Familial Platelet Disorder With Predisposition To Acute Myeloid Leukemia Research Articles

Unrelated hematopoietic stem cell transplantation for familial platelet disorder/acute myeloid leukemia with germline RUNX1 mutations.

Germline mutations in RUNX1 result in rare autosomal-dominant familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). As genetic analysis is becoming increasingly prevalent, the diagnosis rate of FPD/AML is expected to increase. In this report, we present two pedigrees, one diagnosed molecularly and another highly suspected to be FPD/AML, whose members both received allogeneic hematopoietic stem cell transplantation (HSCT). Both pedigrees had a family history of thrombocytopenia, platelet dysfunction, and hematological malignancies. One family inherited a frameshift mutation (p.P240fs) of RUNX1, a known pathogenic variant. Another family inherited a point mutation (p.G168R) in the runt-homology domain, the clinical significance of which is uncertain at this point. As this mutation was completely absent from all population databases and had a relatively high REVEL score of 0.947, we thought that it would be dangerous to ignore its possible pathogenicity. Consequently, we avoided choosing HSCT donors from relatives of both families and performed HSCT from unrelated donors. In conclusion, our experience with two families of FPD/AML highlights the importance of searching for gene mutations associated with germline predisposition and indicates the necessity of developing a donor coordination system for FPD/AML patients, as well as a support system for families.

The Clinical, Molecular, and Mechanistic Basis of RUNX1 Mutations Identified in Hematological Malignancies.

RUNX1 plays an important role in the regulation of normal hematopoiesis. RUNX1 mutations are frequently found and have been intensively studied in hematological malignancies. Germline mutations in RUNX1 cause familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). Somatic mutations of RUNX1 are observed in various types of hematological malignancies, such as AML, acute lymphoblastic leukemia (ALL), myelodysplastic syndromes (MDS), myeloproliferative neoplasm (MPN), chronic myelomonocytic leukemia (CMML), and congenital bone marrow failure (CBMF). Here, we systematically review the clinical and molecular characteristics of RUNX1 mutations, the mechanisms of pathogenesis caused by RUNX1 mutations, and potential therapeutic strategies to target RUNX1-mutated cases of hematological malignancies.

Strategies for Analysis of Novel Molecular Variants in the RUNX1 Gene As a Cause of Familial Platelet Disorder with Predisposition to Acute Myeloid Leukemia (FPD/AML)

Introduction: Runx 1 is a key transcription factor (TF) in hematopoiesis. Germline mutations in RUNX1 (≈40 described) are associated with FPD/AML, an autosomal dominant disorder characterized by moderate thrombocytopenia, platelet dysfunction and a high risk (40% before 35 yr) of developing acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Overestimating the pathogenicity of new molecular variants detected by high throughput sequencing (HTS) in the diagnosis of inherited thrombocytopenias (IT), even affecting well established candidate genes, could be more negative for the management and quality of life of patients than lacking such diagnosis (Lentaigne, Blood 2016). This is especially relevant for variants in TF such as Runx1, due to the risk of developing myeloid malignancies. Objective: To evaluate the pathogenicity of new variants in RUNX1 identified in patients enrolled in the Spanish multicentric project "Functional and molecular characterization of patients with Congenital Platelet Disorders" (TPC-GT-PH-SETH). Methods: Three unrelated women aged 27 (P1), 46 (P2) and 50 (P3) years, were included in TPC-GT-PH-SETH because of their history of mild thrombocytopenia (≈105pl/uL) with normal volume (MPV) and moderate bleeding. A daughter and a brother of P2 died after developing MDS and LAM, respectively, and a sister of P3 had breast cancer. Only P1 and P2 had previously showed moderate functional platelet abnormalities (reduced aggregation, P-selectin secretion and number of δ-granules). By HTS (Bastida, Haematologica 2018) we identified new heterozygous variants in RUNX1: P1, de novo c.802C<T [p.Gln.268*]; P2, c.586A>G [p.Thr196Ala], segregated with thrombocytopenia and neoplasia in the family; P3, c.476A>G; [p.Asn159Ser], no segregation with thrombocytopenia, with 4 of 6 carriers in the family having normal platelet numbers. For this study, we obtained blood from patients and three controls, and isolated ultrapure platelets by filtration+immunoselection. Platelet RNA was analyzed using Clariom-D Array (≈540 000 transcripts) (Caparrós-Pérez, PLoS One 2017). In P1 we isolated CD34+ cells from blood, differentiated them in vitro to megakaryocytes (Mks), and evaluated the formation of proplatelets. Results: Analysis of transcriptomes showed that when considering the 13 genes well recognized as targets of RUNX1 (including MYL9, MYH9, ALOX12), the percentage of those with altered expression in patients, vs. controls, was 69.2% and 61.5% in P1 and P2, respectively, and only 7.7% in P3. Previously, the transcriptome of a single patient with RUNX1 variant (83aa deletion) (Sun, JTH 2007) has reported the infraexpression of 100 genes. Our cases showed low expression of 74.7%, 67.7% and 7.1% (P1, P2 and P3, respectively) of those genes. Principal component analysis of the array intensity signals, grouped together controls and the P3 case, while the P1 and P2 cases were clustered together. Additionally, in vitro culture of CD34-Mks from patient P1 showed reduced pro-platelet formation. Conclusion: Platelet phenotyping, family segregation studies, platelet transcriptome and in vitro pro-platelet formation, support the pathogenicity of Runx 1 variants p.Gln268* and p.Thr196Ala, but not of p.Asn159Ser. Platelet transcriptome analysis is a useful tool in the pathogenic characterization of new molecular variants affecting TF genes in patients with IT, such as RUNX1 and can help to identify new target genes of these TF. Disclosures No relevant conflicts of interest to declare.

