A novel mutation of HFE explains the classical phenotype of genetic hemochromatosis in a C282Y heterozygote

Background & Aims: Most patients with genetic hemochromatosis are homozygou s for a single mutation of the HFE gene (C282Y). There is a second mutation , H63D, but its role in iron overload is less conclusive. The aim of this s tudy was to investigate the basis of iron overload in a patient with classi cal hemochromatosis who was only heterozygous for C282Y and negative for H6 3D. Methods: Genotype for the C282Y, H63D, and S65C mutations of HFE was de termined in patient RFH, his family members, and 365 controls. The HFE gene was sequenced in patient RFH. Allele-specific reverse-transcription polyme rase chain reaction was performed to investigate RNA splicing. Allele frequ ency was determined by allele-specific oligonucleotide hybridization. Resul ts: The patient is compound heterozygous for C282Y and a novel splice site mutation (IVS3 + 1G --> T). His sister has an identical genotype and elevat ed serum ferritin and transferrin saturation. The novel mutation functional ly alters messenger RNA splicing, causing obligate skipping of exon 3. Howe ver, the IVS3 + 1G --> T mutation was found to be rare and was not detected in 630 control European chromosomes. Conclusions: IVS3 + 1G --> T in the c ompound heterozygous state with C282Y results in iron overload that can pro gress to a severe phenotype of classical hemochromatosis. The demonstration of IVS3 + 1G --> T highlights the possibility of other rare HFE mutations, particularly in C282Y heterozygotes with iron overload.