A novel RUNX1 mutation in familial platelet disorder with propensity to develop myeloid malignancies

Abstract Familial platelet disorder with associated myeloid malignancies (FPDMM) is a rare autosomal dominant disease caused by germline RUNX1 mutations. FPDMM patients have defective megakaryocytic development, low platelet counts, prolonged bleeding times, and a life-long risk (20-50%) of developing hematological malignancies. FPDMM is a rare genetic disease in need of comprehensive clinical and genomic studies. In early 2019 we launched a longitudinal natural history study of patients with FPDMM at the NIH Clinical Center and by May 2021 we have enrolled 98 patients and 100 family controls from 55 unrelated families. Genomic data have been generated from 56 patients in 24 families, including whole exome sequencing (WES), RNA-seq, and single-nucleotide polymorphism (SNP) array. We have identified 21 different germline RUNX1 variants among these 24 families, which include lost-of-function mutations throughout the RUNX1 gene, but pathogenic/likely pathogenic missense mutations are mostly clustered in the runt-homology domain (RHD). As an important form of RUNX1 germline mutations, five splice site variants located between exon 4-5 and exon 5-6 were identified in 6 families, which led to the productions of novel transcript forms that are predicted to generate truncated RUNX1 proteins. Large deletions affecting the RUNX1 gene are also common, ranging from 50 Kb to 1.5Mb, which were detected in 8 of the 55 enrolled families. Besides RUNX1, copy number variation (CNV) analysis from both SNP array and WES showed limited CNV events in non-malignant FPDMM patients. In addition, fusion gene analysis did not detect any in-frame fusion gene in these patients, indicating a relatively stable chromosome status in FPDMM patients. Somatic mutation landscape shows that the overall mutation burden in non-malignant FPDMM patients is lower than AML or other cancer types. However, in 13 of the 44 non-malignant patients (30%), somatic mutations were detected in at least one of the reported clonal hematopoiesis of indeterminate potential (CHIP) genes, significantly higher than the general population (4.3%). Moreover, 85% of our patients who carried CHIP mutations are under 65 years of age; in the general population, only 10% of people above 65 years of age and 1% of people under 50 were reported to carry CHIP mutations. Among mutated genes related to clonal hematopoiesis, BCOR is the most frequently mutated gene (5/44) in our FPDMM cohort, which is not a common CHIP gene among the general population. Mutations in known CHIP genes including SF3B1, TET2, and DNMT3A were also found in more than one patient. In addition, sequencing of 5 patients who already developed myeloid malignancies detected somatic mutations in BCOR, TET2, NRAS, KRAS, CTCF, KMT2D, PHF6, and SUZ12. Besides reported CHIP genes or leukemia driver genes, 3 unrelated patients carried somatic mutations in the NFE2 gene, which is essential for regulating erythroid and megakaryocytic maturation and differentiation. Two of the NFE2 mutations are nonsense mutations, and the other is a missense mutation in the important functional domain. NFE2 somatic mutations may play important roles in developing malignancy because 2 of the 3 patients already developed myeloid malignancies. For multiple patients in our cohort, we have sequenced their DNA on multiple timepoints. We have observed patients with expanding clones carrying FKBP8, BCOR or FOXP1 mutations. We have also observed a patient with relatively stable clone(s) with somatic BCOR, DNMT3A, and RUNX1T1, who have been sampled over more than four years. We will follow these somatic mutations through sequencing longitudinally and correlate the findings with clinical observations to see if the dynamic changes of CHIP clones harboring the mutations give rise to MDS or leukemia. In summary, the genomic analysis of our new natural history study demonstrated diverse types of germline RUNX1 mutations and high frequency of somatic mutations related to clonal hematopoiesis in FPDMM patients. These findings indicate that monitoring the dynamic changes of these CHIP mutations prospectively will benefit patients' clinical management and help us understand possible mechanisms for the progression from FPDMM to myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

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A Novel Inherited Single-Nucleotide Mutation in 5′-UTR in the Transcription Factor RUNX1 in Familial Platelet Disorder with Propensity To Develop Myeloid Malignancies.

Blood ◽

10.1182/blood.v108.11.1917.1917 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1917-1917 ◽

Cited By ~ 2

Author(s):

Keita Kirito ◽

Toru Mitsumori ◽

Takahiro Nagashima ◽

Masae Kunitama ◽

Kei Nakajima ◽

...

Keyword(s):

