Congenital Thrombocytopenia Research Articles

MECOM-associated syndrome: a heterogeneous inherited bone marrow failure syndrome with amegakaryocytic thrombocytopenia

AbstractHeterozygous mutations in MECOM (MDS1 and EVI1 complex locus) have been reported to be causative of a rare association of congenital amegakaryocytic thrombocytopenia and radioulnar synostosis. Here we report on 12 patients with congenital hypomegakaryocytic thrombocytopenia caused by MECOM mutations (including 10 novel mutations). The mutations affected different functional domains of the EVI1 protein. The spectrum of phenotypes was much broader than initially reported for the first 3 patients; we found familial as well as sporadic cases, and the clinical spectrum ranged from isolated radioulnar synostosis with no or mild hematological involvement to severe bone marrow failure without obvious skeletal abnormality. The clinical picture included radioulnar synostosis, bone marrow failure, clinodactyly, cardiac and renal malformations, B-cell deficiency, and presenile hearing loss. No single clinical manifestation was detected in all patients affected by MECOM mutations. Radioulnar synostosis and B-cell deficiency were observed only in patients with mutations affecting a short region in the C-terminal zinc finger domain of EVI1. We propose the term MECOM-associated syndrome for this heterogeneous hereditary disease and inclusion of MECOM sequencing in the diagnostic workup of congenital bone marrow failure.

Congenital hypoplastic bone marrow failure associated with a de novo partial deletion of the MECOM gene at 3q26.2

Congenital hypoplastic bone marrow failure is a rare condition in neonates. The genetics and mechanisms behind are largely obscure.Here we characterize a neonate presenting with congenital thrombocytopenia and anemia. During the first 2–4 weeks after birth the neonate developed severe neutropenia while the lymphoid lineages were unaffected. The neonate was without dysmorphic signs. A de novo mono-allelic constitutional microdeletion of 175.1 kb at 3q26.2 affecting exon 2 of MECOM, involving MDS1 but not EVI1, was identified as the only copy number alteration by oligo-based array-CGH analysis. Expression analysis showed profoundly reduced expression of multiple MECOM transcripts in the bone marrow cells. Whole exome sequencing detected no pathogenic mutations in genes known to be associated with inherited bone marrow failure syndromes. The patient was successfully treated with hematopoietic stem cell transplantation at 5 months of age.Interstitial deletions encompassing the 3q26.2 region are very rare. A literature search revealed two previous cases with microdeletions involving this region, and the cases were associated with congenital thrombocytopenia and anemia, but unaffected lymphopoiesis. Together these data indicate that MECOM may be important for normal myeloid hematopoiesis in humans but dispensable for lymphoid differentiation. We suggest that partial deletion in MECOM may be a primary event associated with congenital pancytopenia.

Congenital thrombocytopenia

Congenital thrombocytopenias are a genetically and clinically heterogeneous group of platelet disorders that are characterized by thrombocytopenia since birth. Although very rare, these disorders are encountered in routine clinical practice. Since 2011, the application of next generation sequencing has identified more than 15 new genes, which, when mutated, can cause congenital thrombocytopenia. Currently, at least 35 different forms have been identified. Although thrombocytopenia is congenital, some forms present an elevated risk of developing additional complications. In MYH9 disorders, patients often develop nephritis, deafness, and cataract. In a subgroup of congenital thrombocytopenias, with defects in the TPO/MPL signaling pathways or transcription factors, enhanced or reduced hematopoietic cell proliferation properties often culminates in the development of hematological malignancy or bone marrow failure. Proper and early genetic diagnosis is essential for the treatment of congenital thrombocytopenia.

A Severe Case of Congenital Thrombotic Thrombocytopenia Purpura Resulting From Compound Heterozygosity Involving a Novel ADAMTS13 Pathogenic Variant.

We report a 9-year-old Chinese girl with congenital thrombotic thrombocytopenic purpura found to be a compound heterozygote for 2 pathogenic variants in the ADAMTS13 gene, including a novel variation. The girl suffered from recurrent, life-threatening episodes of thrombocytopenia and hemolysis, and laboratory testing showed ADAMST13 enzyme activity of <5%. Sequencing of the ADAMTS13 gene revealed a previously reported missense variant, c.1787C>T (p.Ala596Val), and a novel duplication defined as c.1007_1025dup19 (p.Asp343Leufs*53); the duplication is predicted to result in a premature stop codon and protein truncation. We propose that this novel variant is partly responsible for the patient's early-onset and severe phenotype.

Hereditary thrombocytopenias: a growing list of disorders.

The introduction of high throughput sequencing (HTS) techniques greatly improved the knowledge of inherited thrombocytopenias (ITs) over the last few years. A total of 33 different forms caused by molecular defects affecting at least 32 genes have been identified; along with the discovery of new disease-causing genes, pathogenetic mechanisms of thrombocytopenia have been better elucidated. Although the clinical picture of ITs is heterogeneous, bleeding has been long considered the major clinical problem for patients with IT. Conversely, the current scenario indicates that patients with some of the most common ITs are at risk of developing additional disorders more dangerous than thrombocytopenia itself during life. In particular, MYH9 mutations result in congenital macrothrombocytopenia and predispose to kidney failure, hearing loss, and cataracts, MPL and MECOM mutations cause congenital thrombocytopenia evolving into bone marrow failure, whereas thrombocytopenias caused by RUNX1, ANKRD26, and ETV6 mutations are characterized by predisposition to hematological malignancies. Making a definite diagnosis of these forms is crucial to provide patients with the most appropriate treatment, follow-up, and counseling. In this review, the ITs known to date are discussed, with specific attention focused on clinical presentations and diagnostic criteria for ITs predisposing to additional illnesses. The currently available therapeutic options for the different forms of IT are illustrated.

