Expression of FPDMM biomarkers and assesment of acquired somatic variants. (A) Integrin α₂β₁ expression, as assessed by flow cytometry using anti-integrin α₂ (CD49b) and anti-integrin β₁ (CD29) fluorescein isothiocyanate–conjugated antibodies. CD49b and CD29 binding are reported as median fluorescence intensity of platelets (gated for their forward scatter and side scatter values, and for their positivity to the platelet marker CD42b) that bound CD49b and CD29, relative to the total platelet population after setting of nonspecific binding. Data are presented as means ± SEM from 4 independent experiments for controls, and n = 1 for the propositus and the brother. (B) MYH10 expression was assessed by nested polymerase chain reaction (PCR). Total RNA was isolated from platelets and HeLa cells (used as a positive control because they express MYH10) using the TRIzol reagent (Invitrogen). A total of 500 ng of total RNA served as the template for complementary DNA (cDNA) synthesis. The RNA was treated with DNase (Invitrogen), and cDNA was then synthesized using the iScript kit (Bio-Rad, Hercules, CA). Specific primers were designed to amplify human MYH10 (primer set for primary PCR was MYH10 forward [For]: 5′-AGTTCAAGGCCACCATCTCA-3′; MYH10 reverse [Rev]: 5′-TGGAAGAGAAGCTGATGGGG-3′; primer set for secondary PCR was MYH10 For: 5′-AGTTCAAGGCCACCATCTCA-3′; MYH10 Rev: 5′-GCTCATCCTCAACCTGCATG-3′). The PCR was performed using the AccuPrime GC-rich DNA Polymerase kit (Thermo Fisher). The PCR was performed as follows: denaturation at 95°C for 3 minutes, followed by 35 cycles of 95°C for 30 minutes, 57°C for 30 seconds, and 72°C for 30 seconds and by a final elongation step at 72°C for 7 minutes. (C) Targeted custom NGS. The Myeloid Solution (MYS, SOPHiA GENECTIC, Arrow Diagnostics) was used to investigate hot spot mutations of the full coding sequence of 30 genes typically involved in myeloid neoplasms, including ABL1, ASXL1, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1, and ZRSR2. Libraries were prepared using 200 ng of peripheral blood genomic DNA, following manufacturer’s instructions. Pooled libraries were sequenced using the MiSeq Reagent kit v3 on the Illumina MiSeq Sequencer (Illumina, San Diego, CA). FASTQ files were analyzed with SOPHiA DDM software (version 5.10.54.1). Exonic, splice site, and noncoding target gene variants were taken into consideration and filtered retaining those with a global minor allele frequency <0.01 and with a variant allele frequency (VAF) >1%. For the interpretation of putative germ line variants, we referred to the American College of Medical Genetics and Genomics. Somatic variants were classified by means of the FATHMM web server (http://fathmm.biocompute.org.uk). Benign/likely benign variants were excluded from analysis. Beyond the somatic variants, the germ line RUNX1 c.784C>T p.(Gln262∗) variant was found in the 2 samples. INDEL, insertion/deletion; SNP, single nucleotide variant; NTC, no template control; OD, optical density.