Transcription Factor ◽

Point Mutation ◽

Aberrant Expression ◽

Single Nucleotide ◽

Myeloid Malignancies ◽

Single Nucleotide Mutation ◽

Runx1 Gene ◽

Nucleotide Mutation ◽

Platelet Disorder ◽

Familial Platelet Disorder

Abstract RUNX1 transcription factor plays pivotal roles in the development of definitive hematopoiesis. Allelic loss of the gene causes complete absence of fetal liver hematopoiesis. In addition to normal hematopoiesis, aberrant expression of RUNX1 is also involved in the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Familial platelet disorder with propensity to develop myeloid malignancies (FPD/AML, OMIM 601399) is a rare autosomal dominant disorder characterized by thrombocytopenia, dysfunction of platelets and predisposition to the development of myeloid malignancies. Recent studies revealed that inherited mutation of RUNX1 gene is responsible for the onset of FPD/AML. To date, 12 families of FPD/AML have been reported in the literature, and point mutation in the RUNT domain or loss of heterozygocity (LOH) of the gene has been identified in the pedigree. Here, we report a Japanese family with FPD/AML with a novel mutation of RUNX1 gene. A 38-year-old man was admitted to our hospital because of MDS (RAEB) in August 2003. Cytogenetic analysis revealed abnormal karyotype; 46XY, t (7; 8)(q34; q11). In addition, prolongation of bleeding time and abnormal platelet aggregation were observed. His son and daughter also showed mild bleeding tendency and had mild thrombocytopenia. In April 2006, the daughter developed MDS (RAEB) with trisomy 8 at age 16. After informed consent, blood samples were obtained from all family members and all 9 exons of RUNX1 gene were sequenced. We identified a novel G to T single-nucleotide mutation in the 5′-untranslated region (5′-UTR) in the exon1, corresponding to position 102 of RUNX1 transcripts (NCBI accession no. D43969). This mutation was also found in all the affected individuals but not in the healthy members. To investigate the possibility of hemizygous intragenic deletion of the gene, we performed an array- based comparative genomic hybridization using Affymetrix GeneChip Human Mapping 250K set including 23 SNPs in RUNX1 gene. We found no loss of heterozygosity of RUNX1 gene in the affected members. Because the mutation is located in 5′-UTR, we investigated whether this mutation might affect the expression of RUNX1 transcripts. Transcription of RUNX1 is regulated by two distinct promoter regions, distal and proximal, resulting in the generation of transcripts having different 5′-UTRs. The 5′-UTR of transcripts controlled by distal promoter contains exon1 (distal form), whereas that of transcripts controlled by proximal promoter contains exon3 but not exon1 (proximal form). We analyzed the expression level of both transcripts from bone marrow cells using quantitative RT-PCR. Affected individuals showed 10 to 15 times higher expression of the distal form of RUNX1 transcripts, compared to normal controls (n=3), MDS patients (n=3) and AML patient (n=1). Considering that not only haploinsufficiney but also overexpression of RUNX1 can cause AML, aberrant expression of RUNX1 induced by the point mutation in 5′-UTR may be involved in progression of FPD/AML.

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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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Bone Marrow Morphology Associated With GermlineRUNX1Mutations in Patients With Familial Platelet Disorder With Associated Myeloid Malignancy

Pediatric and Developmental Pathology ◽

10.1177/1093526618822108 ◽

2019 ◽

Vol 22 (4) ◽

pp. 315-328 ◽

Cited By ~ 4

Author(s):

Karen M Chisholm ◽

Christopher Denton ◽

Sioban Keel ◽

Amy E Geddis ◽

Min Xu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

Autosomal Dominant ◽

Germline Mutations ◽

Thrombocytopenic Purpura ◽

Myeloid Malignancy ◽

Platelet Disorder ◽

Familial Platelet Disorder ◽

Runx1 Mutation ◽

Clonal Abnormalities

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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Functional classification of RUNX1 variants in familial platelet disorder with associated myeloid malignancies

Leukemia ◽

10.1038/s41375-021-01200-w ◽

2021 ◽

Author(s):

Melanie Decker ◽

Tim Lammens ◽

Alina Ferster ◽

Miriam Erlacher ◽

Ayami Yoshimi ◽

...

Keyword(s):

Functional Classification ◽

Myeloid Malignancies ◽

Platelet Disorder ◽

Familial Platelet Disorder

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Different mutant RUNX1 oncoproteins program alternate haematopoietic differentiation trajectories

10.1101/2020.08.10.244657 ◽

2020 ◽

Author(s):

Sophie G Kellaway ◽

Peter Keane ◽

Benjamin Edginton-White ◽

Regha Kakkad ◽

Ella Kennett ◽

...

Keyword(s):

Embryonic Stem ◽

Disease Phenotype ◽

Novel Treatments ◽

Platelet Disorder ◽

Individual Consideration ◽

Gene Regulatory ◽

Familial Platelet Disorder ◽

Runx1 Mutation ◽

Genomic Basis ◽

Differential Action

AbstractMutations of the hematopoietic master regulator RUNX1 cause acute myeloid leukaemia, familial platelet disorder and other haematological malignancies whose phenotypes and prognoses depend upon the class of RUNX1 mutation. The biochemical behaviour of these oncoproteins and their ability to cause unique diseases has been well studied, but the genomic basis of their differential action is unknown. To address this question we compared integrated phenotypic, transcriptomic and genomic data from cells expressing four types of RUNX1 oncoproteins in an inducible fashion during blood development from embryonic stem cells. We show that each class of mutated RUNX1 deregulates endogenous RUNX1 function by a different mechanism, leading to specific alterations in developmentally controlled transcription factor binding and chromatin programming. The result is distinct perturbations in the trajectories of gene regulatory network changes underlying blood cell development that are consistent with the nature of the final disease phenotype. The development of novel treatments for RUNX1-driven diseases will therefore require individual consideration.

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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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