pharmaceuticals Article L-Ferritin: One Gene, Five Diseases; from Hereditary Hyperferritinemia to Hypoferritinemia—Report of New Cases Beatriz Cadenas 1,2,3 , Josep Fita-Torró 4 , Mar Bermúdez-Cortés 5 , Inés Hernandez-Rodriguez 6 , José Luis Fuster 5 , María Esther Llinares 5 , Ana María Galera 5 , Julia Lee Romero 7 , Santiago Pérez-Montero 4 , Cristian Tornador 1,4 and Mayka Sanchez 4,8,9, * 1 Whole Genix SL., 08021 Barcelona, Spain; Beatriz.cadenas@wholegenix.com (B.C.); cristian.tornador@wholegenix.com (C.T.) 2 Iron Metabolism: Regulation and Diseases Group, Josep Carreras Leukemia Research Institute (IJC), Campus Can Ruti, Badalona, 08916 Barcelona, Spain 3 Experimental Sciences and Technology Department, Universitat de Vic-Universitat Central de Catalunya, 08500 Vic, Spain 4 BloodGenetics SL, Esplugues de Llobregat, 08950 Barcelona, Spain; josepfita@hotmail.com (J.F.-T.); sperez@bloodgenetics.com (S.P.-M.) 5 Pediatric OncoHematology Service, Clinic University Hospital Virgen de la Arrixaca, Instituto Murciano de Investigación Biosanitaria (IMIB), 30120 Murcia, Spain; mariam.bermudez2@carm.es (M.B.-C.); josel.fuster@carm.es (J.L.F.); mariae.llinares@carm.es (M.E.L.); anam.galera@carm.es (A.M.G.) 6 Hematology Service, University Hospital Germans Trias i Pujol (HGTiP), Institut Català d’Oncologia (ICO), Badalona, 08916 Barcelona, Spain; agnesrh@iconcologia.net 7 Biomedical Engineering Department, University of Texas at Austin, Austin, TX 78712, USA; julialromero@utexas.edu 8 Program of Predictive and Personalised Medicine of Cancer (PMPPC), Institut d’Investigació Germans Trias i Pujol (IGTP), Campus Can Ruti, Badalona, 08916 Barcelona, Spain 9 Iron Metabolism: Regulation and Diseases Group, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya (UIC), 08195 Barcelona, Spain * Correspondence: msanchezfe@uic.es; Tel.: +34-935-042-000  Received: 23 December 2018; Accepted: 19 January 2019; Published: 23 January 2019  Abstract: Ferritin is a multimeric protein composed of light (L-ferritin) and heavy (H-ferritin) subunits that binds and stores iron inside the cell. A variety of mutations have been reported in the L-ferritin subunit gene (FTL gene) that cause the following five diseases: (1) hereditary hyperferritinemia with cataract syndrome (HHCS), (2) neuroferritinopathy, a subtype of neurodegeneration with brain iron accumulation (NBIA), (3) benign hyperferritinemia, (4) L-ferritin deficiency with autosomal dominant inheritance, and (5) L-ferritin deficiency with autosomal recessive inheritance. Defects in the FTL gene lead to abnormally high levels of serum ferritin (hyperferritinemia) in HHCS and benign hyperferritinemia, while low levels (hypoferritinemia) are present in neuroferritinopathy and in autosomal dominant and recessive L-ferritin deficiency. Iron disturbances as well as neuromuscular and cognitive deficits are present in some, but not all, of these diseases. Here, we identified two novel FTL variants that cause dominant L-ferritin deficiency and HHCS (c.375+2T > A and 36_42delCAACAGT, respectively), and one previously reported variant (Met1Val) that causes dominant L-ferritin deficiency. Globally, genetic changes in the FTL gene are responsible for multiple phenotypes and an accurate diagnosis is useful for appropriate treatment. To help in this goal, we included a diagnostic algorithm for the detection of diseases caused by defects in FTL gene. Keywords: ferritin; hereditary hyperferritinemia; hereditary hypoferritinemia; iron metabolism; cataracts syndrome; neurodegenerative disease Pharmaceuticals 2019, 12, 17; doi:10.3390/ph12010017 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2019, 12, 17 2 of 15 1. Introduction Ferritin is an iron-binding protein that stores and releases iron and thus contributes to maintaining and controlling iron homeostasis. Iron is stored in ferritin in the Fe+3 form and released in the Fe+2 form. Tissue ferritin is a multimeric protein formed from the assembly of 24 peptide subunits, known as the light (L) and heavy (H) ferritin subunits. The H subunit has ferroxidase activity and converts iron from Fe+2 to Fe+3 , which enables iron storage; it also serves to regulate pH and increase concentrations of free radicals in the body, which can be extremely damaging to cellular structures and proteins [1]. The L-ferritin subunit helps with electron transport in and out of the ferritin core protein and plays a role in iron release, as Fe+2 , from ferritin. Unlike tissue ferritin, serum ferritin is partially glycosylated and nearly completely iron-free [2,3] and is mainly composed of L-ferritin subunits [4–6]. Focusing on the L-ferritin (FTL) gene, five diseases have been identified as directly resulting from mutations in this gene. These diseases include hereditary hyperferritinemia with cataract syndrome (HHCS), neuroferritinopathy, benign hyperferritinemia (or hyperferritinemia without iron overload), autosomal dominant L-ferritin deficiency and autosomal recessive