Mutations in the SLC40A1 gene result in a dominant genetic disorder [ferroportin 
disease; hereditary hemochromatosis type (HH) IV], characterized by iron 
overload with two different clinical manifestations, normal transferrin 
saturation with macrophage iron accumulation (the most prevalent type) or high 
transferrin saturation with hepatocyte iron accumulation (classical 
hemochromatosis phenotype). In previous studies, the mutational analysis of 
SLC40A1 gene has been performed at the genomic DNA level by PCR amplification 
and direct sequencing of all coding regions and flanking intron-exon boundaries 
(usually in 9 PCR reactions). In this study, we analyzed the SLC40A1 gene at the 
mRNA level, in two RT-PCR reactions, followed by direct sequencing and/or NIRCA 
(non-isotopic RNase cleavage assay). This protocol turned out to be rapid, 
sensitive and reliable, facilitating the detection of the SLC40A1 gene mutations 
in two patients with hyperferritinemia, normal transferrin saturation and iron 
accumulation predominantly in macrophages and Kupffer cells. The first one 
displayed the well-described alteration V162 Delta and the second a novel 
mutation (R178G) that was further detected in two relatives in a pedigree 
analysis. The proposed procedure would facilitate the wide-range molecular 
analysis of the SLC40A1 gene, contributing to better understanding the 
pathogenesis of the ferroportin disease.