Treatment of Congenital Thrombotic Thrombocytopenia Purpura: A New Paradigm.

Congenital thrombotic thrombocytopenia purpura (cTTP) is a very rare disorder worldwide. Standard treatment of recognized cases has been to administer fresh frozen plasma as the source of ADAMTS13, to replenish the absent ADAMTS13 enzyme. An alternative source, a plasma-derived factor VIII concentrate used for hemophilia A, and found to contain this enzyme, was reported to be effective in 1 patient in the United States. We now report details on a US cohort of 8 cTTP patients who have been successfully treated for varying periods with a marketed antihemophilic factor concentrate Koate-DVI. This biological product has been used successfully on demand in varying doses to treat acute exacerbations, as well as prophylactically (3 to 6 U ADAMTS13 every 3 to 21 d). Self-infused at home, in lieu of fresh frozen plasma therapy in the hospital setting, this product has effectively prevented episodes of thrombocytopenia, microangiopathic hemolytic anemia, and the concomitant organ damage in these patients. This specific virus inactivated product can be used to prevent further manifestations of this congenital enzyme deficiency.

Haploinsufficiency for ANKRD11-flanking genes makes the difference between KBG and 16q24.3 microdeletion syndromes: 12 new cases

16q24 deletion involving the ANKRD11 gene, ranging from 137 kb to 2 Mb, have been associated with a microdeletion syndrome characterized by variable cognitive impairment, autism spectrum disorder, facial dysmorphisms with dental anomalies, brain abnormalities essentially affecting the corpus callosum and short stature. On the other hand, patients carrying either deletions encompassing solely ANKRD11 or its loss-of-function variants were reported in association with the KBG syndrome, characterized by a very similar phenotype, including mild-to-moderate intellectual disability, short stature and macrodontia of upper incisors, with inter and intrafamilial variability. To assess whether the haploinsufficiency of ANKRD11-flanking genes, such as ZFPM1, CDH15 and ZNF778, contributed to either the severity of the neurological impairment or was associated with other clinical features, we collected 12 new cases with a 16q24.2q24.3 deletion (de novo in 11 cases), ranging from 343 kb to 2.3 Mb. In 11 of them, the deletion involved the ANKRD11 gene, whereas in 1 case only flanking genes upstream to it were deleted. By comparing the clinical and genetic features of our patients with those previously reported, we show that the severity of the neurological phenotype and the frequency of congenital heart defects characterize the deletions that, besides ANKRD11, contain ZFPM1, CDH15 and ZNF778 as well. Moreover, the presence of thrombocytopenia and astigmatism should be taken into account to distinguish between 16q24 microdeletion syndrome and KBG syndrome. The single patient not deleted for ANKRD11, whose phenotype is characterized by milder psychomotor delay, cardiac congenital malformation, thrombocytopenia and astigmatism, confirms all this data.

Bone marrow morphology and disease progression in congenital thrombocytopenia: a detailed clinicopathologic and genetic study of eight cases

Patients with congenital thrombocytopenia have an increased risk of developing myeloid neoplasms. In these cases, the morphologic distinction between disease at baseline and at progression is challenging. This report analyzes clinicopathologic features of congenital thrombocytopenia with long-term follow-up at one referral center. Records from the last 20 years were searched for cases of congenital thrombocytopenia with bone marrow biopsies and peripheral blood smears. The clinical, morphologic, immunophenotypic, and molecular features were analyzed. Six adult and two pediatric patients were identified (six male, two female). Age range at first biopsy was 1–47 (median, 31) years. Underlying diseases included thrombocytopenia-absent radius syndrome, congenital thrombocytopenia with radial–ulnar synostosis, MYH9-related disorder, shortened telomere syndrome, congenital thrombocytopenia with ANKRD26 mutation, and familial platelet disorder with predisposition to acute myeloid leukemia. Four patients had myelodysplastic/myeloproliferative neoplasm-like marrow changes such as hypercellularity, increased myeloid to erythroid ratio, numerous micromegakaryocytes (highlighted by CD42b), and marrow fibrosis. Two patients had marrow hypoplasia and two had unremarkable marrow morphology. Three patients—all in the myelodysplastic/myeloproliferative neoplasm-like group—developed disease progression characterized by erythroid and myeloid dysplasia, elevated bone marrow blasts, and new cytogenetic abnormalities. Unlike non-familial myeloid neoplasms, congenital thrombocytopenia patients in the myelodysplastic/myeloproliferative neoplasm-like group had a long and indolent clinical course (average age at disease progression, 47 years). In summary, three distinct morphologic types of congenital thrombocytopenia were identified: a hyperplastic myelodysplastic/myeloproliferative neoplasm-like group, a hypoplastic bone marrow failure-like group, and a group with relatively normal marrow morphology. Emergence of cytogenetic abnormalities and dysplasia in non-megakaryocyte lineages correlated with disease progression.