Analysis of the Frequency of Spontaneous, Functionally-Significant Mutations in Genes Associated with Platelet Disorders in >120,000 Healthy Individuals

Inherited platelet disorders (IPD) are increasingly recognized as the cause of clinical bleeding. Advances in genomic technologies have identified a growing number of platelet-associated genes that are currently vetted with phenotypic correlates. These platelet-associated genes are a disparate group, including transcription or related nuclear factors, cytoskeletal proteins, surface receptors, intracellular proteins and granule forming proteins. At present the prevalence of each inherited platelet-associated disorder and the disorders in aggregate are not well defined. We leveraged the recent curating of 123,136 high quality exomes from a cross section of the general population in the form of the genome aggregation database (gnomAD) for analysis. We used the loss-of-function transcript effect estimator (LoFTEE) in conjunction with the gnomAD dataset to study loss of function (LoF) variants in genes of interest. With this set of predicted LoF variants, we generated a LoF frequency for each gene of interest taking into account whether the heterozygote or homozygote state is sufficient and/or necessary for clinical phenotype. These data are analyzed to determine whether each platelet-associated gene is relatively tolerant or intolerant to LoF mutations in the context of their clinical phenotypes.By this analysis, we found approximately 800 novel LoF variants in platelet-related genes in this population of >120,000 individuals. Affected genes known to cause disease phenotype in the heterozygous state (n=33) accounted for 27% of the mutations analyzed. With these data, we calculated the frequency of IPD in the general population secondary to LoF mutations and estimated the relative impact of dominant versus recessive cases of IPD. We demonstrate that the majority of manifest cases of IPD will be due to the dominantly inherited, haploinsufficient IPDs. The transcription factor gene subset (9 of the IPD associated genes) was the most intolerant to LoF variants based on ratio of observed vs. expected number of variants (pLI measurement). Interestingly, the severity of the platelet dysfunction and resultant bleeding from LoF mutations in this subset of genes is not directly related to their intolerance of these mutations. For instance, heterozygous LoF of RUNX1 result in a mild-moderate bleeding disorder; however, a pLI of 0.819 indicates this gene is moderately to very intolerant of LoF variants. These same LoF variants in RUNX1 predispose to myelodysplastic syndromes with a high risk of myeloid leukemia in the form of familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML), and likely this is driving LoF intolerance. Cytoskeletal protein encoding genes represent another subset of mostly LoF intolerant platelet-related genes.Intracellular protein encoding genes and granule protein genes have varied tolerance and platelet-associated receptor protein genes as a subgroup were most tolerant of haploinsufficiency. There were some genes with similar clinical bleeding phenotypes that had divergent tolerance to LoF. For instance, GP9 and GP1BB both cause Montreal Platelet Syndrome in the haploinsufficient state and had moderate intolerance to LoF mutations (pLI GP9 = 0.804; pLI GP1BB = 0.575). In contrast, while LoF mutations in GP1BA cause the same bleeding phenotype, this gene is much more tolerant to haploinsufficiency (pLI = 0.0002). These data indicate that perhaps there is another unidentified adverse condition associated with GP9 and GP1BB that is driving increased haploinsufficiency intolerance.In summary, we present a comprehensive analysis of known platelet-associated genes, the frequency of LoF mutations in these genes and their relative tolerance of the haploinsufficient state. These data generate an incidence of IPDs of ~0.18% in the general population. Importantly, these data also inform the driving mechanisms of LoF intolerance as there are defective genes resulting in similar bleeding phenotypes, but divergent tolerance to haploinsufficiency, indicating that further investigation is warranted for additional biology. DisclosuresLambert:CSL: Consultancy; Rigel: Consultancy; Sysmex: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Summus: Consultancy; Bayer: Membership on an entity's Board of Directors or advisory committees; Shionogi: Consultancy; Educational Concepts in Medicine: Consultancy. Poncz:Incyte Corporation: Consultancy, Research Funding.

