METHODS
Patients . We enrolled 4 families with history of hematological disorders. Each family comprised two siblings, of which at least one was diagnosed and treated in our Institute. For routine clinical management, patients underwent bone marrow (BM) evaluation including morphological, immunophenotypic and cytogenetic analysis. The main patient characteristics are reported in Table 1. All patients gave written informed consent to their participation to the diagnostic and treatment program, according to the IEO ethical committee approval.
Molecular analyses . We extracted DNA from BM aspirates or peripheral blood draws for the analysis of somatic variants, and from buccal swabs for the analysis of germline variants, using the QIAamp DNA mini kit following the manufacturer’s instructions (Qiagen). As previously described, we performed two genomic characterizations: one for routine clinical practice and one for research purposes. For clinical assessment, we used a commercially available diagnostic NGS assay, the Oncomine Myeloid Research Assay (40 key DNA target genes and 29 driver genes in a broad fusion panel). Data analysis was performed with IonReporter software applying the last release of Myeloid workflow (ThermoFisher Scientific). For research purposes, we used the Myelo-panel, a custom gene panel for analysis of 256 cancer-predisposing genes, purposely designed to stratify patients for possible targeted therapies and to identify germline variants associated with predisposition to develop myeloid neoplasms. The 256 cancer-predisposing genes include: 79 susceptibility genes, the most frequently associated with risk of development of hematological tumors; 38 AML drivers, with a key role in leukemogenesis; 113 actionable genes, with a concrete clinical manageability; 26 genes belonging to more than 1 of these categories and 126 pharmacogenomics single nucleotide polymorphisms (SNPs), allelic variants associated with susceptibility to certain drug (see Supplementary Table S1 for gene list).
Library preparation was performed using Ion Torrent Ion AmpliSeq Library Kit 2.0 and libraries were sequenced with Ion S5 system according to the manufacturer’s recommendations (ThermoFisher Scientific). Variant analysis was performed with Ion Reporter software and filtering as previously described. Variants passing our filters were annotated in terms of pathogenicity using different computational tools, including ClinVar, CancerVar, Intervar, Varsome and RENOVO.