Bone marrow pathologic abnormalities in familial platelet disorder with propensity for myeloid malignancy and germline RUNX1 mutation

Germline mutations in RUNX1 result in autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM). To characterize the hematopathologic features associated with a germline RUNX1 mutation, we reviewed a total of 42 bone marrow aspirates from 14 FPDMM patients, including 24 cases with no cytogenetic clonal abnormalities, and 18 with clonal karyotypes or leukemia. We found that all aspirate smears had ≥10% atypical megakaryocytes, predominantly characterized by small forms with hypolobated and eccentric nuclei, and forms with high nuclear-to-cytoplasmic ratios. Core biopsies showed variable cellularity and variable numbers of megakaryocytes with similar features to those in the aspirates. Granulocytic and/or erythroid dysplasia (≥10% cells per lineage) were present infrequently. Megakaryocytes with separate nuclear lobes were increased in patients with myelodysplastic syndrome (MDS) and acute leukemia. Comparison to an immune thrombocytopenic purpura cohort confirms increased megakaryocytes with hypolobated eccentric nuclei in FPDMM patients. As such, patients with FPDMM often have atypical megakaryocytes with small hypolobated and eccentric nuclei even in the absence of clonal cytogenetic abnormalities; these findings are related to the underlying RUNX1 germline mutation and not diagnostic of MDS. Isolated megakaryocytic dysplasia in patients with unexplained thrombocytopenia should raise the possibility of an underlying germline RUNX1 mutation.

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Pre-Donor Evaluation of an HLA Matched Sibling Identifies a Novel Inherited RUNX1 Mutation Encoding a Missense Mutation Found Outside of the RUNT Domain in Familial Platelet Disorder

Blood ◽

10.1182/blood.v116.21.2709.2709 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2709-2709 ◽

Cited By ~ 4

Author(s):

Jacqueline S Garcia ◽

Jozef Madzo ◽

Devin Cooper ◽

Sarah A Jackson ◽

Kenan Onel ◽

...

Keyword(s):

Bone Marrow ◽

Amino Acid ◽

Dna Binding ◽

Amino Acid Change ◽

Single Amino Acid ◽

Wild Type ◽

Single Amino Acid Change ◽

Platelet Disorder ◽

Familial Platelet Disorder ◽

Runx1 Mutation

Abstract Abstract 2709 Introduction: RUNX1 is a critical transcription factor in the regulation of normal hematopoiesis. Inherited RUNX1 mutations have been identified as the culprit genetic lesion in Familial Platelet Disorder (FPD; OMIM 601399), a rare autosomal dominant condition with a propensity to myeloid malignancy. The spectrum of RUNX1 mutations causing the FPD/acute myeloid leukemia (AML) syndrome includes frameshift and termination mutations detected throughout the gene, and missense mutations clustered within the highly conserved RUNT homology domain (RHD), which is responsible for both DNA binding and heterodimerization with CBFβ/PEBP2β, the non-DNA binding regulatory subunit. We present a new FPD/AML pedigree with a novel missense mutation leading to a single amino acid change, L56S. This L56S mutation is the first reported point mutation in this syndrome to be found outside of the RHD. Patients and Methods: Our new pedigree involves a 41-year-old man (proband) diagnosed with myelodysplastic syndrome (MDS, specifically refractory anemia with excess blasts type-2) with a normal karyotype. He was initiated on azacitidine, which was administered on a seven-day treatment schedule every four weeks. Bone marrow biopsy analysis after six monthly cycles of azacitidine showed persistent MDS, with similar findings after a total of ten monthly cycles. Given his lack of a clinical response, his young age and good performance status, he was referred to The University of Chicago for allogeneic hematopoietic stem cell transplantation (HCT). Routine pre-transplant evaluation revealed mild thrombocytopenia (platelets = 123,000 K/μl) in his HLA-matched brother. In addition, his father was reported to have thrombocytopenia. Clinical concern for an inherited condition initiated the investigation for a RUNX1 mutation in the family. Results: We sequenced full-length cDNA synthesized from leukocyte-derived RNA collected from the proband's sibling with thrombocytopenia, and detected a novel missense germline mutation in exon 4 at nucleotide position 371, causing a T to C mutation leading to a single amino acid change in the RUNX1 protein, L56S. This amino acid substitution is located N-terminal to the RHD (aa 76–209). RUNX1 sequencing of the proband with MDS demonstrated the same mutation. The RUNX1 RHD and the transactivation domain remain intact in this mutant. Initial transactivation assays using a luciferase reporter assay performed in triplicate demonstrated similar levels of activation as wild-type RUNX1. Corresponding Western blot analysis showed similar levels of protein expression of both wild-type RUNX1 and mutant RUNX1 transfected cell lines using an anti-RUNX1-antibody. Current studies include determination of the transactivation ability of mutant RUNX1 with its heterodimerization partner, CBFβ/PEBP2β, testing the DNA binding ability of this RUNX1 mutant by electrophoretic mobility shift assay, and analysis of the RUNX1 cDNA for an acquired biallelic mutation in leukocytes collected from the proband's bone marrow aspirate at the time of diagnosis of bone marrow malignancy. Conclusions: FPD/AML is likely an underreported condition. Clinical suspicion for this inherited syndrome may be raised by the presence of mild to moderate thrombocytopenia in healthy siblings, and should lead to prompt screening for germline RUNX1 mutations to confirm an inherited predisposition and to prevent siblings carrying RUNX1 mutations from being selected as HCT donors. In vitro studies of identified RUNX1 mutations may elucidate potential mechanisms involved in the pathogenesis of the FPD/AML syndrome. Disclosures: No relevant conflicts of interest to declare.