L-ferritin deficiency. HHCS (OMIM#600886, ORPHA163) is associated with mutations located in the iron responsive element (IRE) at the 50 untranslated region (UTR) of the L-ferritin mRNA, which results in the disruption of binding with iron regulatory proteins (IRP1 and 2), this is known as the IRP-IRE post-transcriptional regulatory system [7–9]. Mutations in this RNA motif result in the loss of ferritin translation repression and excess ferritin production, even though iron levels remain normal. Ferritin overproduction leads to deposits in the lens of the eye, resulting in the development of cataracts [10]. Up to now, there are 47 known mutations associated with HHCS [11]. In 2001, Curtis and collaborators [12] described for the first time neuroferritinopathy (OMIM#606159, ORPHA:157846), an autosomal dominant condition characterized by normal to low serum ferritin levels, progressive chorea or dystonia, and subtle cognitive deficits. Neuroferritinopathy is classified as a member of the group of diseases known as neurodegeneration with brain iron accumulation (NBIA). So far, there have been ten reported mutations causing this condition, mostly located at the C-terminal region of the FLT gene [13]. FTL mutations diminish ferritin’s ability to store iron so, in an attempt to control free iron levels, neurons produce more ferritin, resulting in iron and ferritin accumulation in the basal ganglia of the brain and leading to movement and cognitive disabilities [14]. Benign hyperferritinemia or genetic hyperferritinemia without iron overload (OMIM#600886, ORPHA:254704) is another FTL mutated disorder where patients have high (greater than 90%) glycosylated serum ferritin levels. There are three known mutations in the N-terminal region of the FTL gene that cause benign hyperferritinemia [15]. Despite serum ferritin hyperglycosylation, no other harmful effects have been detected in patients with this disorder [16]. Finally, two variants in FTL have been reported causing L-ferritin deficiency, i.e., hypoferritinemia (OMIM#615604, ORPHA:440731). Mutation Glu104Ter was described in a single patient with inheritance in autosomal recessive mode and consists of a G > C nucleotide substitution in exon 3 (c.310G > T). This mutation causes a complete lack of translation of the FTL gene with subsequently undetectable levels of serum ferritin. This patient presented with seizures and restless leg syndrome [17]. The FTL mutation Met1Val, resulting in a change at the start codon (c.-1A > G), has also been described in a single case; however, this mutation was inherited in an autosomal dominant manner. The patient presented with decreased levels of serum ferritin, but no history of iron deficiency anemia or neurologic dysfunction [18]. In this study, we report the identification of two novel mutations in the FTL gene detected by gene sequencing. One mutation is associated with a diagnosis of HHCS and the other with a diagnosis of dominant L-ferritin deficiency. We also describe an additional dominant L-ferritin deficiency case with a previously described (Met1Val) mutation in the FTL gene. Moreover, we have performed an extensive review of all reported variants in the FTL gene linked with the previously described five conditions to help in the understanding of the phenotypes. Pharmaceuticals 2019, 11, x FOR PEER REVIEW 3 of 15 with a previously described (Met1Val) mutation in the FTL gene. Moreover, we have performed an extensive review of all reported variants in the FTL gene linked with the previously described five Pharmaceuticals 2019, 12, 17 3 of 15 conditions to help in the understanding of the phenotypes. 2.2.Results Results Wecompletely We completelysequenced sequenced the the entire entire coding coding region, region, intron-exon intron-exon boundaries boundaries and and 5′50 and and3′ 30regulatory regulatoryregions for for regions the the FTL FTL gene gene eithereither by Sanger sequencing by Sanger or by next sequencing generation or sequencing by next generation (NGS). sequencing (NGS). 2.1.Case 2.1. CaseStudies Studies 2.1.1. 2.1.1.Family Family1—A 1—ACase Casewith withAutosomal AutosomalDominant DominantL-Ferritin L-FerritinDeficiency Deficiency Proband ProbandII.1 II.1from fromfamily family1 1(Figure (Figure1A1AandandTable Table1)1)is isa four-year-old a four-year-oldfemale femaleofofSpanish Spanishorigin origin referred referred to the department of Pediatric OncoHematology Service of the Clinic UniversityHospital to the department of Pediatric OncoHematology Service of the Clinic University Hospital Virgen Virgen dedelala Arrixaca Arrixaca because becauseofof refractory refractory hypoferritinemia hypoferritinemia (serum (serumferritin 4–9 ferritin ng/mL) 4–9 ng/mL)unresponsive unresponsive totooral iron supplementation, without any accompanying sign or symptoms. oral iron supplementation, without any accompanying sign or symptoms. Physical examination Physical examination was wasnormal normalwith withnormal