The Significance of the Next Generation Targeted Sequencing in the Precise Diagnosis of Pediatric Acquire Aplastic Anemia and Inherited Bone Marrow Failure Syndromes

▪BackgroundDiagnosis of inherited bone marrow failure syndromes (IBMFs) depend on classic clinical manifestation including early onset, physical anomalies, family history of cancer and/or bone marrow failure and chromosome breakage testing (MMC and/or DEB), mutation analyses and bone marrow chromosome analyses. At present, more than 70 pathogenic gene mutations had been identified. However, in some patients, physical anomalies is absent or delayed, and were misdiagnosed as acquired aplastic anemia(AA). Genetic analysis is very important to establish a precise diagnosis, predict cancer risk, direct treatment and genetic counseling. In this study, we focus on the application of next generation targeted sequencing in precise diagnosis of pediatric acquired AA/IBMFs, and the association between genetic abnormalities and clinical and laboratory characteristics.MethodsWe designed a targeted sequencing assay to test a panel of 417 genes. The panel contain reported gene associated with IBMFs and other diseases need be differentiated. Pediatic patients (≤14 year old) with suspected diagnosis of AA/IBMFs were enrolled. Peripheral blood (PB) DNA was used to genetic analysis and oral epithelia cells or PB DNA from their parents were used to identify somatic mutations and unreported polymorphism. All the results were validated by Sanger sequencing.ResultsThe average coverage of targeted region was 98.15%, and the average sequencing depth was 315.9×. Totally, 283 patients were enrolled, including 176 clinically diagnosed acquired AA, 51 Fanconi anemia (FA), 8 dyskeratosis congenital(DKC), 30 Diamond-Blanckfan anemia(DBA), 15 congenital neutropenia(CN), and 3 congenital thrombocytopenia. Totally, 19% subjects had IBMFs related genetic mutations. In the patients who were clinically diagnosed as acquired AA patients, about 7% had IBMFs related disease-causing genetic mutations. Finally, 7 patients were genetically diagnosed as FA, 2 were DKC, 1 was WAS and 1 was SDS. In patients who were clinically diagnosed as FA, 33.4% had FANC related gene mutations. Telomere associated gene mutations were detected in 75% of clinical diagnosed DKC. For patients clinically diagnosed as DBA and CN, 36.7% and 20% were detected disease-causing mutations. After genetic screening, 2 patients who had been diagnosed as FA were modified as WAS and 1 DBA was modified as SDS.Only 26% genetic diagnosed IBMFs patients had family history of bone marrow failure, leukemia, tumor or physical anomalies. Compared with acquired AA, patients with genetic diagnosed FA were more likely to have physical anomalies of short stature and development retardation, Cafe au lait spots and finger or toe malformation(P<0.001).However, 46% patients with IBMFs did not have any type of physical anomalies. Moreover, there were only 24% patients with genetic diagnosed IBMFs had positive results of MMC induced chromosome breakage test or SCGE, and both the examinations could not differentiate subtype of IBMFs. FANCD2 mono-ubiquitination test were performed recently. However, even in the genetically confirmed FA, the positive rate was only 18% (2/11). And, there were positive results in some acquired AA patients.For FA patients with definitely genetic mutations, 62.5%(15/24) were compound heterozygous mutations,37.5%(9/24) were homozygous mutations.Mutational frequencies of FANC were: FANCA 65%, FANCD2 23%, FANCG 9%, FANCI 9% and FANCB 4%. For the mutated type, the frequencies of missense, frameshift, nonsense, splicing mutation were 42%, 26%, 16%, 16%. In our study, there were 4 undetermined patients met the clinical diagnostic criteria of FA, and having heterozygous damaged mutations in FANC genes.ConclusionIn conclusion, our IBMFs associated genes targeted sequencing assay is an effective strategy for precise diagnosis of bone marrow failure diseases, especially for those without family history or physical anomalies. However, nearly half of the clinically diagnosed IBMFs patients in our study were not detected the disease-causing mutations. This may be due to the mutations in the intron area, or large fragment deletion, which cannot be detected by targeted sequencing. And the novel gene involved in IBMFs need further study. DisclosuresNo relevant conflicts of interest to declare.

Utility of Whole Exome Sequencing in Diagnosis of Pediatric Platelet Disorders: A Subanalysis of the Pediseq Study

