A novel RUNX1 exon 3 - 7 deletion causing a familial platelet disorder.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare inherited disorder confirmed with the presence of a pathogenic germline RUNX1 variant and is thought to be heavily underdiagnosed. RUNX1 has also been found to be mutated in up to 10% of adult AML cases and other cell malignancies. We performed targeted next-generation sequencing and subsequent MLPA analysis in a kindred with multiple affected individuals with low platelet counts and a bleeding history. We detected a novel heterozygous exon 3-7 large deletion in the RUNX1 gene in all affected family members which is predicted to remove all of the Runt-homology DNA-binding domain and a portion of the Activation domain. Our results show that the combination of targeted NGS and MLPA analysis is an effective way to detect copy number variants (CNVs) which would be missed by conventional sequencing methods. This precise diagnosis offers the possibility of accurate counseling and clinical management in such patients who could go onto develop other cell malignancies.

Genetic screening of children with suspected inherited bleeding disorders.

INTRODUCTION: Genetic screening using high-throughput DNA sequencing has become a tool in diagnosing patients with suspected inherited bleeding disorders (IBD). However, its usefulness and diagnostic efficacy in children is unclear. AIM: To evaluate the diagnostic efficacy of genetic screening for IBD in children and downstream further testing. METHODS: After informed consent, children (<18 years) with suspected IBD underwent genetic screening with 94 selected genes. RESULTS: A total of 68 heterozygous class 3-5 variants were detected in 30 children, 2.3 variants per patient. Directed specific functional testing was performed after genetic screening in a subset of patients. Adhering to the ACMG guidelines, the results of functional testing together with family history and previous publications classified three variants as likely disease causing (class 4) and two variants as disease causing (class 5), all in children with thrombocytopenia. The overall diagnostic rate was 16.7% (5/30). Children with thrombocytopenia had a significantly higher rate of significant genetic findings, 5/9 (55.6%) vs. 0/21 (0%; P = .0009). CONCLUSION: We conclude that performing genetic screening in children is an effective tool especially for children with inherited thrombocytopenia and has the possibility to diagnose platelet disorders adequately early in life. Children with bleeding diathesis, normal coagulation work-up and without thrombocytopenia are unlikely to be diagnosed by genetic screening. Ethical issues such as incidental findings, variants associated with cancer and the interpretation of the genetic results into clinical practice remain problematic.

GENYOi005-A: An induced pluripotent stem cells (iPSCs) line generated from a patient with Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) carrying a p.Thr196Ala variant.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder caused by mutations in RUNX1. We generated an iPSC line (GENYOi005-A) from a FPDMM patient with a non-previously reported variant p.Thr196Ala. Non-integrative Sendai viruses expressing the Yamanaka reprogramming factors were used to reprogram peripheral blood mononuclear cells from this FPDMM patient. Characterization of GENYOi005-A included genetic analysis of RUNX1 locus, Short Tandem Repeats profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and differentiation studies in vitro and in vivo. This iPSC line will provide a powerful tool to study developmental alterations of FPDMM patients.

Bone Marrow Morphology Associated With Germline RUNX1 Mutations in Patients With Familial Platelet Disorder With Associated Myeloid Malignancy.

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.

Congenital Combined Deficiency of the Vitamin K-dependent Clotting Factors (VKCFD): A Novel Gamma-glutamyl Carboxylase (GGCX) Mutation.

Congenital combined vitamin K-dependent clotting factors deficiency (VKCFD) is a very rare autosomal recessive bleeding disorder. Here we report a case of a girl with novel variant in the gamma-glutamyl carboxylase (GGCX) gene leading to VKCFD. A 3-month-old girl presented to our hospital with a history of bleeding from puncture site. Laboratory evaluation showed markedly prolonged partial thromboplastin time and activated partial thromboplastin time. Activities of vitamin K-dependent factors were all low. Genetic analysis revealed a homozygous currently unreported variant in the GGCX gene further supporting a diagnosis of VKCFD type 1. VKCFD due to GGCX mutation has an overall good prognosis.

RUNX1 deficiency (familial platelet disorder with predisposition to myeloid leukemia, FPDMM).

