Iron overload is associated with acquired and genetic conditions, the most 
common being hereditary hemochromatosis (HH) type-I, caused by HFE mutations. 
Here, we conducted a hospital-based case-control study of 41 patients from the 
São Miguel Island (Azores, Portugal), six belonging to a family with HH type-I 
pseudodominant inheritance, and 35 unrelated individuals fulfilling the 
biochemical criteria of iron overload compatible with HH type-I. For this 
purpose, we analyzed the most common HFE mutations- c.845G>A [p.Cys282Tyr], 
c.187C>G [p.His63Asp], and c.193A>T [p.Ser65Cys]. Results revealed that the 
family's HH pseudodominant pattern is due to consanguineous marriage of 
HFE-c.845G>A carriers, and to marriage with a genetically unrelated spouse that 
is a -c.187G carrier. Regarding unrelated patients, six were homozygous for 
c.845A, and three were c.845A/c.187G compound heterozygous. We then performed 
sequencing of HFE exons 2, 4, 5 and their intron-flanking regions. No other 
mutations were observed, but we identified the -c.340+4C [IVS2+4C] splice 
variant in 26 (74.3%) patients. Functionally, the c.340+4C may generate 
alternative splicing by HFE exon 2 skipping and consequently, a protein missing 
the α1-domain essential for HFE/ transferrin receptor-1 interactions. Finally, 
we investigated HFE mutations configuration with iron overload by determining 
haplotypes and genotypic profiles. Results evidenced that carriers of HFE-c.187G 
allele also carry -c.340+4C, suggesting in-cis configuration. This data is 
corroborated by the association analysis where carriers of the complex allele 
HFE-c.[187C>G;340+4T>C] have an increased iron overload risk (RR = 2.08, 95% CI 
= 1.40-2.94, p<0.001). Therefore, homozygous for this complex allele are at risk 
of having iron overload because they will produce two altered proteins--the 
p.63Asp [c.187G], and the protein lacking 88 amino acids encoded by exon 2. In 
summary, we provide evidence that the complex allele HFE-c.[187C>G;340+4T>C] has 
a role, as genetic predisposition factor, on iron overload in the São Miguel 
population. Independent replication studies in other populations are needed to 
confirm this association.