Autosomal Dominant Thrombocytopenia Research Articles

Thrombocytopenia 4 (THC4): Six novel families with mutations of the cytochrome c gene.

Thrombocytopenia 4 (THC4) is an autosomal-dominant thrombocytopenia caused by mutations in CYCS, the gene encoding cytochrome c (CYCS), a small haeme protein essential for electron transport in mitochondria and cell apoptosis. THC4 is considered an extremely rare condition since only a few patients have been reported so far. These subjects presented mild thrombocytopenia and no or mild bleeding tendency. In this study, we describe six Italian families with five different heterozygous missense CYCS variants: p.Gly42Ser and p.Tyr49His previously associated with THC4, and three novel variants (p.Ala52Thr, p.Arg92Gly, and p.Leu99Val), which have been classified as pathogenic by bioinformatics and segregation analyses. Moreover, we supported functional effects of p.Ala52Thr and p.Arg92Gly on oxidative growth and respiratory activity in a yeast model. The clinical characterization of the 22 affected individuals, the largest series of THC4 patients ever reported, showed that this disorder is characterized by mild-to-moderate thrombocytopenia, normal platelet size, and function, low risk of bleeding, and no additional clinical phenotypes associated with reduced platelet count. Finally, we describe a significant correlation between the region of CYCS affected by mutations and the extent of thrombocytopenia, which could reflect different degrees of impairment of CYCS functions caused by different pathogenetic variants.

The Y49H cytochrome c variant enhances megakaryocytic maturation of K-562 cells

Five pathogenic variants in the gene encoding cytochrome c (CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. Previous studies of peripheral blood CD34+ or CD45+ cells from subjects with the G42S CYCS variant showed an acceleration in megakaryopoiesis compared to wild-type (WT) cells. To determine whether this result reflects a common feature of the CYCS variants, the c.145T>C mutation (Y49H variant) was introduced into the endogenous CYCS locus in K-562 cells, which undergo megakaryocytic maturation in response to treatment with a phorbol ester. The c.145T>C (Y49H) variant enhanced the megakaryocyte maturation of the K-562 cells, and this effect was seen when the cells were cultured at both 18 % and 5 % oxygen. Thus, alteration of megakaryopoiesis is common to both the G42S and Y49H CYCS variants and may contribute to the low platelet phenotype. The Y49H CYCS variant has previously been reported to impair mitochondrial respiratory chain function in vitro, however using extracellular flux analysis the c.145T>C (Y49H) variant does not alter mitochondrial bioenergetics of the K-562 cells, consistent with the lack of a phenotype characteristic of mitochondrial diseases in CYCS variant families. The Y49H variant has also been reported to enhance the ability of cytochrome c to trigger caspase activation in the intrinsic apoptosis pathway. However, as seen in peripheral blood cells from G42S CYCS variant carriers, the presence of Y49H cytochrome c in K-562 cells did not significantly change their response to an apoptotic stimulus.

Altered conformational dynamics contribute to species-specific effects of cytochrome c mutations on caspase activation

Variants in the gene encoding human cytochrome c (CYCS) cause mild autosomal dominant thrombocytopenia. Despite high sequence conservation between mouse and human cytochrome c, this phenotype is not recapitulated in mice for the sole mutant (G41S) that has been investigated. The effect of the G41S mutation on the in vitro activities of cytochrome c is also not conserved between human and mouse. Peroxidase activity is increased in both mouse and human G41S variants, whereas apoptosome activation is increased for human G41S cytochrome c but decreased for mouse G41S cytochrome c. These apoptotic activities of cytochrome c are regulated at least in part by conformational dynamics of the main chain. Here we use computational and in vitro approaches to understand why the impact of the G41S mutation differs between mouse and human cytochromes c. The G41S mutation increases the inherent entropy and main chain mobility of human but not mouse cytochrome c. Exclusively in human G41S cytochrome c this is accompanied by a decrease in occupancy of H-bonds between protein and heme during simulations. These data demonstrate that binding of cytochrome c to Apaf-1 to trigger apoptosome formation, but not the peroxidase activity of cytochrome c, is enhanced by increased mobility of the native protein conformation.