Background: Inherited Platelet Disorders (IPD) are individually rare disorders that have many different molecular causes. Diagnosis of IPD is often complicated by the need for complex testing that is not readily available at many centers and the lack of available testing to define the molecular cause of some disorders. While some platelet disorders are sufficiently defined by functional characterization, recent data suggests that some platelet disorders may predispose to significant other complications including cancer predisposition, myelofibrosis or hearing loss. Therefore, it may be important to establish a molecular diagnosis to better counsel families about necessary follow up and possible risks. The goal of this study was to determine the diagnostic yield of whole exome sequencing in a cohort of 22 pediatric patients with clinical presentation suggesting an underlying genetic cause (or positive family history of platelet disorder with no prior genetic diagnosis).Methods: Peripheral blood was collected from patients identified as likely to have an inherited platelet disorder after informed consent. Samples were also obtained from parents and siblings for co-segregation and variant calling. Genomic DNA was extracted manually using the Gentra Puregene Blood Kit. Exome capture was performed using the Agilent SureSelect v4 and 100 base paired end sequencing was done on an IlluminaHiSeq 2000 with 100X average coverage. Sequencing reads were generated in FASTQ format and mapped to human genome GRCh37 (hg19) and Novoalign v2.08 was used for optimal alignment. Disease-related variants were extracted from HGMD to identify variants that might be missed. Variant filtering and pathogenicity classification was performed using a customized pipeline and manual curation. We identified 53 genes of interest and on average across all exomes with an indication for a platelet disorder, bases were sequenced at a minimum depth of 15X to be considered covered within an exon. 80.4% of exons were 100% covered with this technology completely, while 10.4% of exons were partially covered (>40 to <100% bases) and 9.2% of exons were not covered (<40% of bases covered at a minimum of 15X depth).Results: 22 patients were enrolled over a 12-month period. Overall, 82% of patients had variants identified in platelet related genes on whole exome sequencing with 64% of patients returning at least one variant of uncertain significance (14) and 23% (5) patients returning definite positive results. One patient referred for further work up carried the initial diagnosis of ITP, but had macrothrombocytopenia since early childhood and bleeding out of proportion to the platelet count. Flow cytometry and functional studies performed on referral suggested possible Bernard Soulier Syndrome and sequencing confirmed homozygous pathogenic mutation in GP9. One patient with congenital thrombocytopenia and history of intracranial hemorrhage with a similarly affected sibling had confirmed pathogenic MYH9 mutation, allowing clinicians to offer prenatal diagnosis during a third pregnancy. One patient with a significant bleeding phenotype was a compound heterozygote for two novel RASGRP2 variants, but the functional significance of those variants is uncertain and further studies are underway to determine whether these variants are causative. 18% of patients (4) had negative sequencing results (no reportable variants in platelet related genes identified). Conclusions: Whole exome sequencing can be a powerful diagnostic tool in identifying the molecular cause of disease in a cohort of patients with suspected inherited platelet disorders. The majority of patients, however, will receive results of uncertain significance and centers that undertake this testing will require an infrastructure to allow for further functional evaluation, which will help in reclassification of these variants, and ensure that results are correctly interpreted. Clinicians who undertake ES to diagnose IPD need to understand limitations of the test as well as the full significance of results that may be returned. DisclosuresLambert:Novartis: Consultancy.

Clinical Spectrum of Germline Mutations with Predisposition to Myeloid Neoplasms- 2016 World Health Organization Classification Update

▪Background: The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms has specifically categorized germline mutations that are associated with myeloid clonal evolution (Arber et al. Blood 2016). This group consists of myeloid neoplasms with an isolated germline predisposition (CEBPA, DDX41), myeloid neoplasms associated with congenital thrombocytopenia (ETV6, RUNX1, ANKRD26) and germline myeloid neoplasms with multi-organ dysfunction (GATA2, chromosomal breakage disorders, telomere biology disorders etc). We carried out this study to describe the clinical spectrum of germline disorders with predisposition to myeloid neoplasms as categorized by the 2016 WHO classification revision.Methods: After Institutional Review Board (IRB) approval, the adult and pediatric bone marrow failure syndrome database (1990-2016) and the electronic medical record were queried for germline disorders involving GATA2, CEBPA, DDX41, ETV6, RUNX1, ANKRD26, Down syndrome and Noonan syndrome. Chromosomal breakage assays (Diepoxybutane/Mitomycin-C), flow-fluorescent in-situ hybridization (FISH) for telomere length assessment, Fanconi anemia complementation assays and Sanger/Next Generation sequencing (NGS) for the aforementioned germline disorders with myeloid predisposition were carried out in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories. These disorders were then classified based on the 2016 WHO classification revision.Results: 54 individuals (37 families) were included in the study. Eleven (20%) patients belonging to 5 families were identified as having germline mutations with a preexisting platelet disorder: ETV6 (n=1), ANKRD26 (n=5), RUNX1 (n=5). Forty-three (79%) patients (32 families) had inherited syndromes with multi-organ dysfunction: GATA2 (n=11, 26%), bone marrow failure syndromes (n=14, 33%) and telomere biology disorder (n=14, 33%). There was one patient with neurofibromatosis with a germline PTPN11 mutation who developed juvenile myelomonocytic leukemia, while there were three patients with Down syndrome; 2 with transient abnormal myelopoiesis and one who developed acute megakaryocytic leukemia. The clinical phenotype, prevalence and characteristics of myeloid clonal evolution and outcomes are presented in Table 1. No patients with germline CEBPA or DDX41 mutations were identified.Patients with germline platelet disorders did not have any prominent non-hematological manifestations. Erythrocytosis (20%) with long-standing thrombocytopenia (100%) was a unique feature associated with ANKRD26 mutations. Non-hematologic clues such as human papillomavirus (HPV)-driven warts, primary lymphedema (Emberger syndrome) and frequent atypical infections with monocytopenia were seen in patients with germline GATA2 mutations, and preceded myeloid clonal evolution (morphologic, cytogenetic and molecular). Notably, the age at presentation and penetrance of myeloid transformation was variable, with individuals from the same family developing symptoms during the first decade of life and others remaining asymptomatic to date (fifth decade). Somatic ASXL1 mutations were detected in all 3 (100%) patients with GATA2 mutations and in one patient with ANKRD26 mutation that developed myeloid clonal evolution. In our study myeloid clonal evolution was seen in 40% with RUNX1 mutations, 27% with GATA2 mutations, and 20% with ANKRD26 mutations. We could not calculate the same for bone marrow failure syndromes as the total number of cases seen are still being assessed. Outcomes with allogeneic stem cell transplantation were favorable in appropriately selected patients (Table 1).Conclusion: The 2016 WHO revision to the classification of myeloid neoplasms highlights the importance of recognition and molecular characterization of germline mutations (syndromic and non-syndromic) with risk for myeloid clonal evolution. While some of these disorders (GATA2, Fanconi anemia, telomere biology disorders) may have important non-hematological clues, many present with isolated thrombocytopenia (RUNX1, ETV6). The age and frequency of myeloid evolution is highly variable. Acquisition of somatic ASXL1 mutations at the time of clonal myeloid transformation highlights the role of epigenetic dysregulation in disease evolution. [Display omitted] DisclosuresKenderian:Novartis: Patents & Royalties, Research Funding.

