Identification of a Novel SLC40A1 Arg88Ile Mutation in a Patient with Familial Iron Overload Treated By Phlebotomy

INTRODUCTION: The cellular iron exporter ferroportin, encoded by the SLC40A1 gene, plays a key role in systemic iron regulation by mediating the absorption of dietary iron from duodenal enterocytes and the release of macrophage iron stores into the plasma. SLC40A1 mutations result in a clinically heterogeneous iron overload disorder exhibiting autosomal dominant transmission. Mutations that impair iron export function result in a classical ferroportin disease phenotype characterized by hyperferritinemia, normal transferrin saturation, and macrophage iron loading, while mutations that impair the regulation of ferroportin by hepcidin result in a non-classical form of disease exhibiting high transferrin saturation and hepatocellular iron loading. Here we report the clinical phenotype of a male patient with a personal and family history of iron overload who was found to harbor a novel SLC40A1 mutation.

CLINICAL HISTORY: A 39-year-old male of Italian descent came to clinical attention after laboratory evidence of iron overload was detected at the time of a routine physical exam. Serum ferritin was markedly elevated at 5018 ng/mL, while transferrin iron saturation was within the normal range at 42%. A complete blood count revealed hemoglobin 16.1 g/dL, hematocrit 48.7%, and MCV 96.7 fL. Liver function tests revealed mild elevation of transaminases (AST 50 U/L, ALT 114 U/L). HBV and HCV serologies, as well as an anti-nuclear antibody screen, were negative. Rheumatoid factor, ceruloplasmin, and alpha-fetoprotein were within the normal range. Genetic testing for the HFE C282Y, H63D, and S65C variants was negative. Abdominal ultrasound revealed a somewhat course and echogenic liver. The patient's past medical and surgical histories were non-contributory. Family history was notable for a 63-year-old father with non-HFE hemochromatosis treated by phlebotomy, as well as a possible history of iron overload in the paternal grandmother. There was no known family history of hepatocellular carcinoma. The patient reported consuming ≤ 5 alcoholic beverages per week. Review of systems was negative, and no organomegaly was detected on physical exam. The patient began undergoing phlebotomy approximately every 2 weeks, which now has been well tolerated for almost two years. His most recent ferritin level was within the normal range (328 ng/mL).

METHODS: Using genomic DNA extracted from peripheral blood as template, all coding regions and intron-exon boundaries of SLC40A1 were amplified by polymerase chain reaction and analyzed by bidirectional Sanger sequencing. Sequence chromatograms were analyzed using Sequencher software. This study was approved by the Yale University Human Investigation Committee (protocol #010412377).

RESULTS: A heterozygous single nucleotide substitution (c.263G>T, encoding p.Arg88Ile) was identified in exon 3 of the SLC40A1 gene (nomenclature per Ensembl reference transcript ENST00000261024). Ferroportin is a predicted multipass transmembrane protein, and Arg88 resides between two predicted transmembrane domains. Protein sequence alignments reveal that amino acid 88 is conserved as an arginine in ferroportin homologs in species as evolutionarily distant as Xenopus laevis and Danio rerio, suggesting that this residue is required for normal ferroportin function. The p.Arg88Ile variant has not been reported in the 1000 Genomes Project or the Exome Aggregation Consortium, demonstrating that it is not a polymorphism in the general population. The mutation predictor algorithms PolyPhen2, SIFT, and MutationTaster all strongly predicted this mutation to be damaging.

DISCUSSION: The ferroportin variant detected in this case (p.Arg88Ile) represents the third non-synonymous substitution at ferroportin residue 88 detected in patients with iron overload phenotypes. p.Arg88Gly was identified in a 38-year-old male who became anemic under a phlebotomy program (Cunat S., et al., Clin Chem 2007), while p.Arg88Thr was detected in multiple affected members of a single kindred in whom serial phlebotomy was well tolerated (Bach V, et al. Blood Cells Mol Dis 2006). Collectively, these findings suggest that the particular amino acid substitution at residue 88 may influence the degree of cellular iron sequestration. Future work will assess the effect of the Arg88Ile substitution on ferroportin function.