A Cytochrome c Variant Enhances Megakaryocytic Maturation

Mutations in genes which play a role in the processes of megakaryopoiesis and platelet production cause thrombocytopenia. Five pathogenic variants in the gene encoding cytochrome c ( CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. The role of cytochrome c in megakaryopoiesis and platelet formation is unknown, and it is unclear how mutations in cytochrome c cause thrombocytopenia. The goal of this project is to understand how CYCS mutations cause thrombocytopenia. We are using the human leukemic K562 cell line as a model system to study the impact of CYCS mutations on megakaryocyte differentiation and mitochondrial function. One of the CYCS pathogenic variants (c.145T>C; p.Tyr49His) was introduced into K562 cells using the CRISPR-Cas9 base-editing method. K562 cells have three copies of CYCS-containing chromosome 7 and we isolated CYCS WT/WT/WT, CYCS WT/WT/Y49H, CYCS WT/Y49H/Y49H, CYCS Y49H/Y49H/Y49H clonal lines, confirmed by sequencing and mass spectrometry. K562 cells undergo megakaryocyte differentiation in response to PMA. We monitored the expression of megakaryocyte markers CD61 and CD41a by flow cytometry in response to PMA in all clonal lines. The K562- CYCS Y49H/Y49H/Y49H line showed faster maturation compared to all other clonal lines and the parental K562 cells. We assessed mitochondrial function using the Seahorse XFe96 Analyzer. Our results show that the mutation does not impact mitochondrial respiratory function. Finally, we have introduced the Y49H mutation into WTC11 iPSCs. Investigations of the impact of the Y49H variant on apoptosis showed that there is no significant differences in apoptosis between WT and the clonal line carrying the Y49H variant. Our results suggest that the pathogenic c.145T>C; p.Tyr49His CYCS variant enhances megakaryocyte maturation.

Analysis of clinical characteristics and treatment efficacy in two pediatric cases of ANKRD26-related thrombocytopenia

ANKRD26-related thrombocytopenia (ANKRD26-RT or THC2, MIM 188 000), an autosomal dominant thrombocytopenia, is unresponsive to immunosuppressive therapy and susceptible to hematological malignancies. A large number of pediatric patients are diagnosed with immune thrombocytopenia (ITP) every year; however, thrombocytopenia of genetic origin is often missed. Extensive characterization of ANKRD26-RT will help prevent missed diagnosis and misdiagnosis. Furthermore, identification of ANKRD26-RT will help in the formulation of an accurate diagnosis and a treatment plan. In our study, we report cases of two Chinese pediatric patients with ANKRD26-RT and analyze their clinical characteristics, gene mutations, and treatment modalities. Both patients were 1-year-old and presented with mild bleeding (World Health Organization(WHO) score grade 1), different degrees of platelet reduction, normal mean platelet volume, and megakaryocyte maturation impairment not obvious. Genetic tests revealed that both patients had ANKRD26 gene mutations.Patient 1 had a mutation c.-140C>G of the 5’ untranslated region (UTR), and patient 2 had a mutation of c.-127A>T of 5’UTR. Both patients were treated with eltrombopag, and the treatment was no response, with no adverse reactions.

