SLC40A1- R178G mutation and ferroportin disease
Authors: Matthaios Speletas, Elias Onoufriadis, Anna Kioumi, Anastasios E. Germenis
Journal: Journal of Hepatology (2011)

Ferroportin disease: A systematic meta-analysis of clinical and molecular findingsJournal of HepatologyVol. 53Issue 5PreviewClassical ferroportin disease is characterized by hyperferritinemia, normal transferrin saturation, and iron overload in macrophages. A non-classical form is characterized by additional hepatocellular iron deposits and a high transferrin saturation. Both forms demonstrate autosomal dominant transmission and are associated with ferroportin gene (SLC40A1) mutations. SLC40A1 encodes a cellular iron exporter expressed in macrophages, enterocytes, and hepatocytes. The aim of the analysis is to determine the penetrance of SLC40A1 mutations and to evaluate in silico tools to predict the functional impairment of ferroportin mutations as an alternative to in vitro studies. Full-Text PDF Open AccessReply to: SLC40A1-R178G mutation and ferroportin diseaseJournal of HepatologyVol. 55Issue 3PreviewClearly, the observation that R178G was not found in 253 ethnically matched bone marrow donors, supports Dr. Speletas’ and his colleagues’ notion that R178G is a rare sequence variant. The concept that R178G is a disease causing mutation is challenged by the apparently incomplete penetrance of this mutation in the family reported. Additional clinical information provided in the letter above further supports the notion that the index case and her mother are affected by an iron storage disease that can be regarded as a genuine ferroportin disease. Full-Text PDF Open AccessSLC40A1-R178G or R178Q and ferroportin disease? A call for vigilance in mutation reportingJournal of HepatologyVol. 59Issue 2PreviewI refer to a previous Letter to the Editor and its reply [1] regarding the excellent meta-analysis on ferroportin disease performed by Mayr et al. in the Journal of Hepatology in November 2010 [2]. The SLC40A1-R178G mutation is discussed and whether it is a disease-causing mutation with incomplete penetrance or a polymorphism. Upon further inspection of the original report by Speletas et al., describing the R178G mutation in a Greek family, published in the journal Blood Cell Molecules and Diseases in 2008 [3], I have come to the conclusion that an error was made in the designation of this mutation. Full-Text PDF Open AccessReply to: “SLC40A1-R178G or R178Q and ferroportin disease? A call for vigilance in mutation reporting”Journal of HepatologyVol. 59Issue 2PreviewWe appreciate the observation by Dr. Wallace uncovering our error in citing unwittingly the nomenclature of amino acid alteration of the mutation g.963G>A (according to the NCBI reference sequence NM_014585 ) of SLC40A1 gene, as R178G (Arg178Gly) instead of R178Q (Arg178Gln). An Erratum has already been submitted in concern to our initial publication [1]. We have to emphasize that all the data presented in both our initial publication describing this alteration and in the Letter to the Editor [2] are absolutely correct. Full-Text PDF Open Access In their excellent systematic meta-analysis on the ferroportin disease, recently published in the Journal of Hepatology, Mayr et al. present in detail all the described SLC40A1 gene alterations and their relationship with disease phenotype [[1]Mayr R. Janecke A.R. Schranz M. Griffiths W.J. Vogel W. Pietrangelo A. et al.Ferroportin disease: a systematic meta-analysis of clinical and molecular findings.J Hepatol. 2010; 53: 941-949Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar]. The prevalence of these alterations in the general population is also presented, indicating that a few of them are polymorphisms with an unknown, as yet, biological significance.Amongst these alterations, the authors include the mutation SLC40A1-R178G, described by us in a Greek family with ferroportin disease [[2]Speletas M. Kioumi A. Loules G. Hytiroglou P. Tsitouridis J. Christakis J. et al.Analysis of SLC40A1 gene at the mRNA level reveals rapidly the causative mutations in patients with hereditary hemochromatosis type IV.Blood Cells Mol Dis. 2008; 40: 353-359Crossref PubMed Scopus (20) Google Scholar], despite the fact that they refer regarding this mutation (Table 3), obviously by mistake, to another publication. To this end, it is interesting to present information derived from the 2-year follow-up of the affected members of this family, as well as the data missing from the literature population that we have acquired in the mean time, indicating that the SLC40A1-R178G mutation is not a polymorphism. Instead, they support our position that it represents a novel mutation resulting in classical ferroportin disease with a mild, and rather variable, phenotype.In our previous publication, the SLC40A1-R178G mutation was found to be responsible for a classical