NEXT GENERATION TARGETED DEEP SEQUENCING OF HEMOCHROMATOSIS GENES IN IRON OVERLOADED PATIENTS: A PILOT STUDY

INTRODUCTION: Hereditary Hemocromatosis (HH) is a common and genetically heterogeneous disorder with relevant geographical differences. While in people of North European descent nearly all cases (up to 95%) are associated with homozygosity for the C282Y mutation in the HFE gene, in the Mediterranean area up to one third of patients do not carry this genotype. Other genes associated with HH include HFE2, HAMP, TFR2 and SLC40A1, but mutations are generally “private”and require sequencing. The advent of high−performance next generation sequencing (NGS) techniques, whose costs are rapidly declining, represents a possible approach for future molecular diagnosis in patients with iron overload. This pilot study was aimed to explore this approach in patients attending a tertiary care center for iron overload disorders. MATERIALS AND METHODS: We re−sequenced targeted regions (340Kb) of 5 HH−related genes (HFE, HFE2, HAMP, TFR2, SLC40A1) in 27 patients attending to our Regional Referral Centre for Iron Overload Disorders. Of them, some had known mutations in the HH genes (3 C282Y homozygotes, 1 C282Y/H63D compound heterozygote, 2 with mutations in TFR2, 3 with mutations in SLC40A1), previously determined by standard techniques or traditional sequencing. 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=27) of subjects with normal serum iron parameters were included as controls. Exon capture was performed by a new technology, i.e. the HaloPlex Target Enrichment System (Agilent), which produces circularized fragments suitable for Illumina paired−end sequencing (using the Illumina HiSeq 1000 platform). Raw reads were filtered by quality and then aligned against human reference HG19 version 37 using BWA with standard parameters. On average we had a 354x sequence coverage for captured regions. These alignments were used to detect variants in each sample using Genome Analysis ToolKit (GATK): on average, GATK found about 1500 variants in each sample. The putative effects of the called variants were evaluated using the Variant Effect Predictor by the EnsEMBL consortium. RESULTS: In the iron overloaded patients, a total of 49 potentially deleterious variants were found in the 5 selected genes (2 missense and 1 splicing variants for HFE, 10 missense variants for HFE2, 0 variants for HAMP, 28 missense and 5 splicing variants for TFR2, 2 missense and 1 splicing variants for SLC40A1). Comparison with control samples as well as the functional relevance of these variants and their possible pathogenic role in each individual patient is currently under further investigation. CONCLUSIONS: high−coverage selective exon capture is a powerful methodology for rapid DNA analysis in patients with iron overload of uncertain origin. Due to the high number of variants found, proper bioinformatic analyses are needed to reveal variants that are interesting for further clinical investigation. Anyway, as costs for NGS analyses keep to decline, they are expected to become useful tools in the near future for clinicians.