ETV6 Represses TNF during Stress Hematopoiesis and Regulates HSC Self Renewal

INTRO: ETS Variant Transcription Factor 6 (ETV6), formerly known as Translocation ETS Leukemia (TEL), functions primarily as a transcriptional repressor and is highly expressed in mouse and human HSPCs where it is essential for the development and maintenance of adult hematopoiesis. Nevertheless, it has remained poorly understood how ETV6 regulates the HSC compartment. In 2015, our group and others identified heterozygous pathogenic germline ETV6 variants in families with autosomal dominant thrombocytopenia and predisposition to hematologic malignancies (most often B-acute lymphoblastic leukemia), a condition now known as thrombocytopenia 5 (T5). METHODS: To elucidate the mechanisms by which germline ETV6 variants impact hematopoiesis and promote leukemogenesis, we generated a knock-in mouse model harboring Etv6 R355X, the murine equivalent of a human T5-associated variant (R359X). We used complementary in vitro and in vivo assays to comprehensively characterize the numbers and functions of Etv6R355X/+ and Etv6+/+ hematopoietic progenitors. RESULTS:Etv6R355X/+ mice display several of the cardinal features of T5, including abnormally rounded platelets with increased area compared to platelets from wild-type Etv6+/+ littermates. Further, Etv6R355X/+ platelets exhibit significantly delayed and incomplete clot formation as measured through clot retraction assays. Together, these findings indicate that this novel model will serve as a viable means to study the human disease. We observe that 3-month-old Etv6R355X/+mice maintain normal frequencies and numbers of lineage-sca1+cKit+ (LSK) cells in the bone marrow (BM); however, at 12 months of age, they exhibit a significant decrease in frequency in this compartment. At each of these timepoints, there are variable shifts in the proportions and/or numbers of certain HSC subpopulations. To examine whether the Etv6R355X/+mutation impacts HSC function, we carried out in vitrosEtv6R355X/+BM cells generate more colonies than wild-type cells in the first two rounds of plating, but significantly fewer or no colonies by the fourth plating. Similarly, Etv6R355X/+LSK cells exhibit significantly impaired engraftment when challenged via competitive transplantation, with near complete failure to reconstitute irradiated recipients by the tertiary transplant. Etv6R355X/+LSKs home to the bone marrow comparably to Etv6+/+cells, indicating that homing does not account for the defect in repopulation capacity. To investigate how ETV6 functions in HSCs, we performed RNA-seq of Etv6R355X/+LSKs and flow cytometry-purified HSPC subpopulations from 12-month-old mice, which revealed upregulation of genes associated with an active stem cell phenotype, downregulation of genes supporting stem cell quiescence, and upregulated expression of Tnf with enrichment for the Hallmark TNFa Signaling via NFKB geneset. Subsequent experiments using CUT&RUN of Etv6+/+LSK cells revealed several known and novel ETV6 target genes, including Tnf, a cytokine implicated in regulating stress hematopoiesis. The sites bound by ETV6 at the Tnf locus aligned with ATAC-seq peaks generated from HPC5 cells, a mouse bone marrow progenitor cell line, suggesting that this is a region of open chromatin and active transcriptional regulation. Corroborating these findings, examination of Etv6R355X/+HSPCs at 6 weeks post-transplant revealed increased cell cycling and intracellular TNF production. CONCLUSION: From these studies, we conclude that ETV6 regulates HSC maintenance. Further, it binds to a regulatory sequence within the Tnf locus, whereby it represses TNF expression. The loss or reduction of this repression in Etv6R355X/+HSCs is associated with increased cell cycling under conditions of hematopoietic stress and a loss of self-renewal capacity. Studies are ongoing to determine how these changes promote leukemogenesis and contribute to the other features of T5.

Defective VWF secretion due to expression of MYH9-RD E1841K mutant in endothelial cells disrupts hemostasis

AbstractMutations in MYH9, the gene encoding the heavy chain of nonmuscle myosin IIa (NMII-A), cause MYH9-related disease (MYH9-RD), which is an autosomal-dominant thrombocytopenia with bleeding tendency. Previously, we showed that NMII-A in endothelial cells (ECs) is critical for hemostasis via regulating von Willebrand factor (VWF) release from Weibel-Palade bodies (WPBs). The aim of this study was to determine the role of the expression of MYH9 mutants in ECs in the pathogenesis of the MYH9-RD bleeding symptom. First, we expressed the 5 most common NMII-A mutants in ECs and found that E1841K mutant-expressing ECs secreted less VWF than the controls in response to a cyclic adenosine monophosphate (cAMP) signaling agonist. Then, we generated 2 knockin mouse lines, 1 with Myh9 E1841K in ECs and the other in megakaryocytes. Endothelium-specific E1841K mice exhibited impaired cAMP-induced VWF release and a prolonged bleeding time with normal platelets, whereas megakaryocyte-specific E1841K mice exhibited macrothrombocytopenia and a prolonged bleeding time with normal VWF release. Finally, we presented mechanistic findings that E1841K mutation not only interferes with S1943 phosphorylation and impairs the peripheral distribution of Rab27a-positive WPBs in Ecs under quiescent condition but also interferes with S1916 phosphorylation by disrupting the interaction with zyxin and CKIIα and reduces actin framework formation around WPBs and subsequent VWF secretion under the stimulation by a cAMP agonist. Altogether, our results suggest that impaired cAMP-induced endothelial VWF secretion by E1841K mutant expression may contribute to the MYH9-RD bleeding phenotype.

ANKRD26-Related Thrombocytopenia and Predisposition to Myeloid Neoplasms.

