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.