<i>SLC40A1</i> c.1402G→A Results in Aberrant Splicing, Ferroportin Truncation after Glycine 330, and an Autosomal Dominant Hemochromatosis Phenotype

Abstract

Background/Aims: To determine the molecular basis of a mild hemochromatosis phenotype in a man of Scottish-Irish descent. Methods: We sequenced genomic DNA to detect mutations of HFE, SLC40A1, TFR2, HAMP, and HFE2. RNA isolated from blood mononuclear cells was used to make cDNA. RT-PCR was performed to amplify ferroportin from cDNA, and amplified products were visualized by electrophoresis and sequenced. Results: The proband was heterozygous for the novel mutation c.1402G→A (predicted G468S) in exon 7 of the ferroportin gene (SLC40A1). Located in the last nucleotide before the splice junction, this mutation results in aberrant splicing to a cryptic upstream splice site located at nt 990 within the same exon. This causes truncation of ferroportin after glycine 330 and the addition of 4 irrelevant amino acids before terminating. The truncated ferroportin protein, missing its C-terminal 241 amino acids, would lack all structural motifs beyond transmembrane region 7. The patient was also heterozygous for the common HFE H63D polymorphism, but did not have coding region mutations in TFR2, HAMP, or HFE2. Conclusions: We conclude that this patient represents a unique example of hemochromatosis due to a single base-pair mutation of SLC40A1 that results in aberrant splicing and truncation of ferroportin.

The Spectra of Disease-Causing Mutations in the Ferroportin 1 (SLC40A1) Encoding Gene and Related Iron Overload Phenotypes (Hemochromatosis Type 4 and Ferroportin Disease)

“…Apart from this type of variation, only the p.Val162 single-amino-acid in-frame deletion has been consistently associated with iron overload phenotypes. The p.Gly468Ser substitution can be considered separately, in that the underlying c.1402G>A transition disrupts the exon 7 donor site, resulting in the partial use of an exonic cryptic splice site and the generation of a truncated reading frame [27,28], while the Gly468 to Ser amino acid change by itself has no obvious effect on the ability of SLC40A1 to export iron [27].…”

Type 4B hereditary hemochromatosis associated with a novel mutation in the SLC40A1 gene

“…[ 7 – 9 ] It is assumed that the phenotypes of patients with SLC40A1 mutations vary depending on the mutation. [ 10 ] Mutations leading to ferroportin that is either not present normally at the cell surface or has defective iron export activity (loss of function) are associated with iron deposition in Kupffer cells and low transferrin saturation, [ 10 ] and define type 4A. Those mutated ferroportin that cannot be internalized after hepcidin binding (gain of function) are associated with iron deposition in hepatocytes and elevated transferrin saturation, [ 10 ] and define type 4B.…”

“…[ 10 ] Mutations leading to ferroportin that is either not present normally at the cell surface or has defective iron export activity (loss of function) are associated with iron deposition in Kupffer cells and low transferrin saturation, [ 10 ] and define type 4A. Those mutated ferroportin that cannot be internalized after hepcidin binding (gain of function) are associated with iron deposition in hepatocytes and elevated transferrin saturation, [ 10 ] and define type 4B. A systemic meta-analysis of 176 individuals reported that SLC40A1 mutations, namely, V162del, D157N, D181V, G80V, Q182H, and R489K, may impair ferroportin's iron export function, and are common mutations for type 4A HH.…”

The global prevalence of HFE and non-HFE hemochromatosis estimated from analysis of next-generation sequencing data

“…However, further functional studies or larger population studies are required to unequivocally link these variants to impaired ferroportin function and iron overload in individuals carrying these variants. The other six SLC40A1 variants identified in the ExAC data set (c.-59_-45del, p.Ile180Thr, p.Gly204Ser, p.Gly267Asp, p.Arg371Gln, and p.Gly468Ser) have all been associated with iron overload in patients and/or families, [26][27][28][29][30][31][32][33] although ferroportin containing the p.Ile180Thr variant seemed to behave similarly to the wild type when analyzed in functional assays. 30 The SLC40A1 p.Val162del variant has been reported as a cause of ferroportin disease more frequently (in nine publications) than any other SLC40A1 mutation (Supplementary Table S1 online); however, we did not detect it in any of the genomic sequence databases.…”