This review describes ANKRD26-related thrombocytopenia (RT) from a molecular, clinical, and laboratory perspective, with a focus on the clinical decision-making that takes place in the diagnosis and management of families with ANKRD26-RT. ANKRD26-related thrombocytopenia (ANKRD26-RT) is a non-syndromic autosomal dominant thrombocytopenia with predisposition to hematologic neoplasm. The clinical presentation is variable with moderate thrombocytopenia with normal platelet size and absent to mild bleeding being the hallmark which makes it difficult to distinguish from other inherited thrombocytopenias. The pathophysiology involves overexpression of ANKRD26 through loss of inhibitory control by transcription factors RUNX1 and FLI1. The great majority of disease-causing variants are in the 5' untranslated region. Acute myeloid leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia have been reported to occur in the context of germline variants in ANKRD26, with the development of somatic driver mutations in hematopoietic regulators playing an important role in malignant transformation. In the absence of clear risk estimates of development of malignancy, optimal surveillance strategies and interventions to reduce risk of evolution to a myeloid disorder, multidisciplinary evaluation, with a strong genetic counseling framework is essential in the approach to these patients and their families. Gene-specific expertise and a multidisciplinary approach are important in the diagnosis and treatment of patients and families with ANKRD26-RT. These strategies help overcome the challenges faced by clinicians in the evaluation of individuals with a rare, non-syndromic, inherited disorder with predisposition to hematologic malignancy for which large data to guide decision-making is not available.

3036 – ETV6 REPRESSES TNF EXPRESSION IN HSCS DURING STRESS HEMATOPOIESIS AND REGULATES HSC SELF-RENEWAL

ETV6 encodes an essential transcription factor during normal hematopoiesis. Our group and others identified heterozygous pathogenic germline ETV6 variants in families with autosomal dominant thrombocytopenia and predisposition to hematologic malignancies. To elucidate the mechanisms by which germline ETV6 variants impact hematopoiesis, we generated a knock-in mouse model harboring Etv6 R355X, the murine equivalent of a human disease variant. Except for reduced MPP4 cells, 3-month-old Etv6R355X/+ mice maintain normal numbers of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). However, by 12 months, they exhibit a decrease in all HSPC compartments. RNA-sequencing of Etv6R355X/+ HSPCs at 12 months identified 402 significant differentially-expressed genes. Among these, we observe upregulation of “active stem cell genes” (e.g., Myc, Cdk6) and downregulation of “self-renewal genes” (e.g., Procr, Mpl, Jun). Moreover, we observe a significant loss of self -renewal potential when challenging Etv6R355X/+ HSPCs with repetitive stressors: First, through serial competitive transplantation, we observe a failure of mutant cells to reconstitute the bone marrow by the tertiary transplant. Next, following serial 5FU treatment, we find that Etv6R355X/+ mice exhibit inferior survival compared to Etv6+/+ controls. To determine which ETV6 target genes contribute to these phenotypes, we performed ETV6 CUT&RUN, ATACseq and HiC using the HPC5 stem cell line, as well as ETV6 CUT&RUN using primary mouse HSPCs and human CD34+ cells. Through these studies, we identify TNF as a putative ETV6 target gene. Notably, using intracellular staining and flow cytometry, we find elevated TNF expression in Etv6R355X/+ HSPCs post-transplant. Together, these data suggest that ETV6 regulates HSPC self-renewal and represses TNF signaling during stress hematopoiesis. ETV6 encodes an essential transcription factor during normal hematopoiesis. Our group and others identified heterozygous pathogenic germline ETV6 variants in families with autosomal dominant thrombocytopenia and predisposition to hematologic malignancies. To elucidate the mechanisms by which germline ETV6 variants impact hematopoiesis, we generated a knock-in mouse model harboring Etv6 R355X, the murine equivalent of a human disease variant. Except for reduced MPP4 cells, 3-month-old Etv6R355X/+ mice maintain normal numbers of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). However, by 12 months, they exhibit a decrease in all HSPC compartments. RNA-sequencing of Etv6R355X/+ HSPCs at 12 months identified 402 significant differentially-expressed genes. Among these, we observe upregulation of “active stem cell genes” (e.g., Myc, Cdk6) and downregulation of “self-renewal genes” (e.g., Procr, Mpl, Jun). Moreover, we observe a significant loss of self -renewal potential when challenging Etv6R355X/+ HSPCs with repetitive stressors: First, through serial competitive transplantation, we observe a failure of mutant cells to reconstitute the bone marrow by the tertiary transplant. Next, following serial 5FU treatment, we find that Etv6R355X/+ mice exhibit inferior survival compared to Etv6+/+ controls. To determine which ETV6 target genes contribute to these phenotypes, we performed ETV6 CUT&RUN, ATACseq and HiC using the HPC5 stem cell line, as well as ETV6 CUT&RUN using primary mouse HSPCs and human CD34+ cells. Through these studies, we identify TNF as a putative ETV6 target gene. Notably, using intracellular staining and flow cytometry, we find elevated TNF expression in Etv6R355X/+ HSPCs post-transplant. Together, these data suggest that ETV6 regulates HSPC self-renewal and represses TNF signaling during stress hematopoiesis.