normalweight weightandandsize sizeforforage. age.At Atthe theage ageofofsix, six,the theproband probandcomplained complainedofof recurrent recurrentsevere severeheadaches. headaches.Cerebral CerebralCT CTand andMRIMRIwere werenormal normalexcept exceptfor fora asmall small(non-specific) (non-specific) subcortical subcortical area of gliosis in the right frontal lobe. This was considered an incidentalfinding area of gliosis in the right frontal lobe. This was considered an incidental findingbyby pediatric pediatric neurologists neurologistswho who established a provisional established diagnosis of a provisional primary headache diagnosis of primary and recommended headache and treatment with flunarizine. recommended treatment with flunarizine. Figure Figure 1.1.Pedigree Pedigreetrees treesfrom from three three studied studied families families affected affected from from dominant dominant L-ferritin L-ferritin deficiency deficiency and and HHCS. HHCS. Squares Squares indicate indicate malesmales and circles and circles females. females. Probands Probands are pointed are pointed with an with an arrow. arrow. Filled Filled symbols indicate symbolsaffected members indicate affectedand asterisksand members indicate subjects asterisks with genetic indicate subjectsstudies done at BloodGenetics with genetic studies done at SL. Mutations are named according the HGVS nomenclature. BloodGenetics SL. Mutations are named according the HGVS nomenclature. Mutation Mutation analysis analysisrevealed revealedthe the presence of T > presence ofATchange in the intron > A change of the FTL in the 3intron 3 of gene, the FTLat position gene, at 375 + 2 (NM_000146.3:c.[375 position + 2T > A];[=], 375 + 2 (NM_000146.3:c.[375 + 2T >HGSV A];[=],nomenclature). HGSV nomenclature). This variant was also This variant wasfound in a also found heterozygous state in the father of the proband (I.1). Human Splicing Finder software in a heterozygous state in the father of the proband (I.1). Human Splicing Finder software predicted predicted that this variant that alters the this variant wild alters thetype wildsplicing donor site, type splicing donoraffecting mRNA mRNA site, affecting splicing.splicing. This mutation is novelis This mutation and has and novel not been previously has not reported either been previously in the reported literature either in theorliterature in any public or indatabase (ENSEMBL, any public database NCBI, 1000Genomes, public HGMD). However, another variant without (ENSEMBL, NCBI, 1000Genomes, public HGMD). However, another variant without a known a known clinical significance exists at the clinical same position, significance existsc.375 + 2T at the same> C,position, reportedc.375in the+SNP 2T >database as rs1371561306 C, reported in the SNP and with aas database very low allele frequency rs1371561306 and with a(MAF very low= 0.000008 reported in allele frequency TOPMED (MAF database). = 0.000008 reported in TOPMED database). Pharmaceuticals 2019, 12, 17 4 of 15 Table 1. Genetic and clinical features of the probands. Case Family 1 Family 2 Family 3 Reference Values Patient II.1 II.1 II.1 - Gender F F M - Age at diagnosis (years) 4 2 67 - Hb (g/dL) 12.2–13.3 13.1–13.7 14.0 13.5–17.5 (M). 12.1–15.1 (F) MCV (fL) 78–84 80 90.2 80–95 Ferritin (ng/mL) 4–9 2–7 3037 12–300 (M), 12–200 (F) Transferrin sat (%) 12.9 17.2–26.2 22.0–41.0 25–50 Iron (µL/dL) n/a 61.95 46 49–226 c.375 + 2T > A p.Met1Val c.-164_158del7 - Mutation Novel Previously reported [18] Novel - Disease L-ferritin deficiency L-ferritin deficiency HHCS - Inheritance AD AD AD - The following abbreviations were used: HB, hemoglobin; MCV, mean corpuscular volume; TF sat, transferrin saturation; F, female; M, male; n/a, not available. The mutation nomenclature used follows the HGVS guidelines. Pharmaceuticals 2019, 12, 17 5 of 15 2.1.2. Family 2—A Case with Autosomal Dominant L-Ferritin Deficiency Proband II.1 from family 2 (Figure 1B and Table 1) is an asymptomatic two-year-old girl evaluated at the department of Pediatric OncoHematology Service of the Clinic University Hospital Virgen de la Arrixaca for further investigation of mild neutropenia and eosinophilia. The patient had been previously diagnosed with a small ventricular septal defect. During her follow-up, we did not find neutropenia or other hematological anomalies, but rather marked hypoferritinemia without anemia. Hypoferritinemia was unresponsive to oral iron supplementation. She occasionally complained of mild asthenia and an occasional mild headache, which were found to be tension headaches after a full evaluation by a pediatric neurologist. The proband´s mother had low serum ferritin levels (<6 ng/mL), low transferrin saturation (9.6%) with normal levels of transferrin. Sequencing analysis of this proband (Figure 1B, II.1) showed an A > G substitution at position 1 in the heterozygous state, causing the start codon methionine to change into valine. This mutation was previously reported in 2004 [18] and was described in the SNP database as rs139732572 with a very low allele frequency (MAF = 0.000008 reported in the ExAc database). This variant has been classified in ClinVar database as pathogenic (Variation ID 96689), causing L-ferritin deficiency in dominant inheritance mode. Here we report the second case of a patient with hypoferritinemia and this same mutation in the FTL gene (NM_000146.3:c[1A > G];[=], NP_000137.2:p(Met1Val);(=), HGSV nomenclature). 