Targeted next‐generation sequencing of familial platelet disorder with predisposition to acute myeloid leukaemia

Familial platelet disorder with propensity to acute myeloid leukaemia (FPD-AML) is a rare, autosomal dominant disorder characterized by quantitative and qualitative platelet abnormalities with a propensity to develop a myelodysplastic syndrome (MDS) or AML. FPD-AML kindred are defined by germ-line mutations of RUNX1 (Song et al, 1999), which encodes a transcription factor essential for definitive haematopoiesis and myeloid cell differentiation, commonly dysregulated by translocations, mutations or amplification in de novo and secondary MDS and acute leukaemias. Most germ line RUNX1 mutations are unique to the individual FPDAML pedigree with variability observed in the MDS or AML phenotype and the incidence of leukaemic transformation of affected individuals (Nickels et al, 2013). The spectrum of somatic genetic events associated with progression to MDS or AML have not been fully appreciated but acquisition of cytogenetic abnormalities, single gene defects that occur in de novo MDS and AML, and bi-allelic RUNX1 mutations have all been demonstrated (Minelli et al, 2004; Preudhomme et al, 2009; Shiba et al, 2012). More recently, mutations of CDC25C have been identified in approximately half of affected FPD-AML patients. CDC25C mutations appear to disrupt a critical cell cycle check point in pre-leukaemic clones, allowing subsequent acquisition of further sub-clonal mutations (Yoshimi et al, 2014). Emerging next-generation sequencing (NGS) technologies, platforms and diseasetargeted panels allow the simultaneous identification of numerous mutational events. Such a targeted NGS approach was applied to a known RUNX1 mutated FPD-AML kindred to identify additional molecular events that co-operate with the germ line RUNX1 mutation in driving leukaemic transformation. A 56-year-old male and a 45-year-old female sibling both presented with AML with myelodysplastic features (Fig 1, I–3 and I–7 respectively). At diagnosis, I-3 had trisomy 8 and I-7 had monosomy 7. The eldest son of patient I-7 (II-1, Fig 1) has thrombocytopenia. The history of familial thrombocytopenia coupled with development of AML suggested a diagnosis of FPD-AML, which was confirmed by Sanger sequencing identification of a heterozygous RUNX1 p.Arg166X mutation in the leukaemic blasts and constitutional buccal scrapes of both affected patients. Patient I-3 underwent a reduced intensity conditioning allogeneic stem cell transplant (ASCT) from RUNX1 wild type sibling donor I-5. Patient I-7 underwent a myeloblative ASCT from RUNX1 wild type sibling donor I-6. Both patients achieved 100% donor chimerism by day-100 post-ASCT. For NGS, amplicon libraries were generated from AML diagnostic bone marrow DNA of I-3 and I-7 using the Ion Ampliseq AML Panel (Thermo Fisher Scientific, Life Technologies, Paisley, UK), a four primer-pool panel that generates 237 amplicons to allow interrogation of 19 commonly mutated genes implicated in AML. Amplicons cover the entire coding region of DNMT3A, CEBPA, GATA2, TET2, TP53 and mutational hot spot regions of ASXL1, BRAF, CBL, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, NPM1, NRAS, PTPN11, RUNX1 and WT1. Sequencing was performed on an Ion PGM with data analysed and reviewed using Torrent Browser and Ion Reporter 4 2 software (Thermo Fisher Scientific, Life Technologies). Criteria to allow confident calling of somatic mutations were a minimum target coverage of 500X, the presence of a mutation at >5% and a predicted change in amino acid sequence. Sanger sequencing was also performed of exon 8 of CDC25C, encompassing the mutation hotspot previously described (Yoshimi et al, 2014). In addition to confirmation of the heterozygous RUNX1 p.Arg166X mutation, targeted NGS demonstrated the presence of further mutations in the genes known to disrupt epigenetic (ASXL1, IDH1, TET2) and transcription factor (CEBPA, RUNX1) function in AML (Table I). Although