A CRISPR-Cas9 Generated Etv6 mutant Mouse Exhibits Decreased Platelet Counts and Functional Platelet Defects

We and others recently described families with autosomal dominant thrombocytopenia, variable platelet function defects, and a predisposition to leukemia caused by heterozygous germline mutations in ETV6 . One of these mutations in ETV6, encoding an amino acid change at position 214 (P214L), was described in five independent families. The P214L mutation, located in the central domain of ETV6, was shown to disrupt nuclear localization of the ETV6 protein and to block megakaryocyte maturation of human cells overexpressing the mutant protein in vitro . Additionally, RNA seq of platelets from patients with the ETV6-P214L mutation showed downregulation of cytoskeletal transcripts and transcripts involved in platelet production and function. To further study the effect of the P214L mutation on ETV6 and on platelet production and function, we have generated a CRISPR-Cas9 knock-in mouse with the equivalent mutation to the human P214L in the mouse Etv6 ortholog (in the corresponding amino acid P216 in mice). CRISPR-Cas9 knock-in mice were generated by designing short guide RNAs specific to the site of Etv6 encoding the proline to be mutated. Additionally, a ssDNA oligo was designed encoding the mutation (cca>cta, P>L) and flanking homologous sequence to provide a template for recombination. Heterozygous and homozygous mice were viable. Heterozygous by heterozygous crosses yielded ratios not significantly different from expected Mendelian ratios: wild type (WT) 18.0%, heterozygous 63.0%, and homozygous 19.1% (p=0.18, Chi-squared test). Complete blood counts were obtained by retro-orbital bleeds and showed a mild but significant decrease in platelet counts in homozygous mice (WT 967.3 x 103/uL, heterozygous 840 x 103/uL, homozygous 807.5 x 103/uL, p=0.03). Western blot indicated normal expression of Etv6 protein in mouse bone marrow. Immunofluorescence and confocal imaging of megakaryocytes from these mice showed an increased percentage of small, immature megakaryocytes in homozygous mice compared to WT mice. Flow cytometry analysis suggested normal distribution of the hematopoietic stem cell compartment. These data are consistent with the patient phenotype and in vitro data, which showed that the P214L mutation resulted in variable thrombocytopenia, immature megakaryocytes, and normal ETV6 protein expression.Considering the downregulation of transcripts related to platelet function in patients with the P214L mutation and the variable platelet function defects reported in patients with germline ETV6 mutations, we characterized the platelet function of Etv6 mutant mice. Platelets from homozygous mice showed decreased platelet activation upon high (10 ug/ml) and low (2 ug/ml) collagen stimulation as measured by light transmission aggregometry (p=0.041 and 0.019, respectively). Additionally, homozygous mice had decreased markers of platelet activation (thrombin and convulxin) including granule secretion (P-selectin exposure) and aIIbb3 activation (JONA binding).In conclusion, we have generated a CRISPR-Cas9 knock-in mouse with the orthologous mutation to the human P214L recurrent germline mutation. Mice homozygous for this mutation recapitulate the human phenotype of decreased platelet counts and impaired megakaryocyte maturation. Additionally, mice homozygous for this mutation show a significant defect in platelet activation, further supporting that Etv6 is important for platelet function. Although complete knock-outs of Etv6 are embryonic lethal and Etv6 is expressed in the hematopoietic stem cell, we did not find a significant hematopoietic stem cell defect in our mutant mice. This finding suggests that the most important function of Etv6 is further downstream in hematopoiesis. The Etv6 P214L mouse represents a reliable model for studying the role of Etv6 in megakaryocyte biology, platelet function, and potentially future studies in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Heterogeneous Landscape of RUNX1 Related Disorder: Four Families with Inherited Thrombocytopenias and Novel Genetic Variants in RUNX1 Associated to Relevant Clinical Complications

