Targeted Next Generation Sequencing of the Five Hemochromatosis Genes in Italian Patients with Iron Overload and Non-Diagnostic First Level Genetic Test: A Pilot Study

Hereditary Hemochromatosis (HHC) is one of the most common genetic diseases in Caucasians, as it can involve up to 5 per 1,000 (0.5 percent) individuals. Early diagnosis is essential, and has greatly improved since the discovery of the HFE gene in 1996, with subsequent development of a simple genetic test for the common mutations C282Y and H63D. Indeed, the majority of HHC patients are C282Y homozygotes or C282Y/H63D compound heterozygotes, the genotypes that are considered diagnosticfor “classic” HFE-HHC. However, HHC is a genetically heterogeneous disease in which near 30 percent of patients do not have such genotypes, and are currently classified as having “non-HFE” HHC.Mutations in at least four other genes involved in modulation of the hepcidin-ferroportin axis, i.e. HAMP, HJV, TFR2, and SLC40A1 have been reported to contribute to the disease expressionin such patients. At variance with HFE, mutations in thesegenes are typically “private”, i.e. limited to just few individuals, making unfeasible the use of standardized tests for the molecular diagnosis. Recent breakthrough of new techniques collectively known as “Next Generation Sequencing” (NGS) allow DNA sequencing with unprecedented rapidity at constantly declining costs. This project was aimed to develop a targeted NGS-based testfor sequencing the five hemochromatosis genes,and then to explore its performance in unravelingpotentially pathogenic variants in HHC patients, particularly in those cases that remained unexplained after first level genetic test. We studied 47 patients with relevant biochemical signs of iron overload (IO) and non-diagnostic first level genetic test suggesting a possible “non-HFE” HHC. This was defined as the absence of the C282Y and H63D mutations, or simple heterozygosity for each of them, or even H63D homozygosis.The five HHC genes were captured by Halo-Plex™ technology, and then sequenced using a NGS platform (IlluminaHiSeq 1000). Sequenced reads were aligned against human reference HG19 and analyzed by GoldenHelix™ software to annotate all the variants possibly involved in the disease. 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”. Among the identified variants 8 were apparently new, i.e. not reported in the following databases: dbSNPs, 1000 genome, OMIM and ESP Databases. Importantly, new rare variants that explained good phenotype/genotype correlation in patients with negative first level genetic and considered to be potentially pathogenic were detected. 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 HHC 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 HHC remains a difficult task, requiring further functional studies and national/international collaborative efforts. This project is also expected to significantly contribute to better characterization of the molecular basis of HHC, and will include the possibility to perform Whole Exome Sequencing (WES) in few cases with currently unexplained HHC-like phenotypes.