A novel SLC40A1 p.Y333H mutation with gain of function of ferroportin: A recurrent cause of haemochromatosis in China

Abstract

Background & Aims:Haemochromatosis type 4, also known as ferroportin disease, is an autosomal dominant genetic disorder caused by pathogenic mutations in the SLC40A1 gene, which encodes ferroportin 1 (FPN1). We have identified a novel SLC40A1 p.Y333H mutation in our previous study. In the present study, we tried to investigate the frequency and pathogenicity of the SLC40A1 p.Y333H mutation in haemochromatosis in China. Methods:Patients were analysed for SLC40A1 p.Y333H as well as mutations in the other classic haemochromatosis-related genes by Sanger sequencing. To analyse iron export capacity of the SLC40A1 p.Y333H mutant, the 293T cells were transfected with the SLC40A1 p.Y333H construct and then treated with hepcidin after exposure to ferric ammonium citrate. Cellular localization of mutant FPN1, expression of FPN1 and intracellular ferritin were analysed by immunofluorescence and Western blotting.Results: Of 22 unrelated cases with primary iron overload, three cases (3/22, 13.6%) harboured the SLC40A1 p.Y333H, with no missense mutations identified in any other classical haemochromatosis-related genes including HFE, HJV, HAMP and TFR2.Pedigree analysis showed that three probands and the son of one proband had haemochromatosis of stage 3, while the son of another proband with age of 16 showed elevated transferrin saturation but normal serum ferritin level. In vitro studies showed the mutant p.Y333H ferroportin was resistant to hepcidin, affecting the See Editorial on page 1014 | 1121 ZHANG et Al.

Iron Matryoshka—Haemochromatosis nested in Ferroportin Disease?

“…Zhang, Xu, Li and coworkers also highlights why functional testing of novel mutations is important. The specific p.Tyr333His mutation identified in this study was predicted to be functionally relevant by all bioinformatics tools applied, but only the integration of comprehensive clinical‐genetic and functional analysis show that this mutation causes a ‘gain of ferroportin function’ by mediating hepcidin resistance . How do these findings affect patient management and what is the contribution to our current understanding of haemochromatosis?…”

“…The findings presented by Drs. Zhang, Xu, Li and coworkers show that in a Chinese cohort of patients with phenotypic haemochromatosis one SLC40A1 mutation (p.Tyr333His) determining hepcidin resistance is the commonest genetic cause of hepatic iron overload . Unlike classical ferroportin disease, these patients presented with high transferrin saturation, progressive fibrosis, diabetes and arthropathy.…”

A recurrent ABCC2 p.G693R mutation resulting in loss of function of MRP2 and hyperbilirubinemia in Dubin-Johnson syndrome in China

“…To create the ABCC2 wild-type plasmid, we amplified ABCC2 from human cDNA and cloned it into Hind III/Not I sites of the pcDNA3.1 vector along with the N-terminal flag tag [8]. The ABCC2 p.G693R constructs were generated using the Gene Tailor Site-Directed Mutagenesis System (Invitrogen, Waltham, MA, USA).…”

Twenty Years of Ferroportin Disease: A Review or An Update of Published Clinical, Biochemical, Molecular, and Functional Features

“…For Cys326Tyr, Cys326Ser, Tyr501Cys and Asp504Asn, the blocked internalization of ferroportin on hepcidin exposure has been attributed to the impaired binding of hepcidin to ferroportin in the extracellular gate, while for Tyr64Asn and His507Arg—of which the location in relation to this gate varies between the available models—the impaired ubiquitination of hepcidin, which was most likely due to interference with conformation, is the most likely mechanism (Figure 4B) [30]. The Tyr333Ala, Tyr333His (both hepcidin-resistant variants in a functional test) and Tyr333Phe (hepcidin-sensitive) GOF variants display both membrane localization and intact iron export, but impaired ubiquitination after normal hepcidin binding; however, there are no data on configural changes due to presence of these variants [30,45].…”