BackgroundFamilial platelet disorder with propensity to myeloid malignancy (FPD/AML) is a rare autosomal dominant disease with incomplete penetrance and variable expressivity. It is characterized by thrombocytopenia and/or platelet functional defects. Although bleeding tendency is usually mild to moderate, an important hallmark of FPD/AML is the increased risk of myeloid neoplasms, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The 2016 revision to the WHO classification system for myeloid neoplasms has incorporated a section on “myeloid neoplasms with germline predisposition” including RUNX1, ANKRD26 and ETV6 genes, among others. There is a great phenotypic heterogeneity and diagnosis is challenging due to specific test is not available. Now, high-throughput sequencing (HTS) is revolutionizing genetic diagnosis of human diseases, including platelet disorders and is extremely helpful to identify this disorder.AimsTo assess the clinical, platelet phenotype and their genetic defect of 4 unrelated families with RUNX1 related thrombocytopenia (RUNX1 -RT) in our cohort of 82 consecutive unrelated probands with inherited platelet disorders (IPDs).MethodsWe studied 5 patients from 4 unrelated families presenting with lifelong thrombocytopenia with normal platelet size. Clinical records were reviewed and bleeding scored with ISTH-BAT. Platelet phenotyping included peripheral blood smear and platelet aggregation. Patients DNAs were analyzed by HTS using a 71-gene panel related to IPDs. Causative variants were elucidated after a workflow algorithm filter based in quality, consequence-damaging, minor allele frequency <0.05 and in silico prediction tools. Finally, variants were qualified according to guidelines of the American College of Medical Genetics and Genomics and Association for Molecular Pathology.ResultsThe median age was 28 years old (1-54), median platelet counts was 90x103/mm3 (35-108x103/mm3) and bleeding score was 3 (2-5). Clinical and family history, and laboratory findings in these patients are shown in Figure 1. Family 1 (F1) showed an autosomal dominant thrombocytopenia (P1, P2, brother and their mother). In this family, a extremely history of hematologic malignancies are presented: one brother underwent to allogeneic transplant from unrelated donor due to a high-risk MDS del(7q) and one daughter of P1 died at the age of 13 years old due to a refractory AML. In F2, a lifelong thrombocytopenia, bleeding and family history of breast cancer was characteristic; but, in F3, only a lifelong thrombocytopenia was referred. Finally, P5 showed mental retardation, corpus callosum hypoplasia, facial and cardiac abnormalities and severe thrombocytopenia. HTS revealed three novel variants affected to RUNX1 (F1, P3 and P4) (Figure 1) and one sporadic germ line RUNX1 deletion involving chromosome 21q22, which, was confirmed by in-situ hybridization, resulting in RUNX1 haploinsufficiency. The missense variants affected to runt homology domain may lead to dominant-negative effects and were associated with higher propensity to develop AML, but it usually needed a second alteration associated, as occurs in our families (F1 and F2).ConclusionHTS facilitates genetic confirmation of FPD/AML diagnosis, and may help investigating phenotype-genotype relationships in this heterogeneous landscape. An earlier diagnosis of this disorder improves the genetic counselling and appropriate therapeutic management.FundingGerencia Regional de Salud [GRS 1370/A/16]; ISCIII & Feder (PI14/01956), CIberer CB15/00055, Sociedad Española de Trombosis y Hemostasia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity.

Mutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and the molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with an exchange between conformational states proposed to regulate changes in function. Here, we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. Three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with the increased overall flexibility of cytochrome c, particularly movement of the Ω loops. Crystal structures of Y48H and G41T complement these studies which overall suggest that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an 'induced fit' mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics. Thus, the mechanism by which the variants increase peroxidase activity is not related to the conformational dynamics of the native hexacoordinate state of cytochrome c. Recent molecular dynamics data proposing conformational mobility of specific cytochrome c regions underpins changes in reduction potential and alkaline transition pK was not fully supported. These data highlight that conformational dynamics of cytochrome c drive some but not all of its properties and activities.

ETV6 germline mutations cause HDAC3/NCOR2 mislocalization and upregulation of interferon response genes.

ETV6 is an ETS family transcription factor that plays a key role in hematopoiesis and megakaryocyte development. Our group and others have identified germline mutations in ETV6 resulting in autosomal dominant thrombocytopenia and predisposition to malignancy; however, molecular mechanisms defining the role of ETV6 in megakaryocyte development have not been well established. Using a combination of molecular, biochemical, and sequencing approaches in patient-derived PBMCs, we demonstrate abnormal cytoplasmic localization of ETV6 and the HDAC3/NCOR2 repressor complex that led to overexpression of HDAC3-regulated interferon response genes. This transcriptional dysregulation was also reflected in patient-derived platelet transcripts and drove aberrant proplatelet formation in megakaryocytes. Our results suggest that aberrant transcription may predispose patients with ETV6 mutations to bone marrow inflammation, dysplasia, and megakaryocyte dysfunction.

