Targeted next-generation sequencing in the molecular diagnosis of hereditary hemochromatosis

Abstract

Hereditary hemochromatosis (HH) is a genetic disorder of iron metabolism characterized by excessive iron accumulation and considerable phenotypic variability, often making diagnosis challenging. This study aimed to identify pathogenic variants associated with HH using targeted next-generation sequencing (NGS). In addition, we investigated rare and novel genetic variants to improve diagnostic accuracy and support more personalized patient management strategies. A targeted NGS panel encompassing 12 genes involved in HH and iron homeostasis was applied. Variant interpretation followed the American College of Medical Genetics and Genomics (ACMG) guidelines, with all findings confirmed by PCR–Sanger sequencing. Structural analyses were conducted exclusively for variants of uncertain significance (VUS) using AlphaFold3 and visualized in PyMOL. Pathogenic variants were detected, notably the established HJV- p.Gly320Val variant, supporting a diagnosis of juvenile HH. An ultra-rare variant of uncertain significance, ERFE -c.478G > A (p.Ala160Thr), was also identified, with structural modeling indicating potential alterations that could affect protein function. Additionally, the established SLC40A1 -p.Arg178Gln variant was detected in a control sample, validating the robustness of the methodology. Overall, these results highlight the utility of targeted NGS and structural modeling in improving diagnostic accuracy and enabling precision medicine approaches in HH.