Detection of a rare mutation in the ferroportin gene through targeted next generation sequencing.

Hereditary haemochromatosis (HH) is a disorder characterised by increase of serum iron parameters and gradual iron accumulation in parenchymal organs. Since the discovery of the genetic defects of the most common form of hereditary haemochromatosis (type 1 haemochromatosis [HFE]), many observations have shown that not only rare/familial mutations in HFE can be present but also that mutations in other genes (transferrin receptor 2 [TFR2], hepcidin [HAMP], hemojuvelin [HJV], and ferroportin [SLC40A1]) can lead to rarer genetic forms of iron overload, referred as non-HFE haemochromatosis1. The genetic heterogeneity is particularly evident in the Italian population where only two-thirds of haemochromatosis patients are HFE C282Y homozygotes2, requiring expensive and time-consuming gene specific genotyping to define the molecular diagnosis. Therefore, “second level” genetic tests should be developed for a rapid and simultaneous study of the haemochromatosis genes. The most common non-HFE haemochromatosis is the autosomal dominant haemochromatosis type 4 (HH4), caused by mutations in the SLC40A1 gene, encoding for cellular iron exporter ferroportin. Ferroportin acts as receptor of hepcidin, the key regulator of iron metabolism. The hepcidin/ferroportin interaction induces the internalisation of the complex, causing a decrease in iron export and its retention in the cell3. HH4 can be phenotypically classified in two groups: patients may have hyperferritinaemia with normal/low transferrin saturation (type-A HH4, ferroportin disease) or both serum iron parameters increased (type-B HH4, non-classical ferroportin disease)4. This depends on ferroportin impairment that could be classified as “loss-of-function” or “gain-of-function”. In the first case (type-A), the mutated protein is not expressed on the membrane and it is not able to exert its exporting function. In the second case (type-B), the mutated iron exporter becomes resistant to the activity of hepcidin, causing increased iron absorption. Several mutations have been characterised in HH4 patients, spreading along the entire gene sequence4. The position of the correspondent amino acidic change is important to define the HH4 clinical phenotype5. Among the different ferroportin mutations, the p.A69T variant, despite the causal nucleotide change not being annotated in dbSNP, has been described in a 52-year old Italian woman with diabetes and type-B HH4, on the basis of hepatocyte iron overload6, but no iron parameters or clinical history of the patient were described. The pathogenic role of this mutation has been subsequently confirmed through in vitro experiments in which mutant cDNA has been over-expressed7. These studies demonstrated that p.A69T mutated ferroportin has a partial resistance to hepcidin. Here we report an Italian patient with a severe iron overload phenotype in whom a careful clinical characterisation led to a diagnose of p.A69T non-classical ferroportin disease through the combination of capture of technology with a next generation sequencing (NGS) platform.