Establishment of a congenital amegakaryocytic thrombocytopenia model and a thrombocyte-specific reporter line in zebrafish.

Mutations in the human myeloproliferative leukemia (MPL) protein gene are known to cause congenital amegakaryocytic thrombocytopenia (CAMT). The prognosis of this heritable disorder is poor and bone marrow transplantation is the only effective treatment. Here, by using the TALEN (transcription activator-like effector nuclease) technology, we created a zebrafish mpl mutant to model human CAMT. Disruption of zebrafish mpl lead to a severe reduction in thrombocytes and a high bleeding tendency, as well as deficiencies in adult hematopoietic stem/progenitor cells. We further demonstrated that thrombocytopenia in mpl mutant zebrafish was caused by impaired Tpo/Mpl/Jak2 signaling, resulting in reduced proliferation of thrombocyte precursors. These results indicate that mpl mutant zebrafish develop thrombocytopenia resembling the human CAMT. To utilize fully zebrafish to study thrombocyte biology and thrombocytopenia disorders, we generated a transgenic reporter line Tg(mpl:eGFP)smu4, in which green fluorescent protein (GFP) expression was driven by the mpl promoter. Detailed characterization of Tg(mpl:eGFP)smu4 fish confirmed that the thrombocyte lineage was specifically marked by GFP expression. In conclusion, we generated the first transmissible congenital thrombocytopenia zebrafish model mimicking human CAMT and a thrombocyte-specific transgenic line. Together with Tg(mpl:eGFP)smu4, mpl mutant zebrafish provide a useful tool for drug screening and study of thrombocytopoiesis.

Harris Platelet Syndrome in Patients of Non-Indian Origin.

Inherited giant platelet disorders are a subgroup of congenital thrombocytopenias characterized by decreased platelet counts along with macroplatelets and variable bleeding symptoms. Harris platelet syndrome, a newly described rare entity, is a subtype of inherited giant platelet disorders and is characterized by mild-to-severe thrombocytopenia, macroplatelets, and no bleeding manifestations. This entity was observed incidentally in healthy blood donors from India in the early 2000s, and the reported cases to date have without exception originated from the same region of the Indian subcontinent. We herein report the occurrence of Harris platelet syndrome in patients from a different ethnogeographic origin.

Clinical and laboratory characteristics in congenital ANKRD26 mutation-associated thrombocytopenia: A detailed phenotypic study of a family

The clinical and laboratory characteristics of patients with non-syndromic, autosomal dominant thrombocytopenia secondary to germ line ANKRD26 mutations appear to be heterogeneous. Except for a targeted molecular genotyping approach, there is no distinct clinical or laboratory phenotype that has been specifically associated with this particular gene mutation. Such heterogeneity could be due to variations in mutation and genetic background in different families. To understand the phenotypic heterogeneity, we thoroughly studied one affected family using the International Society for Thrombosis and Haemostasis bleeding assessment tool and both clinically validated standard and esoteric platelet testing (electron microscopy (EM) and flow cytometry). We found that decreased platelet aggregation with arachidonic acid and epinephrine agonists was common in affected family members. EM studies demonstrated persistent borderline low mean dense granules per platelet, decreased alpha granules and an increased canalicular network pattern in all affected members. Since these characteristics are subtle or non-pathognomonic, molecular testing for ANKRD26 mutation remains the most reliable test to render a diagnosis and should be considered when evaluating a patient or family with congenital thrombocytopenia, particularly if there is a history of myeloid neoplasms.

Platelet Transmission Electron Microscopy and Flow Cytometry in the Diagnosis of Congenital/Hereditary Qualitative or Quantitative Platelet Disorders

