IntroductionFamilial platelet disorder with a propensity to develop acute myeloid leukemia (FPD/AML) is a rare autosomal dominant disease characterized by thrombocytopenia, abnormal platelet function, and a propensity to develop myelodysplastic syndrome (MDS) and AML. 1,2 Since Song et al reported haploinsufficiency of the RUNX1/CBFA2 gene, 3 more than 20 affected families have been reported. [4][5][6][7][8] Notably, various types of mono-allelic mutations of the RUNX1 gene have been found in patients with AML secondary to FPD. 3,7-9 RUNX1, which is a key regulator of definitive hematopoiesis and myeloid differentiation, is also commonly involved in sporadic cases of MDS and AML, by translocations in AML 10 and by point mutations in AML 11,12 and MDS. 13 Recently, a second RUNX1 alteration has been reported 8 ; however, no additional molecular abnormalities have been found so far.In this regard, recent reports of somatic mutations of the CBL proto-oncogene in myeloid neoplasms are intriguing because these CBL mutations have been shown to result in aberrant tyrosine kinase signaling, which would also lead to the activation of RAS signaling pathways. So far, we and others have reported that CBL mutations occurred in a variety of myeloid neoplasms, including de novo AML, 14,15 MDS,16,17 and myeloproliferative neoplasm, 16,17 especially in chronic myelomonocytic leukemia (CMML) 16,17 and juvenile myelomonocytic leukemia. 18 The importance of CBL mutations for leukemogenesis has substantially increased, which prompted us to search for possible CBL mutations in this pedigree.Here, we reported that CBL mutation developed at the time of diagnosis of CMML, but not during refractory cytopenia, in a Japanese patient with FPD/AML harboring a RUNX1 mutation.
Methods
RUNX1 mutation analysisDNA and RNA were extracted from peripheral blood (PB) of the proband, her sister, and their mother after obtaining informed consent. We performed mutation analysis of the RUNX1 gene by PCR followed by direct sequencing with the use of an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). For further confirmation of deletion mutations, the PCR products were subcloned with the use of a TOPO TA Cloning Kit (Invitrogen) and then sequenced. Mutations were screened from exons 1-8 of the RUNX1 gene.
CBL mutation analysisBecause CBL mutations thus far reported almost exclusively involved exons 8-9 that encode Linker/RING finger domains, we confined our mutation analysis to these exons, which were subjected to direct sequencing. Because the frequency of 11q-acquired uniparental disomy (11q-aUPD) was reported as Ïł 85%-90% in CBL mutations, we also analyzed the sample with Affymetrix GeneChip 250K NspI. [17][18][19] Genome-wide detection of copy number abnormalities or allelic imbalances was performed with CNAG/AsCNAR Version 3.0 software (http:// www.genome.umin.jp), which enabled sensitive detection of copy number neutral loss of heterozygosity (or aUPD). 19 In addition, we examined mutations of the following genes in the proband as previously rep...