Monoallelic loss-of-function THPO variants cause heritable thrombocytopenia

Key Points We report rare monoallelic variants of THPO that alter intracellular trafficking and diminish thrombopoietin secretion. Affected cases have autosomal-dominant thrombocytopenia but no other hematological features.

Comparison of the structural dynamic and mitochondrial electron-transfer properties of the proapoptotic human cytochrome c variants, G41S, Y48H and A51V

Mitochondrial cytochrome c is associated with electron transfer in the respiratory chain and in apoptosis. Four cytochrome c variants have been identified in families that suffer from mild autosomal dominant thrombocytopenia, a platelet disorder associated with increased apoptosis. Three out of the four substitutions, G41S, Y48H and A51V are located on the 40–57 Ω-loop. The G41S and Y48H variants perturb key physicochemical and dynamic properties that result in enhanced functional features associated with apoptotic activity. Herein we characterise the ferric A51V variant. We show by chemical denaturation that this variant causes the native state to be destabilized. Through azide binding kinetics, the population of a pentacoordinate heme form, whereby the Met80 axial ligand is dissociated, is estimated to be of equal magnitude to that found in the Y48H variant. This pentacoordinate form gives rise to peroxidase activity, which despite the similar pentacoordinate population of the A51V variant to that of the Y48H variant, the peroxidase activity of the A51V variant is suppressed. Far-UV circular dichroism spectroscopy and pH jump studies, suggest that a combination of structural and dynamic features in addition to the population of the pentacoordinate form regulate peroxidase activity in these disease variants. Additionally, the steady-state ratio of ferric/ferrous cytochrome c when in turnover with cytochrome c oxidase has been investigated for all 40–57 Ω-loop variants. These studies show that the lower pKa of the alkaline transition for the disease causing variants increases the ferric to ferrous heme ratio, indicating a possible influence on respiration in vivo.

Familial Platelet Disorder/Predisposition to Acute Myeloid Leukemia With Germline RUNX1 Mutation Masquerading as Idiopathic Thrombocytopenic Purpura: Case Study

Abstract A 25-year-old female underwent emergent laparoscopic salpingectomy when she presented with an ectopic pregnancy (G1P0). A bone marrow biopsy performed for moderate persistent thrombocytopenia (60-105 K/μL) showed normocellularity, maturing trilineage hematopoiesis, and mildly increased morphologically normal megakaryocytes reported as consistent with idiopathic thrombocytopenic purpura (ITP). However, on further inquiry, the patient stated that her sister, father, paternal aunt, and paternal grandfather have/had low platelets. Her father had been diagnosed with myelodysplasia (MDS). Her paternal grandmother had died of acute myeloid leukemia (AML). Thus, although her marrow morphology revealed no evidence of overt dysplasia and despite her normal karyotypic study, a sample was sent for RUNX1 sequence analysis. A splice site mutation (c.967 + 2_967 + 5delTAAG) in RUNX1 was detected and initially interpreted as a somatic mutation. However, given the strong family history of thrombocytopenia and MDS/AML, a sample from her father was also sent for RUNX1 sequence analysis, and the identical intronic sequence variant in the RUNX1 gene was detected in him. This confirmed the RUNX1 mutation to be germline. In the context of autosomal dominant thrombocytopenia in the patient’s family, the finding of this variant in the affected patient and her similarly affected father was consistent with a pathogenic role. Since this patient was of reproductive age actively trying to have children, these molecular genetic findings had important implications. She elected to undergo in vitro fertilization in order to choose embryos without the RUNX1 mutation for implantation. Considering that her father and paternal grandmother developed MDS/AML, and because familial platelet disorder with predisposition to AML appears to show “anticipation,” she would require close future monitoring for blastic transformation. This case illustrates the significance of careful history taking and high suspicion index that can lead to simplified targeted cost-effective molecular testing resulting in the correct diagnosis without necessarily employing large gene panels.