ferroportin disease phenotype in a 25-year-old female [[2]Speletas M. Kioumi A. Loules G. Hytiroglou P. Tsitouridis J. Christakis J. et al.Analysis of SLC40A1 gene at the mRNA level reveals rapidly the causative mutations in patients with hereditary hemochromatosis type IV.Blood Cells Mol Dis. 2008; 40: 353-359Crossref PubMed Scopus (20) Google Scholar], who remains in excellent condition being subjected to periodical venesections every three months. Examining the family tree, we detected the same mutation in the proband’s 53-year-old mother, presenting with slight hyperferritinemia and liver hemosiderosis of moderate degree. During the follow-up, her hyperfirretinemia worsened, necessitating therapeutic venesections. The SLC40A1-R178G mutation was also detected in the proband’s 87-year-old grandfather who displayed low transferrin saturation, but not hyperferritinemia. Because of his old age, this patient, the oldest one in the literature diagnosed with ferroportin disease, was not submitted to further evaluation regarding the possible coexistence of iron deficiency. Two years after the diagnosis, he remains alive without needing venesections.Following this atypical presentation of ferroportin disease and considering that the SLC40A1-R178G mutation had not been observed in large comprehensive population studies [3Beutler E. Barton J.C. Felitti V.J. Gelbart T. West C. Lee P.L. et al.Ferroportin 1 (SLC40A1) variant associated with iron overload in African-Americans.Blood Cells Mol Dis. 2003; 31: 305-309Crossref PubMed Scopus (114) Google Scholar, 4Gordeuk V.R. Caleffi A. Corradini E. Ferrara F. Jones R.A. Castro O. et al.Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene.Blood Cells Mol Dis. 2003; 31: 299-304Crossref PubMed Scopus (181) Google Scholar, 5Barton J.C. Acton R.T. Lee P.L. West C. SLC40A1 Q248H allele frequencies and Q248H-associated risk of non-HFE iron overload in persons of sub-Saharan African descent.Blood Cells Mol Dis. 2007; 39: 206-211Crossref PubMed Scopus (29) Google Scholar], we questioned whether it was a functional polymorphism prevalent in the Greek population.To this end, we analyzed 253 bone marrow donors (male/female: 123/130, mean age: 34.7 years, range: 21–60) from Central Greece area. All subjects provided written informed consent, while the study was conducted in accordance with the principles of Helsinki declaration and was approved by the Institutional Review Board of the University Hospital of Larissa, Greece. Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini kit (Qiagen, UK). The detection of the R178G mutation was performed after amplification of the exon 6 of SLC40A1 gene by PCR and subsequent restriction-digestion (RFLP, Restriction Fragment Length Polymorphism) analysis (Fig. 1). The protocol was designed based on the fact that the mutation results in the creation of a DNA sequence that is not recognized by the restriction enzyme HpyAV (New England Biolabs, UK). All PCR and digestion procedures were carried out in the PCR-engine apparatus PTC-200, MJ-Research (Watertown-Massachusetts), while the PCR and digestion products were analyzed in 2.5% TBE agarose gels (Fig. 1). For the confirmation of PCR–RFLP results, randomly chosen PCR products were purified by Qiagen PCR Purification System (Qiagen, UK) and directed sequenced using an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) and a Big Dye pTerminator DNA sequencing kit (Applied Biosystems).No individual carrying the SLC40A1-R178G mutation was found, supporting, in parallel with the findings of the above mentioned family, its causative relation with ferroportin disease.Finally, we would like to emphasize that the specific PCR–RFLP protocol utilized in this study can also be applied for the detection of another mutation found in the 178R amino acid, providing a rapid and reliable approach for the analysis of specific alterations of the SLC40A1 gene.Conflict of interestThe authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. In their excellent systematic meta-analysis on the ferroportin disease, recently published in the Journal of Hepatology, Mayr et al. present in detail all the described SLC40A1 gene alterations and their relationship with disease phenotype [[1]Mayr R. Janecke A.R. Schranz M. Griffiths W.J. Vogel W. Pietrangelo A. et al.Ferroportin disease: a systematic meta-analysis of clinical and molecular findings.J Hepatol. 