RUNX1 Mutations in Inherited and Sporadic Leukemia.

RUNX1 is a recurrently mutated gene in sporadic myelodysplastic syndrome and leukemia. Inherited mutations in RUNX1 cause familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). In sporadic AML, mutations in RUNX1 are usually secondary events, whereas in FPD/AML they are initiating events. Here we will describe mutations in RUNX1 in sporadic AML and in FPD/AML, discuss the mechanisms by which inherited mutations in RUNX1 could elevate the risk of AML in FPD/AML individuals, and speculate on why mutations in RUNX1 are rarely, if ever, the first event in sporadic AML.

Thrombocytopenia and Platelet Ultra-Structural Abnormalities Associated with RUNX1 Haploinsufficiency in Monosomy 21 Mosaicism.

AbstractAbstract 2190RUNX1, also known as core binding factor A2 (CBFA2), is a transcription factor that regulates the expression of several hematopoietic specific genes through a highly conserved DNA-binding region called the RUNT homology domain. The significant role of RUNX1 on megakaryopoiesis and leukemogenesis is supported by the finding that germline, heterozygous mutations in the RUNX1 gene on 21q22.12 forms the genetic basis of the autosomal dominant, familial platelet disorder with predisposition to acute myeloid leukemia (FDP/AML). Patients with FDP/AML present with mild to moderate thrombocytopenia, qualitative platelet defects and a propensity to develop AML. While several platelet abnormalities have been reported in this cohort including abnormalities in platelet aggregation and secretion, Mpl receptors, activation of GPIIb-IIIa, phosphorylation of myosin light chain and deficiency of platelet factor 4 (PF4); to the best of our knowledge, platelet electron microscopy findings have never been described. Furthermore, data on phenotypic manifestations of partial monosomy 21 are sparse and anecdotal. Published reports from literature describe patients with growth restriction, developmental delay, heart defects and dysmorphic facies. Thrombocytopenia, bleeding and even fatal hemorrhage have been noted, though detailed hematological manifestations are not consistently described. It is plausible that deletion of RUNX1 may be responsible for some of the characteristics observed in this cohort.An 18 month old male with a history of unprovoked recurrent subdural hemorrhage was referred to our service for further investigation. Patient was born at 35 weeks of gestation with intra-uterine growth restriction (birth weight 1440 grams). Post-natal history was significant for dysmorphic facies, global developmental delay, seizure disorder, chronic thrombocytopenia (platelet count 70 – 140 × 109/L) and subaortic stenosis. Bleeding diathesis workup including INR, aPTT, von Willebrand antigen, ristocetin co-factor and multimer analysis were normal. PFA-100 showed consistently prolonged closure times with Col/Epi and Col/ADP cartridges and platelet aggregation showed dis-aggregation with ADP, but normal ATP release. Peripheral blood karyotype showed 46, XY, r(21) (p11.2q22.3)[22]/45, XY, −21[8]. Fluorescent-in-situ hybridization (FISH) analysis for the RUNX1 locus showed 2 copies of RUNX1 in 116/200 (58%) nuclei and one signal of RUNX1 (consistent with loss of the ring chromosome 21) in 84/200 (42%) nuclei analyzed. Transmission electron microscopy of patient platelets revealed frequent fused/large alpha granules, with approximately 20% of the platelet population characterized as agranular or having few granules. PF4 in patient total platelet lysates was not significantly decreased as assessed by immunoblotting.To our knowledge this is the first demonstration of ultra-structural platelet abnormalities in a patient with RUNX1 haploinsufficiency. The molecular data from our patient helps elaborate the role of RUNX1 on both thrombocytopenia and platelet developmental abnormalities which also affect platelet function. Further use of electron microscopy in RUNX1 haplodeficient cohort may help better understand and characterize this rare platelet disorder. Disclosures:No relevant conflicts of interest to declare.

Integrative analysis of RUNX1 downstream pathways and target genes

BackgroundThe RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia.ResultsHere we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes.ConclusionThis work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.

Novel Heritable Mutation of the Transcription Factor RUNX1 as a Cause of Autosomal Dominant Familial Platelet Disorder with Predisposition to Acute Myeloid Leukemia (FPD/AML).