2.1.3. Family 3—A Case with HHCS The proband I.1 in family 3 (Figure 1C and Table 1) is a 65-year-old man with a history of enolism and dyslipidemia, showing high levels of serum ferritin (>3000 ng/mL), motive for what he was referred to the Hematology Service at the University Hospital Germans Trias i Pujol (HGTiP). At age of 45, he underwent cataract surgery. Initially, he underwent three therapeutic phlebotomies, but they were suspended due to the development of anemia. Magnetic resonance (MR) imaging showed normal deposits of liver iron (30 µmol/g). The family history suggests the presence of HHCS due to the existence of additional cases of hyperferritinemia (proband´s son and uncle) and cataracts (proband´s mother). The proband’s son (II.1) is a 39-year-old male with history of stage 1 orchiectomized and disease-free seminoma and no surgical removed cataracts. He was contacted by the same hematology service (HGTiP) under suspicion of HHCS because of hyperferritinemia (>2000 ng/mL) and cataracts. The hematological evaluation was normal except for the high ferritin levels, and liver magnetic resonance showed normal levels of hepatic iron (20 µmol/g). The genetic studies performed on family 3 showed the presence a heterozygous deletion c.[-164_-158del7] located in the 50 FTL IRE in the proband (I.1) and his son (II.1), both affected with hereditary hyperferritinemia with cataracts syndrome (Figure 1). Genetic analyses were not available for the mother and the uncle of the proband. This variant consists of a deletion of seven nucleotides (CAACAGT), excising part of the hexanucleotide loop and upper stem of the FTL IRE (Figure 2). Following the traditional nomenclature for FTL IRE mutations, we refer to these mutations as Esplugues +36_42del7 mutation (HGVS nomenclature as NM_000146.3:c.[-164_158del7];[=]). This deletion is predicted to impair the IRE structure. RNA secondary structure modelling of WT and mutated FTL 50 IRE sequences was performed using the Sfold web server, which predicted that -164_-158del7 mutation, located at the hexanucleotide loop, is likely to disturb the WT IRE conformation (Figure 3). In addition, the SIREs web server prediction [19] indicate loss of the IRE structure, as the mutated query returned no results. This mutation has not been previously described in the literature, but other similar IRE deletions have been previously demonstrated to be pathogenic for HHCS [11]. The location and severity of this mutation, together with the clinical manifestations of HHCS present in the affected individuals of this family, indicates that this variant is most probably the genetic cause of disease in this family. Pharmaceuticals 2019, 11, x FOR PEER REVIEW 6 of 15 Pharmaceuticals 2019, 12, 17 6 of 15 Figure 2. Schematic localization of literature reported and new FTL mutations. Mutations described in Figure this 2. Schematic work are in bold localization of literature and new mutations reported are boxed. Theand new FTL domains mutations. of the five alphaMutations helices (Adescribed to E) are in this work are in bold and new mutations are boxed. The domains of the five alpha represented in the protein (NP_000137.2). Mutations are classified as nonsense, frameshift,helices (A to E) missense, are represented in the protein (NP_000137.2). Mutations are classified as nonsense, or splicing. Here we report FTL protein changes using the three-letter amino acid code. frameshift, missense, or splicing. Here we report FTL protein changes using the three-letter amino acid code. Pharmaceuticals 2019, 12, 17 7 of 15 Pharmaceuticals 2019, 11, x FOR PEER REVIEW 7 of 15 Figure 3. WT Figure andand 3. WT mutated mutatedFTL-IRE FTL-IREfold foldprediction. Predictedsecondary prediction. Predicted secondary structure structure of WT of WT and and mutated mutated FTL-IRE using Sfold web server [20]. Deletion in hexanucleotide loop (c.-164_158del7) is FTL-IRE using Sfold web server [20]. Deletion in hexanucleotide loop (c.-164_158del7) is expected expected to to disturb completely the IRE structure. Nucleotides are numbered from the transcription starting disturb completely the IRE structure. Nucleotides are numbered from the transcription starting site. site. Free energy Free energy (∆G)(ΔG) is detailed. is detailed. 2.2. Update 2.2. Update on L-Ferritin on L-Ferritin Mutations Mutations andDiseases and Diseases Mutations Mutations in L-ferritin in L-ferritin gene gene causes causes fivedifferent five differentphenotypic phenotypic diseases, diseases,asassummarized summarized in Table in Table 2. 