2010; 53: 941-949Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar]. The prevalence of these alterations in the general population is also presented, indicating that a few of them are polymorphisms with an unknown, as yet, biological significance. Amongst these alterations, the authors include the mutation SLC40A1-R178G, described by us in a Greek family with ferroportin disease [[2]Speletas M. Kioumi A. Loules G. Hytiroglou P. Tsitouridis J. Christakis J. et al.Analysis of SLC40A1 gene at the mRNA level reveals rapidly the causative mutations in patients with hereditary hemochromatosis type IV.Blood Cells Mol Dis. 2008; 40: 353-359Crossref PubMed Scopus (20) Google Scholar], despite the fact that they refer regarding this mutation (Table 3), obviously by mistake, to another publication. To this end, it is interesting to present information derived from the 2-year follow-up of the affected members of this family, as well as the data missing from the literature population that we have acquired in the mean time, indicating that the SLC40A1-R178G mutation is not a polymorphism. Instead, they support our position that it represents a novel mutation resulting in classical ferroportin disease with a mild, and rather variable, phenotype. In our previous publication, the SLC40A1-R178G mutation was found to be responsible for a classical ferroportin disease phenotype in a 25-year-old female [[2]Speletas M. Kioumi A. Loules G. Hytiroglou P. Tsitouridis J. Christakis J. et al.Analysis of SLC40A1 gene at the mRNA level reveals rapidly the causative mutations in patients with hereditary hemochromatosis type IV.Blood Cells Mol Dis. 2008; 40: 353-359Crossref PubMed Scopus (20) Google Scholar], who remains in excellent condition being subjected to periodical venesections every three months. Examining the family tree, we detected the same mutation in the proband’s 53-year-old mother, presenting with slight hyperferritinemia and liver hemosiderosis of moderate degree. During the follow-up, her hyperfirretinemia worsened, necessitating therapeutic venesections. The SLC40A1-R178G mutation was also detected in the proband’s 87-year-old grandfather who displayed low transferrin saturation, but not hyperferritinemia. Because of his old age, this patient, the oldest one in the literature diagnosed with ferroportin disease, was not submitted to further evaluation regarding the possible coexistence of iron deficiency. Two years after the diagnosis, he remains alive without needing venesections. Following this atypical presentation of ferroportin disease and considering that the SLC40A1-R178G mutation had not been observed in large comprehensive population studies [3Beutler E. Barton J.C. Felitti V.J. Gelbart T. West C. Lee P.L. et al.Ferroportin 1 (SLC40A1) variant associated with iron overload in African-Americans.Blood Cells Mol Dis. 2003; 31: 305-309Crossref PubMed Scopus (114) Google Scholar, 4Gordeuk V.R. Caleffi A. Corradini E. Ferrara F. Jones R.A. Castro O. et al.Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene.Blood Cells Mol Dis. 2003; 31: 299-304Crossref PubMed Scopus (181) Google Scholar, 5Barton J.C. Acton R.T. Lee P.L. West C. SLC40A1 Q248H allele frequencies and Q248H-associated risk of non-HFE iron overload in persons of sub-Saharan African descent.Blood Cells Mol Dis. 2007; 39: 206-211Crossref PubMed Scopus (29) Google Scholar], we questioned whether it was a functional polymorphism prevalent in the Greek population. To this end, we analyzed 253 bone marrow donors (male/female: 123/130, mean age: 34.7 years, range: 21–60) from Central Greece area. All subjects provided written informed consent, while the study was conducted in accordance with the principles of Helsinki declaration and was approved by the Institutional Review Board of the University Hospital of Larissa, Greece. Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini kit (Qiagen, UK). The detection of the R178G mutation was performed after amplification of the exon 6 of SLC40A1 gene by PCR and subsequent restriction-digestion (RFLP, Restriction Fragment Length Polymorphism) analysis (Fig. 1). The protocol was designed based on the fact that the mutation results in the creation of a DNA sequence that is not recognized by the restriction enzyme HpyAV (New England Biolabs, UK). All PCR and digestion procedures were carried out in the PCR-engine apparatus PTC-200, MJ-Research (Watertown-Massachusetts), while the PCR and digestion products were analyzed in 2.5% TBE agarose gels (Fig. 1). For the confirmation of PCR–RFLP results, randomly chosen PCR products were purified by Qiagen PCR Purification System (Qiagen, UK) and directed sequenced using an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) and a Big Dye pTerminator DNA sequencing kit (Applied Biosystems). No individual carrying the SLC40A1-R178G mutation was found, supporting, in parallel with the findings of the above mentioned family, its causative relation with ferroportin disease. Finally, we would like to emphasize that the specific PCR–RFLP protocol utilized in this study can also be applied for the detection of another mutation found in the 178R amino acid, providing a rapid and reliable approach for the analysis of specific alterations of the SLC40A1 gene. Conflict of interestThe authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.