Background and Aim: Molecular diagnosis of HFE-related hereditary hemochromatosis (HH) is typically made by searching for the C282Y and H63D mutations (first level genetic test). However, in the Mediterranean area up to one third of patients with HH phenotype do not have the “diagnostic” genotypes (C282Y homozygosity, or C282Y/H63D compound heterozygosity). This pilot study was designed to develop a “second level” next generation sequencing (NGS)-based test for rapid and simultaneous analysis of the five HH genes (HFE, HFE2, HAMP, TFR2 and SLC40A1). Methodology: we studied 61 patients with relevant biochemical signs of iron overload (IO) and non-diagnostic first level genetic test suggesting a possible “non-HFE” HH. The five HH genes were captured by Halo-Plex™ technology, and then sequenced using aNGS platform (Illumina HiSeq 1000). Sequenced reads were aligned against human reference HG19 and analyzed by GoldenHelix™ software to annotate all the variants possibly involved in the disease. Results: In IO patients a large number of new non-synonymous variants (according to bioinformatics tools based on publicly available databases including the 1000-genomes project) were found. Many of them were relatively frequent and detected also in controls, thus being considered likely “non-pathogenic”, unless clearly enriched in patients. On the other hand, some rare variants (i.e. limited to a single or very few individuals), particularly in SCL40A1, TFR2, and HFE, were found exclusively in patients, and could be considered “potentially pathogenic”. Conclusions: The combination of the Halo-Plex™ approach with NGS platform and GoldenHelix™ algorithm appears a suitable approach for a better molecular characterization of patients with unexplained HH phenotype, and could represent a good option for second level genetic testing in referral centers. However, establishing the clinical relevance of NGS-detected “novel” genetic variants in a prevalently autosomal recessive disorder like HH remains a difficult task, requiring further functional studies and national/international collaborative efforts.
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