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RUNX1 associated Familial Platelet Disorder with Myeloid Malignancy (FPD-MM) in Children: A Novel New Phenotype with Juvenile and Chronic Myelomonocytic Leukemia (JMML/CMML) Characteristics

Blood ◽

10.1182/blood-2018-99-116925 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5504-5504 ◽

Cited By ~ 1

Author(s):

Katherine Regling ◽

Shruti Bagla ◽

Ahmar Urooj Zaidi ◽

Erin Wakeling ◽

Michael C. Chicka ◽

...

Keyword(s):

Chronic Myelomonocytic Leukemia ◽

Comparative Genomic ◽

Molecular Testing ◽

Myeloid Malignancy ◽

Ras Genes ◽

Erythroid Hyperplasia ◽

Platelet Disorder ◽

Familial Platelet Disorder ◽

Myelomonocytic Leukemia ◽

Runx1 Mutation

Abstract Introduction: RUNX1 (aka AML1; 21q22.12) is indispensable in the establishment of definitive hematopoiesis in humans. Activating RUNX1 mutations are associated with both Acute Myeloid and Lymphoblastic Leukemias (AML, ALL). On the other hand, hypofunctioning RUNX1 mutations cause dominantly inherited Familial Platelet Disorder (FPD). RUNX1 FPD has a high risk for progression to pancytopenia, myeloproliferative disorders (MPD) or AML, hence the new WHO category FPD with myeloid malignancy (FPD-MM). Those with MM carry mutations in other genes seen in AML, MDS. It is a relatively rare disorder with ~75 affected kindreds reported worldwide (Sood, et al. Blood 2017). Detailed reviews of pediatric cases are few. Case Histories: We encountered two children with RUNX1 associated thrombocytopenia; the mutations are novel. The first family is that of 14 yr old AAF, presenting with fainting- blood counts are shown in Table 1; fetal hemoglobin (HbF) was elevated; bone marrow was hypercellular with 6% type 1 blasts, extreme paucity of megakaryocytes, erythroid hyperplasia and large numbers of sea blue histiocytes. The high HbF suggested JMML while the monocyte CD16;14 profile (95.6% CD14+ cells) was similar to that seen in the adult type Chronic Myelomonocytic Leukemia (CMML). Her mother has pancytopenia without excess blasts in the marrow. The second case presented with neonatal thrombocytopenia; father has history of excessive bruising. Results: Blood counts and values for HbF are listed in Table 1. Molecular testing: Case1: A Myeloid gene panel showed RUNX1 - NM_001754.4:c.501delT, p.Ser167Argfs*9; PHF6 - NM_032458.2:c.902dupA, p.Tyr301*; CUX1- NM_001202543.1:c.2378delC, p.Pro793Argfs*26. No mutations were noted in PTPN11, CBL or RAS genes, the latter confirmed by JMML panel done at University of California, San Francisco. UCSF panel identified a mutation in SH2B3, a gene linked to erythrocytosis not caused by JAK2 mutations. Her mother has the same RUNX1 mutation, thus identifying a germline mutation of RUNX1 in her and her child but not the PHF6, CUX1 or the SH2B3 mutations seen in her daughter. A half sibling is unaffected and is a potential transplant donor for the mother. Case2: No coding sequence