Aim To identify the causative heritable mutation in a family with autosomal dominant familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML).Method Confirmation of family pedigree, enrolment into ethics committee approved Australian Familial Hematological Cancer Study, procurement of genomic DNA from pedigree members and genetic analysis by sequencing of RUNX1 and CEBPA genes.Results The proband intially presented aged 50 with mild thrombocytopenia initially diagnosed as idiopathic thrombocytopenic purpura when bone marrow examination (including cytogenetics analysis) was normal. Three years later she was referred with severe progressive thrombocytopenia unresponsive to high dose steroids and intravenous immunoglobulin together with mild anemia and neutropenia. Marrow examination revealed subtle dysplasia with monosomy 7 in 12/20 metaphases. A diagnosis of myelodysplastic syndrome (MDS) was made. Three months later (Feb 2007), she progressed to acute myeloid leukemia (AML). The proband's mother had had mild thrombocytopenia with subsequent MDS (aged 70) and died of AML two years later. The proband's only sibling and nephew have mild thrombocytopenia without features of MDS. The proband entered cytogenetic remission following one course of AML induction therapy but continued to show dysplastic features. She then received 2 cycles of consolidation therapy and has undergone unrelated donor allogeneic stem cell transplant (July 2007). Sequencing of PCR products from exons of the RUNX1 gene identified a novel heterozygous mutation in the proband's constitutional DNA: c.958C>T in exon 7, in the transactivation domain, causing a nonsense mutation, p.Arg293X (sequence variation for RUNX1 classified according to GenBank Accession No. NC_000021). The sibling has the same mutation and studies in other relatives are underway.Conclusion This study has identified a novel RUNX1 mutation responsible for FPD/AML in this family. Clinicians should be aware of this rare inherited disorder and the availability of genetic testing. People with mutations may be asymptomatic and genetic testing in such families is essential before considering bone marrow transplantation from a living related donor.

Identification of Cooperating Genetic Alterations in RUNX Leukemia Using the Mouse Model for the Human Familial Leukemia, FPD/AML.

The RUNX1/AML1 gene is essential for hematopoiesis and is most frequently involved in human leukemias. Loss-of-function of RUNX1 is the common underlying mechanism of RUNX leukemias: those carrying t(8;21), inv(16) and RUNX1 point mutations. As the weakest mode, haploinsufficiency of RUNX1/AML1 causes familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). Mice heterozygous for Runx1 null mutation, Runx1+/−, appear genetically equivalent to human FPD/AML patients, but showed no apparent defects and did not develop spontaneous leukemia, implying that additional genetic alterations are required for leukemia. To identify such abnormalities, retroviral insertional mutagenesis was employed by using BXH2 mice that have spontaneously transmittable ecotropic retrovirus. BXH2-Runx1+/− mice showed a shorter disease latency and more myeloid-specific phenotype than wild-type littermates. Therefore, BXH2-Runx1+/− mice appear to serve as the mouse model for FPD/AML to investigate myeloid leukemia. The genes selectively affected in BXH2-Runx1+/− mice by retroviral integration are likely to cooperate with Runx1+/−. c-Kit locus was involved twice in 24 BXH2-Runx1+/− but neither in 17 wild-type littermates nor in 135 inbred BXH2 mice reported in the RTCGD database, suggesting that c-KIT mutation cooperates with RUNX alteration in human leukemia. In fact, constitutively active mutants of c-KIT or functionally equivalent FLT3, were found in 10 out of 25 (40%) of human RUNX leukemias. Together, BXH2-Runx1+/− system appears useful to identify the genes cooperating with RUNX1 abnormalities.

Implications of somatic mutations in the AML1 gene in radiation-associated and therapy-related myelodysplastic syndrome/acute myeloid leukemia

Somatically acquired point mutations of AML1/RUNX1 gene have been recently identified in rare cases of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Moreover, germ line mutations of AML1 were found in an autosomal dominant disease, familial platelet disorder with predisposition to AML (FPD/AML), suggesting that AML1 mutants, as well as AML1 chimeras, contribute to the transformation of hematopoietic progenitors. In this report, we showed that AML1 point mutations were found in 6 (46%) of 13 MDS patients among atomic bomb (A-bomb) survivors in Hiroshima. Unlike acute or chronic leukemia patients among A-bomb survivors, MDS patients exposed relatively low-dose radiation and developed the disease after a long latency period. AML1 mutations also were found in 5 (38%) of 13 therapy-related AML/MDS patients who were treated with alkylating agents with or without local radiation therapy. In contrast, frequency of AML1 mutation in sporadic MDS patients was 2.7% (2 of 74). Among AML1 mutations identified in this study, truncated-type mutants lost DNA binding potential and trans-activation activity. All missense mutations with one exception (Gly42Arg) lacked DNA binding ability and down-regulated the trans-activation potential of wild-type AML1 in a dominant-negative fashion. The Gly42Arg mutation that was shared by 2 patients bound DNA even more avidly than wild-type AML1 and enhanced the trans-activation potential of normal AML1. These results suggest that AML1 point mutations are related to low-dose radiation or alkylating agents and play a role distinct from that of leukemogenic chimeras as a result of chromosomal translocations caused by sublethal radiation or topoisomerase II inhibitors.