4.2. DNA Extraction, PCR Amplification, and DNA Sequencing

Genetic studies were performed with minor differences for all the pedigrees. Genomic DNA was extracted from peripheral blood using the FlexiGene DNA kit (Qiagen) according to manufacturer’s instructions.

Ferritin gene regions (exonic, intron–exon boundaries, and untranslated regions) were sequenced using the Sanger method or next generation sequencing (NGS).

For the Sanger method, the FTL gene was amplified using 50 ng of genomic DNA. Primer sequences and PCR conditions are available upon request. The resulting amplification products were verified on a 2% ethidium bromide agarose gel. The purified PCR products were sequenced using a conventional Sanger method. Sequencing results were analyzed using Mutation Surveyor software (SoftGenetics LLC, PA, USA).

For NGS methods, patients were analyzed using a targeted NGS gene panel (v14) for hereditary hemochromatosis and hyper/hypoferritinemia that included the following nine genes: HFE, HFE2, HAMP, TFR2, SLC40A1, BMP6, FTL, FTH1, and GNPAT.

Briefly, the capture of genomic regions was conducted starting from 225 ng of gDNA using a custom design HaloPlex^TM Target Enrichment 1–500 kb kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. Library quality was determined using the Agilent 4200 TapeStation System. The quantity was measured using a QubitdsDNA assay kit with a Qubitfluorometer (Life Technologies, Carlsbad, CA, USA) and fluorescence was detected using a SpectraMax Gemini EM microplate reader (Molecular Devices). Libraries were sequenced using MiSeq reagent kit v2 (300 cycles) (Illumina, San Diego, CA, USA) on an Illumina or a MiSeq or MiniSeq sequencer (Illumina, San Diego, CA, USA), generating 150-bp paired-end reads. Samples were aligned with the reference human genome GRCh37/hg19 and data analysis was performed using our algorithms. Mutations detected by NGS were confirmed by conventional Sanger sequencing.

Genetic variants are reported following the official Human Genome Variation Sequence (HGVS) nomenclature and refer to NM_ 000146.3 for the Homo sapiens FTL transcript variant and NP_ 000137.2 for the Homo sapiens FTL protein.

Reported mutations in this study have been submitted to the Leiden Open Variation Database (http://www.lovd.nl) or to ClinVar (http://www.ncbi.nlm.nih.gov/clinvar).