2. After an extensive literature search, we have collected all 63 different mutations reported so far for After an extensive literature search, we have collected all 63 different mutations reported so far for these five diseases, including the two novel mutations reported in the present work (Figure 2 and these five diseases, including the two novel mutations reported in the present work (Figure 2 and Supplementary Material: Table S1). Supplementary Material: Table S1). Most of the FTL mutations described correspond to hereditary hyperferritinemia with cataract Most of the syndrome FTL mutations (HHCS), includingdescribed correspond 36 point mutations, to hereditary 9 deletions, and hyperferritinemia 2 insertion-deletions.withAll cataract the syndrome (HHCS), including 36 point mutations, 9 deletions, and 2 insertion-deletions. mutations for HHCS are located at the 5´UTR of the FTL gene (chr19:49468566-49468764), affecting All the mutations for HHCS are located at the 5´UTR of the FTL the primary sequence and structure of FTL-IRE (Figure 2). HHCS is an autosomal dominant disorder, the gene (chr19:49468566-49468764), affecting and sequence primary all reported andvariants structure areofinFTL-IRE heterozygous (Figurestate except is 2). HHCS foranthree cases [11,21,22] autosomal dominant where disorder, homozygous and all mutations reported variants have are in been described. heterozygous Patients state except for with three HHCS do not present cases [11,21,22] clinical where homozygous manifestations mutations have been other than highPatients described. serum ferritin levels and with HHCS congenial do not present bilateral clinicalnuclear cataracts. other than manifestations Neuroferritinopathy is the only NBIA disorder with an autosomal dominant inheritance. It is high serum ferritin levels and congenial bilateral nuclear cataracts. caused by mutation in the FTL gene. Up to now, nine insertions from one to 16 nucleotides located in Neuroferritinopathy is the only NBIA disorder with an autosomal dominant inheritance. It is the exon 4 of FTL gene have been reported to cause this neurodegenerative disorder (Figure 2). These caused by mutation insertions in the alter the FTL gene. reading frame Up to now, of the nine insertions C-terminal from oneatolonger region, generating 16 nucleotides protein withlocated in theadditional FTL gene exon 4 ofaberrant amino acids. In addition, a missense Ala96Thr mutation (rs104894685) has been 2). have been reported to cause this neurodegenerative disorder (Figure Thesealso insertions described to causereading alter the frame of theamino neuroferritinopathy; C-terminal acid 96 region, generating is predicted a longer to be situated protein at the same with additional tertiaryaberrant structureamino region acids. In addition, as the pathogenic a missense insertions [13]. Ala96Thr Mutations in mutation (rs104894685) the C-terminal region of has FTL been disrupt α-helix also described D or E,neuroferritinopathy; to cause which seems to be essential amino foracid the stability of the peptide 96 is predicted [23,24]. to be Clinical situated andsame at the tertiary structure region as the pathogenic insertions [13]. Mutations in the C-terminal regionthe biochemical manifestations of this disease include low serum ferritin levels, iron accumulation in of FTL basal disrupt ganglia α-helix D and or E,progressive which seems and to severe neurological be essential dysfunctions for the stability of with thesubtle peptidecognitive deficits [23,24]. in and Clinical some cases. biochemical manifestations of this disease include low serum ferritin levels, iron accumulation in the basal ganglia and progressive and severe neurological dysfunctions with subtle cognitive deficits in some cases. Pharmaceuticals 2019, 12, 17 8 of 15 Table 2. Summary of diseases caused by mutations in FTL gene. Hereditary Neurodegeneration with L-Ferritin Deficiency, L-Ferritin Deficiency, Disease Hyperferritinemia Cataract Benign Hyperferritinemia Brain Iron Accumulation 3 Dominant Recessive Syndrome First publication [7,8] [15] [12] [18] [17] Inheritance Autosomal dominant Autosomal dominant Autosomal dominant Autosomal dominant Autosomal recessive DOMINANT NEGATIVE Mechanism LOST OF IRP REGULATION (DO NOT PROCEED) HAPLOINSUFICIENCY TOTAL LOSS OF FTL EFFECT Mutation/s Many in the 50 IRE Missense in exon 1 Frameshift in exon 4 p.(M1V; =) p.