br Discussion A familial platelet disorder with a propensity
Discussion A familial platelet disorder with a propensity to myeloid malignancy (FPD/MM) was first reported in 1978 and since then approximately 30 pedigrees with RUNX1 germline mutations have been reported in the literature [9,11]. The RUNX1 gene is composed of 10 exons (1–6, 7A, 7B, 7C and 8). Distinct promoter regions and variable splicing leads to transcription of different isoforms, the longest being 480 serine threonine protein kinase in length. The N-terminal region encodes for the runt homology domain (RHD), which mediates both DNA binding and heterodimerization with its partner, core binding factor (CBF) beta. This region is coded on exons 3 through 5, which correlates to amino acids 50–177, and includes three specific DNA binding sites. The C-terminal region regulates gene transcription and encodes for the transcription activation domain on exons 7B and 8 (amino acids 243–371). Within this domain, there is a specific region known as the DNA-binding inhibitory domain (amino acids 184–291), which is important in down-regulating gene expression and a key site for gene mutations. The Runx1 protein acts by binding with CBF-beta to form a transcription factor complex that is expressed in hematopoietic stem cells and regulates expression of several important hematopoietic genes. Runx1 also plays a role in the transcription of many genes involved in normal platelet function, which explains the mild thrombocytopenia and platelet dysfunction seen in FPD/MM [12,13]. Runx1 is also important in regulating transcription of many tumor suppressor genes [14,15], and has been found to regulate DNA damage repair . Aberrations of the RUNX1 gene are commonly seen in sporadic forms of AML and MDS, with chromosomal translocations more common in AML and point mutations in MDS [17–20]. In FPD/MM, RUNX1 mutations are very heterogeneous and are often specific to individual pedigrees. The most common mutation site involves the RHD domain located near the N-terminus of Runx1, within exons three to five of RUNX1 . The RHD domain is especially prone to mutations as it contains easily mutable primary sequences that are prone to transition-type mutations via methylation of a cytidine residue . Some mutations are more damaging than others and confer a greater risk of transformation to malignancy. Mutations that cause haploinsufficiency are most common; however, mutations that act in a “dominant negative” fashion are particularly high risk and act by competing for DNA binding sites or by preferentially binding to CBF-beta, thereby reducing wildtype Runx1 activity below 50%. As compared to mutations that cause haploinsufficiency, dominant negative mutations have a higher incidence of progression to MDS/AML [7,21]. Likewise, Runx1 mutations at the C-terminus have enhanced capacity to bind DNA due to loss of the DNA-binding inhibitory domain, leading to preferential expression of the mutant allele. When mutated, Runx1 enhances myeloid cell proliferation, blocks cell differentiation, and leads to genomic instability, resulting in leukemogenesis [22–24]. Although RUNX1 mutations are very important in promoting leukemic transformation, they are insufficient to initiate disease by themselves. Secondary mutations are required before patients develop malignancy . An important second hit is that of the otherwise normal wildtype RUNX1 gene, resulting in biallelic mutations in RUNX1 . This has led some to refer to RUNX1 as the “gatekeeper gene” in the pathogenesis of RUNX1-associated acute leukemias . The case presented above is an example of a spontaneous RUNX1 germline mutation associated with a congenital thrombocytopenia that evolved into a myelomonocytic malignancy likely due to a secondary RUNX1 somatic mutation. Reported here for the first time is a c.837G>A nonsense mutation in exon 8 resulting premature stop codon within the DNA-binding inhibitory domain, an important self-regulator of RUNX1 transcription. Truncated Runx1 proteins are a common phenotype resulting in malignant transformation, and mutations in the DNA-binding inhibitory region are known to have a dominant negative effect . Also reported here is a novel second mutation that is an in-frame insertion of six nucleotides within exon 5 of the RUNX1 gene. This second mutation involved the DNA-binding RHD, where mutations resulting in CMML have been reported before . The second mutation is believed to be a secondary somatic mutation because it was mosaic prior to transplant and disappeared after transplant. Also, because of the absence of other genetic mutations (e.g., CEBPA, FLT3, NPM1, PDGFR), it is believed that these two RUNX1 mutations may have exerted a stronger dominant negative effect than either RUNX1 mutation alone, thus leading to malignancy with myelomonocytic differentiation. These hypotheses bear experimental study.