| Summary: | Introduction: Hereditary Hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive intestinal iron absorption and iron deposition in several organs leading to cardiac failure, cirrhosis and hepatocellular carcinoma. Molecular diagnosis of HFE-related HH is typically made by searching for specific genotypes (C282Y homozygosity or C282Y/H63D compound heterozygosity), denominated the “first level genetic test”. However, in the Mediterranean area, up to one third of patients with a clinical diagnosis of hemochromatosis do not present those mutations. This pilot study was designed to develop a “second level genetic test” based on next generation sequencing (NGS) for rapid and simultaneous analysis of 6 HH-related genes (HFE, TfR2, HJV, HAMP, SLC40A1 and FTL). A second objective was to establish genotype/phenotype associations. Patients and Methods: A TruSeq Custom Amplicon (TSCA, by Illumina) kit was designed in order to generate 97 amplicons covering exons, intron/exon junctions and UTRs of the mentioned genes with a cumulative target sequence of 12115bp. Amplicons were sequenced in the MiSeq instrument (IIlumina) using 250bp pared-end reads. Sequences were aligned against human genome reference hg19 using alignment and variant caller algorithms in the MiSeq reporter software. Firstly, some controls presenting known mutations were sequenced in order to validate the test. Subsequently, 88 iron overload patients with a negative first level test were studied according to previous conditions. Results: We found a total of 55 variants in the 6 selected genes. These include novel missense and splicing variants, a mutation that originates a novel translation initiation codon, among others. Novel potentially pathogenic variants were validated by Sanger sequencing and their functional significance are currently under study. An unusual clinical case will be presented. Discussion: The merger between TSCA methodology and NGS technology appears to be an appropriate tool for simultaneous and fast analysis of HH-related genes in a large number of samples. However, establishing the clinical relevance of NGS-detected variants for HH development remains a hard-working task, requiring further functional studies.
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