Scanning mutations of the 5'UTR regulatory sequence of l-ferritin by denaturing high-performance liquid chromatography: identification of newmutations

Background: Hereditary hemochromatosis is a recessive
disorder characterized by iron accumulation in parenchymal
cells, followed by organ damage and failure.
The disorder is mainly attributable to the C282Y and
H63D mutations in the HFE gene, but additional mutations
in the HFE, transferrin receptor 2 (TfR2), and
hepcidin genes have been reported. The copresence of
mutations in different genes may explain the phenotypic
heterogeneity of the disorder and its variable
penetrance.
Methods: We used denaturing HPLC (DHPLC) for rapid
DNA scanning of the HFE (exons 2, 3, and 4), hepcidin,
and TfR2 (exons 2, 4 and 6) genes in a cohort of 657
individuals with altered indicators of iron status.
Results: DHPLC identification of C282Y and H63D HFE
alleles was in perfect agreement with the restriction
endonuclease assay. Fourteen DNA samples were heterozygous
for the HFE S65C mutation. In addition, we
found novel mutations: two in HFE (R66C in exon 2 and
R224G in exon 4), one in the hepcidin gene (G71D), and
one in TfR2 (V22I), plus several intronic or silent substitutions.
Six of the seven individuals with hepcidin or
TfR2 coding mutations carried also HFE C282Y or S65C
mutations.
Conclusion: DHPLC is an efficient method for mutational
screening for the genes involved in hereditary
hemochromatosis and for the study of their copresence.