Identification of Novel Mutations by Targeted NGS Panel in Patients with Hyperferritinemia

Abstract

Background: Several inherited diseases cause hyperferritinemia with or without iron overload. Differential diagnosis is complex and requires an extensive work-up. Currently, a clinical-guided approach to genetic tests is performed based on gene-by-gene sequencing. Although reasonable, this approach is expensive and time-consuming and Next Generation Sequencing (NGS) technology may provide cheaper and quicker large-scale DNA sequencing.

Methods: We analysed 36 patients with non-HFE-related hyperferritinemia. Liver iron concentration was measured in 33 by magnetic resonance. A panel of 25 iron related genes was designed using SureDesign software. Custom libraries were generated and then sequenced using Ion Torrent PGM.

Results: We identified six novel mutations in SLC40A1, three novel and one known mutation in TFR2, one known mutation and a de-novo deletion in HJV, and a novel mutation in HAMP in ten patients. In silico analyses supported the pathogenic role of the mutations.

Conclusions: Our results support the use of an NGS-based panel in selected patients with hyperferritinemia in a tertiary center for iron metabolism disorders. However, 26 out of 36 patients did not show genetic variants that can individually explain hyperferritinemia and/or iron overload suggesting the existence of other genetic defects or gene-gene and gene-environment interactions needing further studies.

Keywords: ferritin; ferroportin; hemochromatosis; hemojuvelin; hepcidin; iron overload; next generation sequencing; transferrin receptor 2.

Figure 1

Luciferase assay in HeLa cell line transfected with WT or mutated ferroportin 5′UTR luciferase constructs. Boxes denote lower quartile, mean and upper quartile, and whiskers show maximum and minimum ranges. (RLU = Relative Luciferase Unit).