(E104X; E104X) Affects the A α-helix near Predicted to cause loss of the Type 50 UTR Start loss Nonsense the N-terminus C-terminal secondary structure High serum ferritin Low serum ferritin High serum ferritin Undetectable serum ferritin Normal serum iron Low transferrin saturation Serum ferritin Normal Transferrin Normal transferrin saturation (17%) Hematological features hyperglycosylation Low serum ferritin saturation Normal red cell counts Normal serum iron Normal hematologic Normal hematologic Normal hematologic Normal hematologic parameters parameters parameters parameters Idiopathic generalized Severe neurological Congenital bilateral nuclear seizures Other features dysfunction cataract Atypical RLS Progressive Gastrointestinal dysphagia hair loss The following abbreviations were used: IRP, iron regulatory protein; IRE, iron responsive element; UTR, untranslated region; RLS, restless leg syndrome; A α-helix, first domain alpha helix of FTL protein. Pharmaceuticals 2019, 12, 17 9 of 15 Three heterozygous mutations in FTL exon 1 have been associated with hyperferritinemia without iron overload where cataracts were absent; this condition has also been named benign hyperferritinemia. The Thr30Ile mutation (rs397514540, ExAc MAF = 0.000008) has been identified in French and British families [15,25]. Two further pathogenic mutations—p.(Ala27-Val) and p.(Gln26Ile)—have been reported in two additional patients [26]. Mutations in exon 1 of FTL are associated with higher than normal serum ferritin glycosylation. These three variants alter the A α-helix near the N terminus of L-ferritin; it has been hypothesized that the aberrant peptide extends the length of the hydrophobic cluster of amino acids at the N terminus, increasing the secretion of L-ferritin [15]. However, the reason for the development of hyperferritinemia and hyperglycosylation associated with these mutant ferritin forms is still not fully elucidated [25]. We have described in this report a novel intronic splicing mutation in the FTL gene (NM_000146.3:c.[375 + 2T > A];[=]) associated also with autosomal dominant L-ferritin deficiency. Including this novel splicing mutation, two heterozygous mutations have been described to cause L-ferritin deficiency with autosomal dominant transmission. Cremonesi and collaborators identified in 2004 a heterozygous A > G substitution in the first nucleotide of FTL, which change the ATG start codon (methionine) into a valine [18]. This mutation is predicted to encode a non-functional and unstable protein. Despite the low serum levels of L-ferritin, the proband presenting this mutation does not show either serious neurological problems (other than headaches) indicating that the molecular mechanism of L-ferritin deficiency with autosomal dominant inheritance is haploinsufficiency. Finally, only one Italian case has been reported to cause L-ferritin deficiency with autosomal recessive inheritance; a homozygous substitution at nucleotide 310 G to T that produces a premature stop codon (E104X) [17]. This amino acid change is predicted to be located in the middle of the α-helix C domain, a critical region for the stability of the protein. In silico analysis predicted that a stop codon at this position produces a truncated protein unable to fold into a functional peptide and, therefore, leads to the generation an L-ferritin subunit with a complete loss of function. This homozygous mutation is associated with a more aggressive phenotype, which is characterized by undetectable ferritin levels, idiopathic generalized seizures and atypical restless leg syndrome. 3. Discussion L-ferritin disease is a clear case of Mendelian disease-related genes that are associated with multiple diseases, including hyperferritinemia linked with cataracts in HHCS, isolated hyperferritinemia (benign hyperferritinemia), hypoferritinemia associated with other symptoms (neurological symptoms as in neuroferritinopathy, or muscular symptoms in autosomal recessive L-ferritin deficiency), or isolated hypoferritinemia (autosomal dominant L-ferritin deficiency). For improving the diagnosis of these different diseases, we have created a clinical diagnostic algorithm of diseases caused by mutations in FTL gene (Figure 4). The underlying mechanisms for these disease patterns are not fully elucidated for all these diseases. In HHCS, it is known that the pathological molecular mechanism is linked to the disruption of the IRP-IRE post-transcriptional regulatory system, with the de-repression of L-ferritin mRNA and the subsequent overproduction of L-ferritin protein that precipitates and deposits in the lens of the eyes, producing cataracts. In HHCS, authors have argued that an association exists between the clinical severity of the disease and the location of the mutation in FTL-IRE [11,27,28]. Higher serum ferritin levels are mostly associated with mutations in the apical loop or the C-bulge area, compared with mutations in the upper or lower stems. These findings are consistent with our cases in family 3, i.e., a father and son with a seven-nucleotide deletion in the IRE of the FTL gene (50 -CAACAGU30 ). The deletion partially affects the hexanucleotide loop of the IRE, and patients show considerably high serum ferritin levels (2071–3037 ng/mL) and develop early bilateral cataracts (Table 1). Mutations in exon 4 of FTL gene that alter the C-terminal sequence and the length of the protein have been reported to cause neuroferritinopathy. These mutations affect the D- or E α-helix (see Figure 2), leading