Molecular Mechanism of Hepcidin Deficiency in a Patient with Juvenile Hemochromatosis.

In eukaryotes protein translation is initiated at an AUG codon typically located within a so-called Kozak sequence (GCCRCC AUG G), a motif that presumably promotes the correct positioning of the mRNA in the ribosome. Recently, we described a patient suffering from juvenile hemochromatosis resulting from a lack of hepcidin production (Blood;15 June 2004; Epub ahead of print). We further revealed that this defect coincided with a G-to-A point mutation at position +14 of the 5′untranslated region (5′UTR) of the HAMP gene. This nucleotide change led to the creation of an ATG codon, out of frame with the physiological pre-hepcidin initiator located at positions +39-41. We speculated that this new ATG, which was embedded within a Kozak consensus sequence, acted as an aberrant translation initiation site that precluded hepcidin synthesis. In order to verify this hypothesis, we cloned in a mammalian expression vector the wild-type and mutant HAMP 5′UTR upstream of a modified EGFP cDNA, replacing the EGFP ATG by the HAMP physiological initiation codon. The resulting vectors were transfected into 293T cells. Presence of the additional ATG in the HAMP 5′UTR led to a 93 % decrease in GFP expression compared with wild-type. To confirm that this effect resulted from aberrant translation initiation at this site, a one-nucleotide deletion was introduced at the very 5′ end of the EGFP coding sequence, in order to place it in frame with the mutant HAMP 5′UTR proximal ATG (GTG in the wild-type sequence). This restored GFP expression to levels obtained when its coding sequence was in frame with the physiological hepcidin ATG of a wild-type HAMP 5′UTR. Our results very strongly suggest that the G to A mutation at position +14 of the HAMP gene 5′UTR blocks hepcidin production by inducing aberrant translational initiation of the pre-hepcidin mRNA. Whether a stable protein is synthesized from the mutated HAMP mRNA, which does not contain a stop codon in frame with the +14 AUG, remains to be determined. It is however unlikely that such protein contributes to the pathology observed in our patient, since the two other cases of hepcidin deficiency reported so far exhibit the same phenotype as our patient in spite of harbouring different mutations in the HAMP gene.