In this review, we discuss disease-causing alterations of RUNT-related transcription factor 1 (RUNX1), a master regulator of hematopoietic differentiation. Familial platelet disorder with predisposition to myeloid leukemia (FPDMM) typically presents with (1) mild to moderate thrombocytopenia with normal-sized platelets; (2) functional platelets defects leading to prolonged bleeding; and (3) an increased risk to develop myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), or T-cell acute lymphoblastic leukemia (T-ALL). Hematological neoplasms in carriers of a germline RUNX1 mutation need additional secondary mutations or chromosome aberrations to develop. If a disease-causing mutation is known in the family, it is important to prevent hematopoietic stem cell transplantation from a sibling or other relative carrying the familial mutation. First experiments introducing a wild-type copy of RUNX1 into induce pluripotent stem cells (iPSC) lines from patients with FPDMM appear to demonstrate that by gene correction reversal of the phenotype may be possible.

An Unexpectedly High Rate of Thrombophilia Disorders in Patients with Superficial Vein Thrombosis of the Lower Extremities.

BACKGROUND: Superficial vein thrombosis (SVT) is a common venous condition. Recent studies have shown that SVT is associated with high frequency of thromboembolic complications: from 22-37% for deep venous thrombosis and up to 33% for pulmonary embolism. Our goal was to assess the prevalence of major hereditary and acquired thrombophilic factors in patients with SVT. METHODS: Sixty-six patients presenting with primary SVT underwent evaluation for thrombophilia: molecular testing for the factor V Leiden and factor II G20210 A (prothrombin) mutations, protein C, protein S, antithrombin deficiency, presence of lupus anticoagulant, as well as anticardiolipin antibody titers. Patients aged less than 18 years, with confirmed deep vein thrombosis, and pregnant women were excluded. RESULTS: 95.5% were Caucasian, and 62.1% were female gender. Age ranged from 21-88 years. Molecular testing showed that 34.2% of patients were heterozygous for factor V Leiden, 23.6% were heterozygous for the factor II mutation, 7.8% had antithrombin deficiency, 2.6% had protein S deficiency, and 2.1% had protein C deficiency. CONCLUSIONS: Our study showed that hereditary and acquired thrombophilias are higher than previously expected and reported.

Myeloid neoplasms with germ line RUNX1 mutation.

Familial platelet disorder with propensity to myeloid malignancies (FPD/AML) is an autosomal dominant disorder characterized by quantitative and/or qualitative platelet defects with a tendency to develop a variety of hematological malignancies. Heterozygous germ line mutations in the RUNX1 gene are responsible genetic events for FPD/AML. Notably, about half of individuals in the family with germ line mutations in RUNX1 develop overt hematological malignancies. The latency is also relatively long as an average age at diagnosis is more than 30 years. Similar to what is observed in sporadic hematological malignancies, acquired additional genetic events cooperate with inherited RUNX1 mutations to progress the overt malignant phase. Reflecting recent increased awareness of hematological malignancies with germ line mutations, FPD/AML was added in the revised WHO 2016 classification. In this review, we provide an update on FPD/AML with recent clinical and experimental findings.

Paradoxical bleeding and thrombotic episodes of dysprothrombinaemia due to a homozygous Arg382His mutation.

We have characterised the pathogenic basis of dysprothrombinaemia in a patient exhibiting paradoxical bleeding and thrombotic defects during pregnancy and postpartum. Genetic analysis revealed that the proband is homozygous for the prothrombin Arg382His mutation, possessing only ~1 % clotting activity. The proband experienced severe bleeding episodes during her pregnancy, which required treatment with prothrombin complex concentrates, and then pulmonary embolism and deep-vein thrombosis at 28 days postpartum, which required treatment with LMWH and fresh frozen plasma. Analysis of haemostatic parameters revealed that the subject had elevated FDP and DD and decreased fibrinogen levels, indicating the presence of hyperfibrinolysis. Thrombin generation and clotting assays with the proband's plasma in the presence of soluble thrombomodulin and tissue-type plasminogen activator indicated a defect in activation of both protein C and thrombin activatable fibrinolysis inhibitor (TAFI). Unlike normal plasma, no TAFI activation could be detected in the patient's plasma. The expression and characterisation of recombinant prothrombin Arg382His indicated that zymogen activation by prothrombinase was markedly impaired and the activation of protein C and TAFI by thrombin-Arg382His was impaired 600-fold and 2500-fold, respectively. The recombinant thrombin mutant exhibited impaired catalytic activity toward both fibrinogen and PAR1 as determined by clotting and signalling assays. However, the mutant activated factor XI normally in both the absence and presence of polyphosphates. Arg382 is a key residue on (pro)exosite-1 of prothrombin and kinetic analysis of substrate activation suggested that the poor zymogenic activity of the mutant is due to its inability to bind factor Va in the prothrombinase complex.

