The functional significance of E 277 K and V 295 A HFE mutations

Summary Hereditary haemochromatosis ( HH ) is an autosomal recessive disorder characterized by excessive intestinal iron absorption resulting in increased pathological body iron stores. It is typically associated with homozygosity for the c.845 G > A (p. C 282 Y ) mutation in the HFE gene. However, other HFE alterations have been reported in affected individuals but their association with the disease is unclear. This study analysed the functional consequences of two HFE mutations, c.829 G > A (p. E 277 K ) and c.884 T > C (p. V 295 A ). Firstly, it was shown that c.829 G > A affects the HFE splicing by diminishing the full length HFE and ivs4_66bp inclusion transcript levels, while increasing the amount of exon 4 skipping transcript. Immunofluorescent techniques showed that the HFE _ E 277 K protein had a diffuse distribution (similar to HFE _ C 282 Y ) while HFE _ V 295 A presented at the cell surface and perinuclear compartments (resembling HFE _wt). Immunoprecipitation assays revealed a decreased association of HFE _ E 277 K and HFE _ V 295 A with both β2‐microglobulin ( B 2 M ; 38 ± 7% and 66 ± 8%, respectively) and transferrin receptor ( TFRC , also termed TFR 1) (58 ± 2% and 49 ± 16%, respectively). Herein, we prove that both mutations partially abrogate HFE association with B 2 M and TFRC , crucial for its correct processing and cell surface presentation. Although E 277 K has a more deleterious effect than V 295 A , we propose that both mutations may play a role in the development of hereditary haemochromatosis.