to significant disruption of the tertiary and quaternary structure of the FTL protein Pharmaceuticals 2019, 12, 17 10 of 15 and producing an unstable and leaky ferritin. Studies have shown that mutant ferritin maintains the normal spherical shell structure and size [29]. However, mutant FTL C-termini, rich in amino acids with iron-binding properties, may be extended and unravel out of the shell. These mutant structures could initiate aggregation, forming ferritin inclusion bodies/precipitates [29,30]. Previous in vitro functional analysis and in vivo mouse models have revealed that these molecular-level defects have two main consequences: (1) the loss of normal protein function, reducing iron incorporation; (2) and the acquisition of toxic function through radical production, ferritin aggregation, and reactive oxygen species (ROS) generation [14,31–33]. It has been demonstrated that mutations in the C terminus have a dominant-negative effect, which explains the dominant transmission of the disorder [34]. In benign hyperferritinemia, missense variations in exon 1 of FTL increase the hydrophobicity of the ferritin N-terminal site. The A-α-helix of the protein is altered and hyperglycosylated, although the precise molecular mechanism is not clear yet. L-ferritin deficiency is characterized by haploinsufficiency of the FTL protein in autosomal dominant L-ferritin deficiency, or complete loss of function in autosomal recessive L-ferritin deficiency. Mutations associated with this condition are predicted to affect protein expression. The inactivation of one allele of FTL, which occurs in the two described heterozygous cases of autosomal dominant L-ferritin deficiency, produces a significant reduction of L-ferritin in serum. The phenotype of this dominant condition is normal apart from low levels of serum ferritin and low transferrin saturation. However, the homozygous mutation Glu104Ter causes a total loss of function of L-ferritin and leads to ferritin missing the FTL chain. Previous mutational studies have suggested that the presence of H homopolymer ferritin in fibroblasts is associated with reduced cellular iron availability and increased ROS production, which triggers cellular damage. These findings were also found in neurons derived from patient fibroblasts and correlate with the neurological phenotype of this more severe condition [17]. In this study, we have identified two new cases of autosomal dominant L-ferritin deficiency, one due to a new mutation in intron 3 of the FTL gene that most probably affects splicing. The T > G substitution in nucleotide 375 + 2 (genomic coordinates g.49469665 in Assembly GRCh37.p13) modifies a highly conserved dinucleotide GT donor site, a key sequence recognized by the spliceosome during splicing. Mutations in splice site junctions are likely to lead to exon skipping or total or partial intron retention in the mRNA transcript in most cases [32]. Our in silico analysis using Human Splicing Finder suggests that our variant would activate a new alternative donor site onwards (375 + 5G and +6T), leading to the insertion of four additional nucleotides in the mRNA sequence between exon 3 and 4 and altering the reading frame of the transcript [33]. Therefore, this change would lead to a premature stop codon formation and the putative generation of a truncated FTL protein of 128 amino acids, if the aberrant mRNA is not detected and degraded by the nonsense mediated decay (NMD) system. According to our bioinformatics prediction, the c.375 + 2T > A mutation will not generate a dominant-negative version of FTL protein, but a truncated FTL protein completely missing the E- α-helix domain and partially lacking the D- α-helix domain. Supported by the clinical manifestations found in this family (low serum ferritin levels, low transferrin saturation and lack of serious neurological or movement abnormalities), the molecular mechanism in this case is most probably due to the loss of function of FTL and it will be not expected that the disease derives in neuroferritinopathy. However, we cannot totally and completely exclude the later development in life of brain iron overload and neuroferritinopathy in these patients by a yet unknown and novel mechanism. Patients with hereditary hyperferritinemia could be misdiagnosed as patients suffering from hereditary hemochromatosis, liver dysfunction or inflammation. Some patients have received unnecessary invasive diagnostic techniques, such as liver biopsy, and are inappropriately treated with venesections and phlebotomies that can cause severe iron-deficiency anemia. On the other hand, clinical manifestations of neuroferritinopathy including tremor, parkinsonism, psychiatric problems, and abnormal involuntary movements, the presence of ferritin-iron precipitation in glia cells Pharmaceuticals 2019, 12, 17 11 of 15 and neurons [30], are often misdiagnosed and treated as Huntington’s or Parkinson’s disease. Potential treatment targets for neuroferritinopathy