Background and Aim. Defects in several proteins involved in iron metabolism can lead to pathological iron accumulation. Autosomal dominant HFE-related Hereditary Hemochromatosis (HH) is the most frequent form of genetically determined iron overloaded (60-90% of cases) disorders, whose penetrance is modulated by both genetic and environmental factors.“Classic” HFE-related HH is due to homozygosity for the C282Y mutation or to compound heterozygosity between the C282Y and the H63D mutation. The search for these two common mutations, easily and cheaply detectable with standard PCR-based techniques, represents the so-called “first level” genetic test for the diagnosis of HH. However, other rare HFE variants have been occasionally reported, and the HH phenotype can also be due to mutations in at least four other genes (HFE2, HAMP, TFR2 and SLC40A1). Of note, mutations in these genes are typically “private” and require sequencing, making the molecular diagnosis of “non-HFE” HH quite difficult and expensive. This pilot study was designed to develop a high-performance next generation sequencing (NGS) technique for the molecular diagnosis of “non-classic” HH, including those cases due to digenic inheritance or to the compound heterozygosity for mutations in any of the five HH genes and concurrent beta-thalassemia trait, a condition relatively frequent in Italy. Methods and Population. The exomic region of 340Kb comprises of 5 HH-related genes (HFE, HFE2, HAMP, TFR2, SLC40A1) was captured and re-sequenced in 61 patients attending our tertiary care center for iron overload disorders. Among them, some had known mutations in the HH genes determined by either standard PCR-based techniques (i.e. the C282Y and the H63D mutation on HFE) or traditional Sanger Sequencing (i.e. mutations in “non-HFE” genes previously described). The remaining had relevant biochemical signs of iron overload that could be considered of “unknown origin” (including some with simple C282Y or H63D heterozygosity). A similar number (n=72) of subjects with normal serum iron parameters were included as controls. Halo-Plex™ Technology, which produces circularized fragments suitable for Illumina paired-end sequencing (using the IlluminaHiSeq 1000 platform) was used to capture the targeted region. Raw reads were filtered by quality and then aligned against human reference HG19 version 37using BWA with standard parameters. On average we obtained 354x sequence coverage for captured regions. These alignments were used to detect variants in each sample using GoldenHelix™ software . Results. In the iron overload patients the known and reported mutations were confirmed, and several new “non-synonymous variants” (according to bioinformatics tools based on publicly available databases including the 1000-genomes project) were found. Of these, 3 were in the HFE gene, 15 in the HFE2 gene, 13 in the SCL40A1 gene, 42 in the TFR2 gene, and 1 in the HAMP gene. Many of these variants were relatively frequent and detected also in controls, thus being likely not pathogenetic. However, we also found some “private” variants potentially pathogenetic that are currently under further characterization. Significance and Future Perspectives. Halo-Plex™ targeted exome capturing technique combined with high resolution NGS technology is a promising approach for increasing the knowledge of the molecular basis of non-HFE HH, with potential implementation as “second level” diagnostic test in those cases that remain unexplained after application of standard first level molecular testing for HFE C282Y and H63D mutations.
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