Clinical application of NGS in the diagnosis of iron overload disorders or hyperferritinemia of genetic origin

Background The liver is central in the pathophysiology and clinical manifestations of most genetic iron-loading disorders. Although HFE- hemochromatosis is the most prevalent condition of hereditary iron overload in Caucasians, a significant portion of patients with clinical suspicion of iron-loading disorder or hyperferritinemia of genetic origin do not have a diagnostic HFE genotype. Aim To investigate, through Next Generation Sequencing (NGS) technology, the presence of pathogenic variants in candidate “iron genes” in patients with clinical suspicion of genetic iron overload disorders and/or hyperferritinemia. Materials and Methods 94 patients with a hemochromatosis-like phenotype (HC-group) and 55 cases with clinical suspicion of hereditary hyperferritinemia (HF-group) were enrolled. A custom AmpliSeqTM NGS panel of 32 genes involved in iron homeostasis was tested using the Ion Torrent PGM platform. Literature information and in silico predictions were used for the prioritization of possibly pathogenic variants. Results In both groups, HFE and CP were respectively the first and second most frequently mutated genes, followed by TFR2 in HC-group and SLC40A1 in HF-group. In HC-group, 43.7% of patients carried a genotype diagnostic for hemochromatosis; in HF-group, 23.6% of patients carried a genotype known to cause hyperferritinemia. Most patients presented single-gene variants: the iron genes that emerged as solely mutated are HFE, HJV, TFR2, TF, and SLC40A1 for HC-group and HFE, SLC40A1, CP, FTL, TMPRSS6, NEO1, and TFRC for HF-group. Novel variants were identified in some of these genes. In HC-group, 40.5% of patients carried only heterozygous variants and always presented at least one variant in HFE, HJV, TFR2, SLC40A1, or TF. Conclusion NGS contributes to the diagnostic workup of patients with non-HFE hemochromatosis or other iron disorders by identifying possible pathogenic variants or disease contributors/modifiers. Our data support the polygenic origin of certain phenotypes and indicate novel variants to take forward for functional or experimental validation. The liver is central in the pathophysiology and clinical manifestations of most genetic iron-loading disorders. Although HFE- hemochromatosis is the most prevalent condition of hereditary iron overload in Caucasians, a significant portion of patients with clinical suspicion of iron-loading disorder or hyperferritinemia of genetic origin do not have a diagnostic HFE genotype. To investigate, through Next Generation Sequencing (NGS) technology, the presence of pathogenic variants in candidate “iron genes” in patients with clinical suspicion of genetic iron overload disorders and/or hyperferritinemia. 94 patients with a hemochromatosis-like phenotype (HC-group) and 55 cases with clinical suspicion of hereditary hyperferritinemia (HF-group) were enrolled. A custom AmpliSeqTM NGS panel of 32 genes involved in iron homeostasis was tested using the Ion Torrent PGM platform. Literature information and in silico predictions were used for the prioritization of possibly pathogenic variants. In both groups, HFE and CP were respectively the first and second most frequently mutated genes, followed by TFR2 in HC-group and SLC40A1 in HF-group. In HC-group, 43.7% of patients carried a genotype diagnostic for hemochromatosis; in HF-group, 23.6% of patients carried a genotype known to cause hyperferritinemia. Most patients presented single-gene variants: the iron genes that emerged as solely mutated are HFE, HJV, TFR2, TF, and SLC40A1 for HC-group and HFE, SLC40A1, CP, FTL, TMPRSS6, NEO1, and TFRC for HF-group. Novel variants were identified in some of these genes. In HC-group, 40.5% of patients carried only heterozygous variants and always presented at least one variant in HFE, HJV, TFR2, SLC40A1, or TF. NGS contributes to the diagnostic workup of patients with non-HFE hemochromatosis or other iron disorders by identifying possible pathogenic variants or disease contributors/modifiers. Our data support the polygenic origin of certain phenotypes and indicate novel variants to take forward for functional or experimental validation.