BackgroundCongenital/hereditary qualitative platelet disorders (CQPD) can be classified by platelet count (eg, normal or decreased) and platelet size as well as by functional defect. Based on mean platelet volume (MPV), congenital thrombocytopenias (CT) are classified as macro-thrombocytopenias (eg, Bernard-Soulier syndrome [BSS], MYH-9 [myosin heavy chain 9, non-muscle]-associated thrombocytopenias, gray platelet syndrome [GPS], etc.); microthrombocytopenias (eg, Wiskott-Aldrich syndrome and variants); and those with normal platelet size. CQPDs with normal platelet count include Glanzmann thrombasthenia (GT), dense granule storage pool disorders (DG-SPD) including Hermansky-Pudlak syndrome (HPS), platelet secretion disorders and many others. Diagnosis has relied on platelet counting and sizing, blood smear light microscopy, platelet aggregation (agg) and platelet function analyzer (PFA-100) studies. Platelet transmission electron microscopy (PTEM) and flow cytometry can provide supplementary information and molecular analysis is also evolving. The objective of this study was to determine the roles of platelet transmission electron microscopy (PTEM) and flow cytometry in the diagnosis of CQPD and CT.MethodsIn this retrospective cohort study, after IRB approval, the electronic medical record system was queried for patients (pt) with a confirmed CQPD or CT seen at Mayo Clinic between 2000 and 2015. A detailed chart review was undertaken.Results54 pt (64% female) met our study criteria; median age was 32 years (range 1 day to 81 years). 18/54 (33%) pt had macrothrombocytopenia: BSS 4 (7%); MYH-9 10 (19%); gray platelet syndrome 4 (7%). 36/54 (66%) had normal MPV: Glanzmann thrombasthenia 6 (11%); storage pool disorders including DG-SPD 10 (19%) and HPS 2 (3%), mild alpha granule deficiency 3 (6%) and York platelet syndrome 4 (7%); platelet secretion disorders 4 (7%); ANKRD26 mutation 3 (6%); congenital amegakaryocytic thrombocytopenia 1 (2%); GATA-1 mutation 1 (2%); RUNX-1 mutation 1 (2%); and Jacobsen syndrome 1 (2%). 44 pt (81%) had a positive bleeding history: epistaxis 52%, cutaneous bleeding 57%, gastrointestinal bleeding 21% and menorrhagia 54%; 10 pt (19%) had a negative bleeding history. Results of standard and esoteric platelet testing are summarized in Table 1.Diagnosis in the 10 pt with glycoprotein deficiency (BSS or GT) was established by agg; flow cytometry was confirmatory but not necessary. Of the 23 pt with SPD, 5 had a negative bleeding history; 4 pt had normal agg and PFA-100 and were diagnosed exclusively by PTEM. In this group, the abnormalities on platelet aggregation and PFA were variable and PTEM allowed for a better definition of the specific abnormalities. For MYH-9 patients, PTEM confirmed leukocyte inclusions that had already been identified on light microscopy. PTEM was the only diagnostic modality able to identify the abnormality in York platelet syndrome. In 5/54 (9%) genetic testing was necessary for diagnosis (GATA-1, ANKRD26 and RUNX-1)ConclusionIn this cohort, standard platelet assays established the diagnosis in the large majority of CQPD and CT. PTEM was essential for confirmation in DG-SPD, mild alpha granule deficiency and York platelet syndrome and useful in combination with molecular testing to establish a diagnosis in selected cases. Guidance on selection of patients for such specialized testing requires further study.Table 1Results of standard and esoteric platelet testing for 54 patients with congenital qualitative platelet disorders and/or thrombocytopeniaDisordern (%)Platelet aggregationPFAPTEMFlow cytometryConfirmation by geneticsDoneAbnormalDoneAbnormalDoneAbnormalDoneAbnormalGlanzmann thrombasthenia6 (11)665510330Bernard-Soulier syndrome & variants4 (7)444430440MYH-9 related disorders10 (19)728355425Dense granule deficiency and HPS12 (22)1281281212400Alpha granule deficiency*7 (13)747677441York platelet syndrome4 (7)101144110TXA2 or PG synthesis/receptor defect4 (7)444330300CATM1 (2)0-0-0-0-1ANKRD263 (6)333233333GATA-11 (2)0-1111111RUNX-11 (2)10110-0-1Jacobsen syndrome1 (2)101111101MYH-9: myosin heavy chain 9, non-muscle (MHY-9) associated thrombocytopenia, HPS: Hermansky-Pudlak Syndrome, TXA2: thromboxane A2, PG: prostaglandin CATM: congenital amegakaryocytic thrombocytopenia. * Includes gray platelet syndrome DisclosuresPardanani:Stemline: Research Funding.

Congenital Thrombotic Thrombocytopenia Purpura - Safer Treatment with Plasma-Derived Viral-Attenuated Clotting Factor