may include an optimized iron chelator to induce the re-solubilization of iron aggregations, in combination with radical scavengers to prevent oxidative ferritin damage [35]. Patients with benign hyperferritinemia could be misdiagnosed as patients with HHCS due to the presence of high ferritin levels and the possible late-onset appearance of cataracts. Apart from the surgical removal of cataracts in HHCS, HHCS and L-ferritin deficiency have no specific therapy. These facts emphasize the importance of a correct and early genetic diagnosis for the subsequent implementation of proper treatment, avoiding detrimental or inappropriate treatments. Following clinical algorithms, such as the one included in this publication (Figure 4) and also HIGHFERRITIN Web Server (http://highferritin.imppc.org/) [36] will surely help in this goal. 4. Materials and Methods 4.1. Patients All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee on 10th July 2015. 4.2. 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) according to manufacturer’s instructions. Ferritin gene regions (exonic, intron–exon boundaries, and untranslated regions) were sequenced using the Sanger method or next generation sequencing (NGS). For the Sanger method, the FTL gene was amplified using 50 ng of genomic DNA. Primer sequences and PCR conditions are available upon request. The resulting amplification products were verified on a 2% ethidium bromide agarose gel. The purified PCR products were sequenced using a conventional Sanger method. Sequencing results were analyzed using Mutation Surveyor software (SoftGenetics LLC, PA, USA). For NGS methods, patients were analyzed using a targeted NGS gene panel (v14) for hereditary hemochromatosis and hyper/hypoferritinemia that included the following nine genes: HFE, HFE2, HAMP, TFR2, SLC40A1, BMP6, FTL, FTH1, and GNPAT. Briefly, the capture of genomic regions was conducted starting from 225 ng of gDNA using a custom design HaloPlexTM Target Enrichment 1–500 kb kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. Library quality was determined using the Agilent 4200 TapeStation System. The quantity was measured using a QubitdsDNA assay kit with a Qubitfluorometer (Life Technologies, Carlsbad, CA, USA) and fluorescence was detected using a SpectraMax Gemini EM microplate reader (Molecular Devices). Libraries were sequenced using MiSeq reagent kit v2 (300 cycles) (Illumina, San Diego, CA, USA) on an Illumina or a MiSeq or MiniSeq sequencer (Illumina, San Diego, CA, USA), generating 150-bp paired-end reads. Samples were aligned with the reference human genome GRCh37/hg19 and data analysis was performed using our algorithms. Mutations detected by NGS were confirmed by conventional Sanger sequencing. Genetic variants are reported following the official Human Genome Variation Sequence (HGVS) nomenclature and refer to NM_ 000146.3 for the Homo sapiens FTL transcript variant and NP_ 000137.2 for the Homo sapiens FTL protein. Reported mutations in this study have been submitted to the Leiden Open Variation Database (http://www.lovd.nl) or to ClinVar (http://www.ncbi.nlm.nih.gov/clinvar). Pharmaceuticals 2019, 12, 17 12 of 15 Pharmaceuticals 2019, 11, x FOR PEER REVIEW 12 of 15 Figure 4. Figure 4. Algorithm for diagnosis Algorithm for of diseases diagnosis of caused by diseases caused by defects defects in in FTL FTL gene. gene. The The following following abbreviations abbreviations were used: F, were used: female; M, F, female; M, male. male. The The mutation mutation nomenclature nomenclature used follows used follows the the HGVS HGVS guidelines. guidelines. Pharmaceuticals 2019, 12, 17 13 of 15 4.3. FTL RNA Fold Predictions RNA folding analysis was carried out to predict the IRE structure of wild-type (WT) and mutated FTL-IRE using the Sfold web server (http://sfold.wadsworth.org/) [20]. DNA sequences used for folding predictions are shown in Supplementary Material Table S2. The SIREs web server tool was also used to predict iron-responsive elements [19]. Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/12/1/17/s1, Table S1: Table summarizing all FTL mutations identified up now in the literature, including the two novel mutations described here. Table S2: DNA sequence for FTL RNA fold predictions. Author Contributions: Conceptualization, M.S. and B.C.; Methodology, C.T., B.C., and M.S.; Software, C.T.; Validation, J.F.-T.; Formal Analysis, J.F.-T.; Investigation, J.F.-T. and B.C.; Resources, S.P.-M., I.H.-R., J.L.F., M.E.L., A.M.G., and M.B.-C.; Data Curation, J.F.-T.; Writing—Original Draft Preparation, B.C., J.L.R., and M.S.; Writing—Review and Editing, M.S., I.H.-R., J.L.F., and M.B.-C.; Visualization, B.C. and M.S.; Supervision, M.S.; Project Administration, M.S.; Funding Acquisition, M.S. Funding: This research was partially supported by grant SAF2015-70412-R from Spanish Secretary of Research, Development and Innovation (MINECO) Spain to M.S. 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