mutations were detected in genes associated with familial thrombocytopenia including ETV6, GATA1 and RUNX1. Array Comparative Genomic Hybridization studies (Prevention Genetics) identified a heterozygous deletion of the entire exon 5 of RUNX1. To understand the complex findings in family 1 additional studies were done- DRAQ5, CD71, Fetal Hb staining showed that NRBC in Case 1 contained predominantly high HBF cells. LIN28B was markedly elevated in the proband but not the mother (HbF- normal); LIN28B expression was normal in Case 2. Treatment/Outcome: In Case 1, low dose decitabine therapy resulted in the control of MPD features with good Hb recovery and normalization of the monocyte CD16;14 profiles. There was no platelet response to decitabine nor to a course of valproic acid. The child died of fulminant acute graft vs host disease affecting the liver following a 4/6 cord mismatch transplantation. Mother continues to show moderately severe pancytopenia requiring frequent transfusion support. The second child is symptom free with mild thrombocytopenia. Discussion: The hybrid JMML/CMML features in the index child are likely caused by the concurrent CUX1/PHF6/SH2B3 mutations. We are unable to establish if these are true de novo mutations as the father was not available for study; she had no full siblings. Neither high HbF nor high LIN28B are known feature of FPD by itself nor CMML or Polycythemia Vera (p Vera). Recently, the high HbF in JMML has been linked to high expression of LIN28B. SH2B3 mutation may have contributed to the high erythroid proliferation observed in our case. Induced CUX1 haploinsufficiency in mice causes MPD akin to CMML and megakaryocytic (Meg) proliferation (An N, et al. Blood 2018). The virtual absence of Megs in our case indicates that the CUX1 mutation was unable to overcome the Meg ploidization defect caused by the RUNX1 mutation. PHF6 mutations occur in T-ALL and AML but have not been linked to high HbF. Conclusions: Normal HbF and normal LIN28B expression in the mother of Case1 and in Case2 indicate that increased LIN28B is linked to the high HbF in Case 1 and that high LIN28B itself is a consequence of the malignant transformation caused by the concurrent CUX1/PHF6/SH2B3 mutations. Disclosures Chitlur: Baxter, Bayer, Biogen Idec, and Pfizer: Honoraria; Novo Nordisk Inc: Consultancy. Ravindranath:AGIOS: Other: Site Investigator for Pyruvate Kinase Deficiency.

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Five New Pedigrees with Inherited RUNX1 Mutations Causing Familial Platelet Disorder with Propensity to Myeloid Malignancy (FPD/AML)

Blood ◽

10.1182/blood.v112.11.5067.5067 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5067-5067

Author(s):

Carolyn J Owen ◽

Cynthia L Toze ◽

Anna Koochin ◽

Donna L. Forrest ◽

Clayton A. Smith ◽

...

Keyword(s):

Young Patients ◽

Trisomy 13 ◽

Degree Relative ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Myeloid Malignancy ◽

