Hemochromatosis type 4 is a rare form of primary iron overload transmitted as an 
autosomal dominant trait caused by mutations in the gene encoding the iron 
transport protein ferroportin 1 (SLC40A1). SLC40A1 mutations fall into two 
functional categories (loss- versus gain-of-function) underlying two distinct 
clinical entities (hemochromatosis type 4A versus type 4B). However, the vast 
majority of SLC40A1 mutations are rare missense variations, with only a few 
showing strong evidence of causality. The present study reports the results of 
an integrated approach collecting genetic and phenotypic data from 44 suspected 
hemochromatosis type 4 patients, with comprehensive structural and functional 
annotations. Causality was demonstrated for 10 missense variants, showing a 
clear dichotomy between the two hemochromatosis type 4 subtypes. Two subgroups 
of loss-of-function mutations were distinguished: one impairing cell-surface 
expression and one altering only iron egress. Additionally, a new 
gain-of-function mutation was identified, and the degradation of ferroportin on 
hepcidin binding was shown to probably depend on the integrity of a large 
extracellular loop outside of the hepcidin-binding domain. Eight further 
missense variations, on the other hand, were shown to have no discernible 
effects at either protein or RNA level; these were found in apparently isolated 
patients and were associated with a less severe phenotype. The present findings 
illustrate the importance of combining in silico and biochemical approaches to 
fully distinguish pathogenic SLC40A1 mutations from benign variants. This has 
profound implications for patient management.