Functional characterization of a novel non-coding mutation "Ghent +49A > G" in the iron-responsive element of L-ferritin causing hereditary hyperferritinaemia-cataract syndrome.

Hereditary hyperferritinaemia-cataract syndrome (HHCS) is a rare disorder usually caused by heterozygous mutations in the iron-responsive element (IRE) in the 5' untranslated region (5'UTR) of the L-ferritin gene (FTL), disturbing the binding of iron regulatory proteins (IRPs) and the post-transcriptional regulation of ferritin expression. Here, the proband of a consanguineous family displayed moderate bilateral cataracts and elevated serum ferritin in the absence of iron overload. The parents and siblings showed variable degrees of mild bilateral cataracts combined with elevated levels of circulating ferritin. Sequencing of FTL identified a novel 5'UTR mutation c.-151A > G, also named "Ghent +49A > G". The zygosity of the mutation, occurring in homozygous and heterozygous state in the proband and other affected family members respectively, correlated well with severity of ophthalmological and hematological manifestations. The substitution is expected to impair the secondary structure of the upper IRE stem. Functional characterization of +49A > G by electrophoretic mobility shift assays demonstrated a reduced binding affinity for IRP1 compared to the wild-type IRE of FTL. Overall, we have expanded the repertoire of deleterious biallelic FTL IRE mutations in HHCS with this novel +49A > G mutation, the zygosity of which correlated well with the disease expression.

The actin-binding protein profilin 2 is a novel regulator of iron homeostasis.

Cellular iron homeostasis is controlled by the iron regulatory proteins (IRPs) 1 and 2 that bind cis-regulatory iron-responsive elements (IRE) on target messenger RNAs (mRNA). We identified profilin 2 (Pfn2) mRNA, which encodes an actin-binding protein involved in endocytosis and neurotransmitter release, as a novel IRP-interacting transcript, and studied its role in iron metabolism. A combination of electrophoretic mobility shift assay experiments and bioinformatic analyses led to the identification of an atypical and conserved IRE in the 3' untranslated region of Pfn2 mRNA. Pfn2 mRNA levels were significantly reduced in duodenal samples from mice with intestinal IRP ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 in HeLa and Hepa1-6 cells reduced their metabolically active iron pool. Importantly, Pfn2-deficient mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus, and midbrain) and reduction of the hepatic iron store without anemia. Despite low liver iron levels, hepatic hepcidin expression remained high, likely because of compensatory activation of hepcidin by mild inflammation. Splenic ferroportin was increased probably to sustain hematopoiesis. Overall, our results indicate that Pfn2 expression is controlled by the IRPs in vivo and that Pfn2 contributes to maintaining iron homeostasis in cell lines and mice.