Platelet Disorder ◽

Familial Platelet Disorder ◽

Inherited Mutations ◽

Runx1 Mutation

Abstract Familial platelet disorder with propensity to myeloid malignancy (FPD/AML) is an autosomal dominant syndrome characterised by platelet abnormalities and a predisposition to myelodysplasia (MDS) and/or acute myeloid leukemia (AML). The disorder, caused by inherited mutations in RUNX1, is uncommon with only 14 pedigrees reported. We screened 10 families with a history of more than one first- degree relative with MDS/AML and detected inherited mutations in RUNX1 in 5 of these pedigrees. Several affected members had normal platelet counts or platelet function, features not previously reported in FPD/AML. The median incidence of MDS/AML among carriers of RUNX1 mutation was 35%. Individual treatments varied but included hematopoietic stem cell transplantation (HSCT) from siblings before recognition of the inherited leukemogenic mutation. Transplantation was associated with a high incidence of complications including early relapse, failure of engraftment and post-transplantation lymphoproliferative disorder. As acquired trisomy 13 and 21 and FLT3-ITD have all been associated with RUNX1 mutation in sporadic MDS/AML, a combination of single nucleotide polymorphism profiling and mutation analysis was performed to determine whether these secondary genetic events were implicated in the onset of overt malignancy in FPD/AML. Five disease (MDS and/or AML) samples from 4 of our pedigrees with FPD/AML were screened and in all cases, these abnormalities were excluded. Therefore, the secondary mutations that promote MDS/AML in individuals with germline RUNX1 mutations are distinct from those reported in sporadic cases and require further investigation. The small size of modern families and the clinical heterogeneity of the FPD/AML syndrome may have resulted in the diagnosis being previously overlooked. Based on our data, FPD/AML may be more prevalent than previously recognized and therefore, it would appear prudent to screen young patients with MDS/AML for RUNX1 mutation, particularly prior to consideration of sibling HSCT.

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Molecular Characterization of a Novel 3' Mutation in RUNX1 in a New Pedigree with Familial Platelet Disorder.

Blood ◽

10.1182/blood.v114.22.1626.1626 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1626-1626

Author(s):

Jane E Churpek ◽

Jacqueline S Garcia ◽

Jozef Madzo ◽

Sarah Jackson ◽

Kenan Onel ◽

...

Keyword(s):

Bone Marrow ◽

Amino Acid ◽

Nonsense Mutation ◽

Myelogenous Leukemia ◽

Truncated Protein ◽

Platelet Disorder ◽

Protein Truncation ◽

Familial Platelet Disorder ◽

Runx1 Mutation

Abstract Abstract 1626 Poster Board I-652 Introduction Familial Platelet Disorder with Propensity to Acute Myelogenous Leukemia (FPD/AML; OMIM 601399) is a rare disorder with an autosomal dominant inheritance pattern characterized by varying degrees of thrombocytopenia, clinical bleeding due to platelet dysfunction, and an increased risk of developing myeloid malignancy. To date, twenty-six families with this disorder have been described and all carry germline RUNX1 mutations as the causative genetic abnormality. The spectrum of RUNX1 mutations includes point mutations within the RUNT domain and frameshift and termination mutations throughout the body of the gene. Here we report identification of a new family with FPD with a novel nonsense mutation resulting in premature protein termination at amino acid 388. Patients and Methods Our four generation pedigree includes a mother (II:4) diagnosed with dysplasia and normal karyotype acute myeloid leukemia now in remission after a matched sibling allogeneic stem cell transplant, and her daughter (III:2) with thrombocytopenia since childhood, excessive bleeding with childbirth, and 5q- syndrome diagnosed at 37 years old. Genomic DNA was obtained from all available family members, and RUNX1 cDNA (transcription variants a through c) was sequenced. In addition, RUNX1 cDNA was analyzed for second mutations in bone marrow samples from both patients at the time of diagnosis of their initial bone marrow malignancy. Results RUNX1 sequencing of germline DNA revealed heterozygosity for a novel nonsense mutation in exon 8 (c.1163C>A), which is predicted to result in premature protein truncation (p.Ser388X). Full sequencing of RUNX1 cDNA from II:4's AML does not show any secondary mutations. Our current efforts include full sequencing of RUNX1 cDNA from III:2's bone marrow malignancy as well as functional studies of the truncated protein. Conclusions We have identified a novel 3' RUNX1 mutation within exon 8, which is predicted to result in premature protein truncation at amino acid 388. To date, this is the most distal mutation identified in an FPD/AML pedigree. The identification of this mutation suggests that the last 100 amino acids, which are known to contain the RUNX1 inhibition domain, contribute an essential function. Further characterization of this RUNX1 mutation and its encoded truncated protein may yield insight into RUNX1's role in leukemogenesis in FPD and de novo AML. Disclosures No relevant conflicts of interest to declare.

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