Congenital Thrombotic Thrombocytopenia Purpura (cTTP) has been considered a very rare disorder. Several international registries have estimated 250 patients worldwide. While the total number of patients in the U.S. is unknown, an ongoing U.S. Registry has recorded only 89 patients. (Singleton et al NORD 2014) Therapy for this disease, for prophylaxis and treatment, has been replacement of the absent ADAMTS13 with fresh frozen plasma (FFP). The major complication has been allergic reactions including anaphylaxis making this treatment unviable for some patients. Virally- inactivated FFP is not readily available in the U.S. A recombinant ADAMTS13 is in initial PK studies.There have been anecdotal reports that a plasma-derived FVIII/vWF biologic (Koate-DVI), double virally inactivated, FVIII replacement product, has been successfully used prophylactically in cTTP patients in lieu of FFP therapy to prevent episodes of TTP (Naik et al J Pediatr Hematol Oncol 2013). No other therapeutic biologic has been able to provide this benefit. Pursuant to that observation, two independent laboratories in the U.S. and Italy conducted analyses of the content of ADAMTS13 in several FVIII concentrates. The ADAMTS13 content in reconstituted concentrate of Koate-DVI was up to 9.08 + 0.70 units/ml, and was substantially higher than other FVIII products and pooled plasma. Table 1 and Table 2We report here a cohort of 10 cTTP patients currently being managed with Koate-DVI prophylaxis. The average age of the patients is 15 years (range 7-22 years). The patients are being treated with a dose range of 25 to 40 IU/kg, at an average of once per week. This dose of Koate-DVI (containing 100 IU FVIII/ml after reconstitution) will provide approximately 2 to 4 IU/kg of ADAMTS13. Patients have been treated for variable lengths of time ranging from less than a year to over 10 years. The patients have responded very well to the Koate-DVI treatment; no severe adverse events or allergic reactions have been reported. We are prospectively comparing the frequency of new TTP episodes in this cohort with the frequency observed prior to initiation of this therapy.We report a unique experience of 10 cTTP patients being successfully managed prophylactically at home with self-infusion of Koate-DVI (Factor VIII concentrate with a long history of viral safety in the treatment of hemophilia A). These patients are receiving a convenient virally-inactivated alternative to FFP without manifesting life-threatening allergic reactions that require immunosuppression and/or hospitalization.A prospective clinical study of the safety and efficacy of Koate-DVI is planned.Table 1ConcentrateADAMTS13 activity U/mLADAMTS13 antigen U/mLKoate-DVI (5 lots)9.08 ± 0.708.42 ± 0.12Product A0.120.13Product B0.220.61Product C2.303.87(Peyvandi et al Am J Hematol 2013)Table 2ConcentrateADAMTS13 Units/mlKoate-DVI5.77Product A0.18Product B0.23Product C1.40(Konkle Personal Communication 2013) DisclosuresAledort:Kedrion BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Baxter Healthcare: Membership on an entity's Board of Directors or advisory committees, Other: DSMB Participation. Off Label Use: Koate-DVI is indicated for the treatment of classical hemophilia (hemophilia A) in which there is a demonstrated deficiency of activity of the plasma clotting factor, factor VIII. This presentation discusses the use of factor VIII concentrate for ADAMTS13 deficiency.. Boggio:CSL Behring: Consultancy, Research Funding; Baxter: Consultancy, Research Funding; Selexys: Research Funding; Bayer: Consultancy, Research Funding; OctaPharma: Consultancy, Research Funding; OPKO: Research Funding; Novo Nordisk: Consultancy, Research Funding. Kobrinsky:Octapharma: Speakers Bureau; CSL Behring: Speakers Bureau; Sanofi: Speakers Bureau; Kedrion Biopharma: Membership on an entity's Board of Directors or advisory committees. Rajasekhar:American Society of Hematology: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; Baxter Healthcare: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees. Shapiro:Baxalta: Research Funding; Shire: Speakers Bureau; BioCryst: Research Funding. Ulsh:Kedrion Biopharma: Employment.

A Novel Splice Site Mutation in the 5'UTR of GATA1 resulting in Abnormal Hematopoiesis

Diamond-Blackfan anemia (DBA) is a rare disorder of impaired erythropoiesis belonging to a broader family of inherited and acquired anemias known as pure red cell aplasias (PRCA). The cardinal feature of DBA is aregenerative anemia accompanied by macrocytosis, reticulocytopenia and decreased or absent erythroid precursors in an otherwise normocellular marrow. Classic DBA is associated with mutations of genes encoding ribosome subunits. Advancements in molecular techniques have prompted discussion regarding the genetic basis of the disease and its prognostic implications as non-ribosomal genes are beginning to be implicated in DBA.Acquired mutations within the X-linked transcription factor GATA1 have been described in Down syndrome-related transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AKML), while germline GATA1 mutations affecting both isoforms are associated with congenital thrombocytopenia. Normal alternative splicing of GATA1 produces a full-length GATA1 isoform (flGATA1) as well as a shortened GATA1 isoform (GATA1s), which lacks the N-terminal domain that activates GATA1-driven erythropoiesis program and recruits flGATA1 to a subset of megakaryocyte and erythroblast genes. Rare germline defects known as "GATA1s mutations" are characterized by an unbalanced production of the GATA1s isoform at the expense of a flGATA1 transcript due to disrupting exon 2 splice sites or the ATG initiation codon. GATA1s mutations had been reported to phenocopy Diamond-Blackfan anemia by causing isolated red blood cell aplasia in some patients, although thrombocytopenia with structural platelet abnormalities and dyserythropoiesis as well as DBA-like picture progressing into MDS had been described in other GATA1s families. Therefore, while GATA1s mutations uniformly disrupt erythropoiesis, the impact of defective flGATA production on megakaryopoiesis in non-trisomy 21 individuals is less clear.Here we describe a child with dyserthropoietic anemia, marked megakaryocyte dysplasia, peripheral thrombocytosis and platelet dysfunction due to a novel disease-causing mutation within the 5' UTR of GATA1. DBA was initially suspected due to progressive macrocytic anemia beginning in infancy. Bone marrow analysis revealed not only a paucity of erythroid precursor cells and dyserythropoiesis, but also prominent megakaryocytosis and megakaryocyte dysplasia, not typically associated with classic DBA. Ribosome mutational analysis was unremarkable, as were cytogenetics and MDS-FISH analysis. Given aregenerative anemia mimicking DBA as well as megakaryocyte dysplasia, the question was raised whether the findings could be associated with a germline mutation in GATA1. Indeed, sequencing revealed a novel, pathogenic mutation in the 5'UTR of GATA1 (c.-21 A>G). We explored the impact of this novel mutation in silico, found it to disrupt a consensus splice site, and further demonstrated that this mutation conferred a striking predominance of the GATA1s isoform at the transcript and protein level. Two-color immunohistochemistry confirmed loss of full-length GATA1 protein in the patient's bone marrow and showed that full-length GATA1 expression is restricted to megakaryocytes and erythroblasts in healthy bone marrow, consistent with dysmegakaryopoiesis and decreased erythropoiesis in our patient.Our findings support a role for the N-terminus of GATA1 in both erythroblast and megakaryocyte maturation and function in vivo. Furthermore, the findings of prominent megakaryocytes in the bone marrow and resultant functional platelet abnormalities may provide subtle clinical clues to differentiate Diamond-Blackfan anemia due to ribosomopathy from dyserythropoiesis secondary to GATA1s mutations. We conclude that GATA1 sequencing, including non-coding GATA1 regions, should be considered in males with congenital multi-lineage dysplasia and/or DBA-like clinical presentation. DisclosuresNo relevant conflicts of interest to declare.

