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.

Haemochromatosis in children: A national retrospective cohort promoted by the A.I.E.O.P. (Associazione Italiana Emato‐Oncologia Pediatrica) study group

“…The recurrent Italian pathogenic variant p.Asp149Thrfs*97 30 was found in the homozygous state in two siblings and in a single proband. Two brothers with TFR2‐HC carried the IVS17+5636G>A variant affecting RNA splicing 31 in homozygosity; one patient was homozygous for the already described p.Tyr250*, 28 one compound was heterozygous for the p.Asp514Metfs*12 and p.Leu224Arg 32 and one was homozygous for the p.Arg698Tyrfs*4 mutation, already described elsewhere 33 …”

Insights into the role of glycerophospholipids on the iron export function of SLC40A1 and the molecular mechanisms of ferroportin disease

“…Based on molecular dynamics (MDs) simulations performed on the experimental 3D structure of human SLC40A1 in the OF conformation, we provide initial clues by focusing on five basic amino acids (Lys90, Arg179, Arg365, Lys366, and Arg371) and undertaking functional studies. Interestingly, the p.Arg179Thr substitution has been reported in an Italian patient with unexplained hyperferritinemia 19 . We investigate this variant and provide a more definitive demonstration of its involvement in FD.…”

Insights into the role of glycerophospholipids on the iron export function of SLC40A1 and the molecular mechanisms of ferroportin disease

“…In vitro evaluation of the p.Arg179Thr substitution, currently classified as a variant of unknown significance We very recently examined the clinical significance of virtually all the missense variations reported in the SLC40A1 gene in literature, taking into account all available phenotypic, familial and functional data (8). We classified the p.Arg179Thr substitution as a variant of unknown significance, as it has been reported in single patient (or Italian origin) without evidence of tissue iron overload (19). We thought it interesting to test its impact on SLC40A1 activity in transiently transfected HEK293T cells.…”

“…Based on molecular dynamics (MD) simulations performed on the experimental 3D structure of human SLC40A1 in the OF conformation, we provide initial clues by focusing on five basic amino acids (Lys90, Arg179, Arg365, Lys366 and Arg371) and undertaking functional studies. Interestingly, the p.Arg179Thr substitution has been reported in an Italian patient with unexplained hyperferritinemia (19). We investigate this variant and provide a more definitive demonstration of its involvement in FD.…”