patients reported so far. The median age at diagnosis was 40 years, and the median diagnostic delay was about 10 years. Clinical and biochemical features of patients highlight substantial phenotypic heterogeneity, as also reported by a recent Italian case series [38], which contributes to the difficulties in the diagnosis of such a rare disease. The complete clinical triad of retinal degeneration, dementia, and diabetes, cited in a previous Japanese series [39], was present only in one 79-year-old patient (proband II.3 from family 1). Our data support that unexplained anemia, often but not necessarily microcytic, in association with low TSAT and paradoxically high ferritin are the best clues for an early diagnosis, which is crucial to prevent irreversible neurological manifestations. This type of anemia can be defined as “atypical microcytic anemia” to differentiate from other common causes of microcytosis, such as iron deficiency anemia or thalassemias [40–42]. With the exception of iron-refractory iron-deficiency anemia (IRIDA), atypical microcytic anemias are accompanied by progressive iron overload. In this regard, we propose a simplified algorithm for the diagnosis of ACP starting from microcytic anemia and hyperferritinemia in Figure 4. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 14 of 17 anemias are accompanied by progressive iron overload. In this regard, we propose a simplified algorithm for the diagnosis of ACP starting from microcytic anemia and hyperferritinemia in Figure 4. J. Mol. Sci. 2020, 21, 2374 Int. 11 of 14 Figure 4. Simplified approach to microcytic anemia + high ferritin. From relatively simple laboratory Figure 4. Simplified approach to microcytic anemia + high ferritin. From relatively simple tests also rare diseases can be suspected. Further investigations should include genetic testing, MRI for laboratory tests also rare diseases can be suspected. Further investigations should include genetic estimation of liver iron content, and eventually liver or bone marrow histopathology. IO: iron overload. testing, MRI for estimation of liver iron content, and eventually liver or bone marrow SCD: sickle cell disease. histopathology. IO: iron overload. SCD: sickle cell disease. In our series, neurological involvement and diabetes were present in 75% and 46.2% of patients, In our substantially respectively, series, neurological involvement in line with a previousand casediabetes were present series [38,43,44]. in 75% Retinopathy and was 46.2% in described of patients, respectively, substantially in line with a previous case series [38,43,44]. 40% of our patients, while it is reported in 76% of Japanese ACP patients [44]. Additional sporadic Retinopathy was described clinical in 40% findings of our in our patients, series included while it is reportedamenorrhea, hypothyroidism, in 76% of abdominal Japanese ACP pain,patients [44]. and chronic Additional sporadic clinical findings in our series included hypothyroidism, diarrhea, but it was not possible to prove a causative role of ACP, which remains uncertain. amenorrhea, abdominal pain, A genetic and of study chronic diarrhea, our patients alsobut it was not confirmed possible genetic to prove a We heterogeneity. causative detectedrole 13of ACP, unique which remains mutations, 10 ofuncertain. which are novel and distributed throughout the entire gene. Indeed, the aminoacidic A genetic changes affected study of our all the patients also six structural confirmed domains, withgenetic heterogeneity. presumable different We detected functional 13 unique effects. The mutations, 10 of which are novel and distributed throughout the entire gene. in-silico modeling provided in our work (and available at the link http://molsim.sci.univr.it/marchetto/ Indeed, the aminoacidic changes affected all the six structural php/gene_detail.php?geneId=CP#tabellaInit) domains,towith is an attempt presumable further different clarify the functional functional role of effects. The these mutations. in‐silico modeling provided in our work (and available at the link http://molsim.sci.univr.it/marchetto/php/gene_detail.php?geneId=CP#tabellaInit) A description of new mutations, their functional study, and genotype–phenotype is an correlations attempt to further clarify the functional role of these mutations. are important for several reasons in ACP, including elucidate pathophysiology, understand why someApatients description developof new brainmutations, their functional iron accumulation study, and others doand not,genotype–phenotype help in understandingcorrelations brain iron are important for several reasons in ACP, including elucidate pathophysiology, metabolism also in other more common neurodegenerative disorders, and hopefully help in developing understand why some patients develop brain new and more efficient treatments. iron accumulation and others do not, help in understanding brain iron metabolism also in other more common neurodegenerative disorders, and hopefully help in developing 5. Conclusionsnew and more efficient treatments. Our study points out that ACP is distributed worldwide and is characterized by heterogeneous 5. Conclusions genotypes and phenotypes, requiring a high level of suspicion for diagnosis. Atypical anemia, often with Our study microcytosis, lowpoints TSAT,outandthat ACP high is distributed ferritin, is the bestworldwide and pre-symptomatic clue for early is characterized by heterogeneous diagnosis, which genotypes and phenotypes, requiring a high level of suspicion for diagnosis. Atypical is crucial to avoid treatment delay and the onset of irreversible neurological damage. Collaborative anemia, often with microcytosis, joint low TSAT, efforts are needed among and high ferritin, specialized is the bestand laboratories cluecenters, for earlyaspre‐symptomatic in our work, for diagnosis, a better which is crucial to avoid treatment delay and the onset of irreversible neurological definition of the molecular and clinical features of ACP, hopefully leading to new pathophysiological damage. Collaborative and jointinsights therapeutical efforts for arethis needed amongincapacitating potentially specialized laboratories disease. and centers, as in our work, for a better definition of the molecular and clinical features of ACP, hopefully leading to new Supplementary Materials: pathophysiological Supplementaryinsights and therapeutical materialsfor canthis be found at http://www.mdpi.com/1422-0067/21/7/2374/ potentially incapacitating disease. s1. Supplementary Materials: Supplementary materials can be found at www.mdpi.com/xxx/s1. Author Contributions: M.S., M.V.C., G.M., A.B. and D.G., designed, analyzed the data, and wrote the manuscript. M.S., C.E.-J., genetic studies in P1, P2, P3, P4, and P5. B.B., P1 clinical and hematological characterization. A.U. and G.K., P1 neurological clinical data. E.J.-B., J.S.-D., N.B.-D., P2 clinical and hematological characterization, V.C., H.H., N.W.W., P3, and P4 clinical and hematological characterization; K.K. and B.K., P5 clinical and hematological Int. J. Mol. Sci. 2020, 21, 2374 12 of 14 characterization. D.G., F.B. and G.M., P6 and P7 clinical and hematological characterization. C.P., genetic studies and data analysis in P6 and P7. E.S., A.M. and A.G., computational in silico modeling studies for all missense mutations. M.D.B., D.M.d.A., P9 and P10 genetic studies, K.Y.F., P9 and P10 clinical and hematological characterization, data analysis. P.B., P4, P10, and P11 genetic studies. J.B.P. and P.E., P4 and P10 clinical and hematological characterization. S.P., P11 clinical and hematological characterization. All authors have read and agreed to the published version of the manuscript. Funding: This work was partially supported by grant SAF2015-70412-R from the Spanish Secretary of Research, Development, and Innovation (MINECO) Spain to M.S., grants for research on iron metabolism by Fondazione Cariverona (2014.0851), and the Veneto Region (PRIHTA no.2014-00000451) to D.G., grants 2014/00984-3 and 2016/08072-9 from Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP) to K.Y.F. and M.D.B., respectively. Acknowledgments: The authors would like to thank all patients and their family members for their participation in this study. We thank Alejandro Negro and Jessica Aranda from Institute Josep Carreras and Josep Fita from BloodGenetics for their technical support with Sanger and NGS sequencing. We thank Paola Capelli, at the Pathology Institute of the University Hospital of Verona for providing the histopathologic figure. 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