ASSESSING THE IMPACT OF A B-ALL-ASSOCIATED GERMLINE ETV6 VARIANT ON MURINE HEMATOPOIESIS AND STEM CELL FUNCTION

The transcription factor ETV6 is required for bone marrow (BM) hematopoiesis and survival of adult hematopoietic stem cells (HSCs), but the mechanisms by which ETV6 regulates these processes remain unclear. We and others reported that germline variants in ETV6 are associated with autosomal dominant thrombocytopenia and occurrence of B-ALL. We identified likely pathogenic variants in 0.8% of 4,405 childhood ALL cases, and functional evaluation of these variants revealed impairment of transcriptional repression activity of ETV6. To investigate the impact of ETV6 variants on hematopoiesis, we generated a CRISPR-Cas9-derived mouse model (Etv6R355X/+) that mirrors an ETV6 R359X variant observed in multiple ALL cases. Flow cytometry of the BM revealed progressive changes in stem and progenitor cells, with 12-month-old ETV6R355X/+ mice showing ∼40% reductions in HSPCs and ∼40% increases in pro-B cells. Lineage-negative, cKit+, Sca1+ (LSK) cells from 3-month-old ETV6R355X/+ mice showed impaired engraftment in a competitive transplant model, as demonstrated by significantly reduced chimerism in primary, secondary, and tertiary recipients. To determine how the ETV6 R355X variant influences the hematopoietic transcriptional landscape, we performed RNA-seq and ATAC-seq from LSK and pro-B cells of 3-month-old WT or Etv6R355X/+ mice. We found 90 and 125 differentially expressed genes in LSK and pro-B cells, respectively. GSEA revealed enrichment of genes associated with hypoxia response and stem cell maintenance. ATAC-seq revealed a preferential shift toward an increase in open chromatin in Etv6R355X/+ cells within regions highly enriched for ETS DNA consensus motifs, indicating a potential loss of ETV6-mediated repression. These findings demonstrate that a pathogenic ETV6 variant can disrupt normal hematopoiesis and alter the transcriptional profile of stem and progenitor cells.

Genome-wide linkage analysis and whole-exome sequencing identifies an ITGA2B mutation in a family with thrombocytopenia.

Hereditary thrombocytopenias can be subclassified based on mode of inheritance and platelet size. Here we report a family with autosomal dominant (AD) thrombocytopenia with normal platelet size. Linkage analysis and whole exome sequencing identified the R1026W substitution in ITGA2B as the causative defect. The same mutation has been previously reported in 7 Japanese families/patients with AD thrombocytopenia, but all of these patients had macrothrombocytopenia. This is the first report of a family with AD thrombocytopenia with normal platelet size resulting from mutation in ITGA2B. ITGA2B mutations should therefore be included in the differential diagnosis of this latter disorder.

Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeletal dynamics in platelets.

MASTL, a Ser/Thr kinase that inhibits PP2A-B55 complexes during mitosis, is mutated in autosomal dominant thrombocytopenia. However, the connections between the cell-cycle machinery and this human disease remain unexplored. We report here that, whereas Mastl ablation in megakaryocytes prevented proper maturation of these cells, mice carrying the thrombocytopenia-associated mutation developed thrombocytopenia as a consequence of aberrant activation and survival of platelets. Activation of mutant platelets was characterized by hyperstabilized pseudopods mimicking the effect of PP2A inhibition and actin polymerization defects. These aberrations were accompanied by abnormal hyperphosphorylation of multiple components of the actin cytoskeleton and were rescued both in vitro and in vivo by inhibiting upstream kinases such as PKA, PKC, or AMPK. These data reveal an unexpected role of Mastl in actin cytoskeletal dynamics in postmitotic cells and suggest that the thrombocytopenia-associated mutation in MASTL is a pathogenic dominant mutation that mimics decreased PP2A activity resulting in altered phosphorylation of cytoskeletal regulatory pathways.

MYH9-Related Thrombocytopenia: Four Novel Variants Affecting the Tail Domain of the Non-Muscle Myosin Heavy Chain IIA Associated with a Mild Clinical Evolution of the Disorder.

MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA). Patients present congenital macrothrombocytopenia and inclusions of NMMHC-IIA in leukocytes, and have a variable risk of developing kidney damage, sensorineural deafness, presenile cataracts and/or liver enzymes abnormalities. The spectrum of mutations found in MYH9-RD patients is limited and the incidence and severity of the non-congenital features are predicted by the causative MYH9 variant. In particular, different alterations of the C-terminal tail domain of NMMHC-IIA associate with remarkably different disease evolution. We report four novel MYH9 mutations affecting the tail domain of NMMHC-IIA and responsible for MYH9-RD in four families. Two variants cause amino acid substitutions in the coiled-coil region of NMMHC-IIA, while the other two are a splicing variant and a single nucleotide deletion both resulting in frameshift alterations of the short non-helical tailpiece. Characterization of phenotypes of affected individuals shows that all of these novel variants are associated with a mild clinical evolution of the disease.