Myelodysplastic syndromes and acute leukemia with genetic predispositions: a new challenge for hematologists.

INTRODUCTION: The determination of an underlying genetic predisposition is not automatically part of the diagnosis of hematological malignancies (HM) in routine practice. However, it is assumed that genetic predispositions to HM are currently underestimated due to great variations in disease phenotype, variable latency and incomplete penetrance. Most of patients do not display any biological or clinical signs besides the overt hematological disease and many of them have a lack of personal or family history of malignancies. Areas covered: Collaborative studies and important advances in molecular testing have led to the discovery of several genes recurrently deregulated in familial HM including RUNX1, CEBPA, GATA2, ANKRD26, SRP72, PAX5, DDX41, ETV6, ATG2B/GSKIP and TERT/TERC. This review summarizes biological and clinical findings encountered within these disorders. Expert commentary: Identify and manage individuals with genetic predisposition is a current challenge for hematologists. Their identification has immediate implications for hematopoietic stem cell transplantation including donor selection and conditioning regimen. Importantly, several features, including familial and personal history as well as molecular and cytogenetic findings, may help clinicians to suspect an underlying genetic predisposition.

Hypercoagulable States: What the Oral Surgeon Needs to Know.

Thrombophilia or hypercoagulable conditions can be thought of as either inherited or acquired. The inherited disorders include deficiencies of antithrombin, protein C, or protein S or the common disorders of factor V Leiden and prothrombin G20210A gene mutation. All these disorders are inherited as autosomal dominant and predispose individuals primarily to venous thrombosis. Acquired thrombophilic conditions are seen in individuals with cancer, phospholipid antibodies, and a whole host of other conditions that alter endothelial function, change blood levels of coagulant or anticoagulant proteins, activate platelets, or have other effects on coagulation proteins, platelet function, or the endothelium.

Haematological spectrum and genotype-phenotype correlations in nine unrelated families with RUNX1 mutations from the French network on inherited platelet disorders.

BACKGROUND: Less than 50 patients with FPD/AML (OMIM 601309) have been reported as of today and there may an underestimation. The purpose of this study was to describe the natural history, the haematological features and the genotype-phenotype correlations of this entity in order to, first, screen it better and earlier, before leukaemia occurrence and secondly to optimize appropriate monitoring and treatment, in particular when familial stem cell transplantation is considered. METHODS: We have investigated 41 carriers of RUNX1 alteration belonging to nine unrelated French families with FPD/AML and two syndromic patients, registered in the French network on rare platelet disorders from 2005 to 2015. RESULTS: Five missense, one non-sense, three frameshift mutations and two large deletions involving several genes including RUNX1 were evidenced. The history of familial leukaemia was suggestive of FPD/AML in seven pedigrees, whereas an autosomal dominant pattern of lifelong thrombocytopenia was the clinical presentation of two. Additional syndromic features characterized two large sporadic deletions. Bleeding tendency was mild and thrombocytopenia moderate (>50 x10(9)/L), with normal platelet volume. A functional platelet defect consistent with a δ-granule release defect was found in ten patients regardless of the type of RUNX1 alteration. The incidence of haematological malignancies was higher when the mutated RUNX1 allele was likely to cause a dominant negative effect (19/34) in comparison with loss of function alleles (3/9). A normal platelet count does not rule out the diagnosis of FPD/AML, since the platelet count was found normal for three mutated subjects, a feature that has a direct impact in the search for a related donor in case of allogeneic haematopoietic stem cell transplantation. CONCLUSIONS: Platelet dysfunction suggestive of defective δ-granule release could be of values for the diagnosis of FPD/AML particularly when the clinical presentation is an autosomal dominant thrombocytopenia with normal platelet size in the absence of familial malignancies. The genotype-phenotype correlations might be helpful in genetic counselling and appropriate optimal therapeutic management.

Vocal Cord Paralysis as the First Sign of Spontaneous Carotid Dissection in a Patient With Extracranial Internal Carotid Artery Aneurysm.

INTRODUCTION: Spontaneous dissection of supra-aortic arteries is an exceptionally rare cause of vocal cord dysfunction. We are reporting a case of spontaneous carotid dissection and internal carotid artery aneurysm presenting as vocal cord paralysis. CASE REPORT: A 44-year-old female was admitted with hoarseness and swallowing disorders. Diagnostic imaging revealed dissection and obliteration of the right internal carotid artery (ICA) 23 mm from the carotid bifurcation. Electromyography revealed unilateral paralysis/paresis of the right vocal cord. Genetic analyses for thrombophilia, methylenetetrahydrofolate reductase, and plasminogen activator inhibitor 1 were found to be at high risk. The patient was discharged after 5 days without any neurological findings, and control angiography revealed complete restitution of the flow in the right ICA one month later. However, a fusiform aneurysm of the distal part of the extracranial right ICA was detected and excluded with endovascular procedure. CONCLUSION: Connective tissue systemic disorders and even mild trauma could initiate the dissection process of neck arteries. Precise diagnosis might be difficult even for an experienced neurologist, however, the final outcome is favorable.

Spectrum of clinical and genetic features of patients with inherited platelet disorder with suspected predisposition to hematological malignancies: a nationwide survey in Japan.

BACKGROUND: Inherited thrombocytopenia (IT) contains several forms of familial thrombocytopenia and some of them have propensity to hematological malignancies. The etiological and genetic features of this heterogeneous syndrome have not yet been elucidated. PATIENTS AND METHODS: We conducted a nationwide survey to collect clinical information and samples from patients with familial thrombocytopenia and/or hematological malignancies in order to obtain a comprehensive understanding of IT. RESULTS: Among the 43 pedigrees with clinical samples, RUNX1 mutations were identified in 8 pedigrees (18.6%). While MYH9 and ANKRD26 mutations were identified in 2 and 1 pedigrees, respectively, no gene mutations were detected in the remaining 32 pedigrees from a panel of previously reported pathogenetic mutations. Clinical data were comparable between FPD/AML and non-FPD/AML probands. CONCLUSIONS: Our study clarified that it is unexpectedly difficult to diagnose FPD/AML based on clinical information alone, and thus, genetic testing is strongly recommended. Our survey also identified some pedigrees with a strong family history of myelodysplastic syndromes of unknown origin. Additionally, there were 14 pedigrees in which three or more members were affected by immune thrombocytopenia (ITP), and a computer-aided simulation suggested that such a distribution almost never happens by coincidence, which implicates a genetic predisposition to ITP.

Targeted gene correction of RUNX1 in induced pluripotent stem cells derived from familial platelet disorder with propensity to myeloid malignancy restores normal megakaryopoiesis.

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease associated with a germline mutation in the RUNX1 gene and is characterized by thrombocytopenia and an increased risk of developing myeloid malignancies. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a patient with FPD/AML possessing a nonsense mutation R174X in the RUNX1 gene. Consistent with the clinical characteristics of the disease, FPD iPSC-derived hematopoietic progenitor cells were significantly impaired in undergoing megakaryocytic differentiation and subsequent maturation, as determined by colony-forming cell assay and surface marker analysis. Notably, when we corrected the RUNX1 mutation using transcription activator-like effector nucleases in conjunction with a donor plasmid containing normal RUNX1 cDNA sequences, megakaryopoiesis and subsequent maturation were restored in FPD iPSC-derived hematopoietic cells. These findings clearly indicate that the RUNX1 mutation is robustly associated with thrombocytopenia in patients with FPD/AML, and transcription activator-like effector nuclease-mediated gene correction in iPSCs generated from patient-derived cells could provide a promising clinical application for treatment of the disease.