Results of the Russian Registry of Primary Immune Thrombocytopenia in Pediatric Patients

Study objectivesWe aim to evaluate disease characteristics and treatment practices of pediatric pts. with Immune thrombocytopenia (ITP) in Russia.Materials and methodsThe ITP Registry was a multicenter, prospective, observational cohort study. Inclusion criteria: diagnosis of primary ITP, informed consent of the patient/guardians. Exclusion criteria: secondary or congenital thrombocytopenia. Data from medical records were registered in the e-CRF in average every 3 months. Descriptive statistics were used. Patients were registered since June 2011 till June 2014.ResultsNinety-three pediatric pts, 46 male (49.5%) and 47 female (50.5%) with a median age 8.4 yrs (range 0.5-17.8) from 5 centers in various regions of Russia were included. The mean observation period reached 17.1 ± 6.5 mo (range1.4 to 28.6 months). Seventy (75.3%) pts had acute and 24.7% pts had insidious disease onset. The presence of trigger factors for ITP development was found in more than half of the cases (in 61.3% of patients), they are listed in Table 1.Table 1TriggersN%No triggers3638.7%Infection4649.5%Vaccination88.6%Other33.2%Total93100%Median disease duration at enrollment was 1.07 years (range 0 to 16.7 yrs). ITP duration shorter than 5 years at the enrollment was reported in 89.2% pts, up to 1 year - in 43 (46.2%), 1- 5 years - in 40 (43%), 5-10 years - in 8 (8.6%), >10 years - in 2 (2.2%) pts. Newly diagnosed ITP was reported in 35 (37.6 %) pts, persistent ITP - in 12 (12.9 %), chronic ITP - in 46 (49.5 %) pts.Median platelets count was 12,0 x 109/L (range 0.0 - 72.0 109/L).Ninety-two (98%) pts experienced hemorrhagic manifestations during the course of ITP: skin hemorrhages - in 98.9%, oral bleeding - in 15.1%, epistaxis - in 36.6%, gastrointestinal bleeding - in 1.1%, intracranial bleeding - in 1.1%, hematuria - in 1.1%, and other hemorrhages - in 9.7% of pts.Relationship between hemorrhagic syndrome and platelet count at the enrollment is provided in table 2.Table 2Relationship between hemorrhagic syndrome and platelet count (at enrollment)Hemorrhage highest grade according to WHOPlatelet count (visit 1)Total pts / %< 30,00030,000 -50,000>50,00003 5.5%3 5.5%49 89.1%55 100%111 40.7%5 18.5%11 40.7%27 100%25 62.5%0 0%3 37.5%8 100%32 66.7%1 33.3%0 0%3 100%Total21 22,6%9 9.7%63 67.7%93 100%Severe course of ITP after enrollment was observed in 12 (13%) pts (of whose 6 (6.5%) had clinically significant hemorrhage at the disease onset and 6 (6.5%) had new clinically significant hemorrhages during follow-up period.Refractory ITP at enrollment was reported in 9 (9.7%) pts and was associated with the resistance to the first-, second- and subsequent lines of therapy. At enrollment 42 (45.2%) pts received specific treatment for ITP. Before enrollment, splenectomy was reported in only 1 (1.1%) 14-years old patient who had a complete response. During the study, splenectomy was performed in 6 (6.6%) pts with chronic ITP; the duration of the disease at the time of splenectomy varied from 2 to 10 years, with average duration of 4.69 years (median - 4.5 years). Complete response to splenectomy was observed in 3 (50%) pts, a partial response - in 2 (33.3%), no response - in 1 (16.7%) patient. Loss of response to splenectomy was not reported. During the study, severe ITP was reported in 8 (8.7%) pts, 41 (44.6%) pt had various hemorrhagic manifestations of ITP at least at 1 visit, grade IV hemorrhagic syndrome was not reported.Thirty-eight (41%) pts received 1-st line treatment: glucocorticosteroids (GCS) - 23 (60.5%) pts, IVIG - 5 (13.2%), alfa-interferons -16 pts (42.1%). Twenty-three pts (24.7%) received second-line therapy: GCS - 1 (4.3%), IVIG -1 (4.3%), immunosupression - 1 (4.3%), rituximab - 2 (8.7%), romiplostim - 11 (47.8%), eltrombopag - 14 (60.9%).ConclusionFor the first time new information on the features of the disease and patterns of management of pediatric pts with primary ITP in Russia was obtained in a prospective study. Interestingly, the preferred therapy for the 2nd or subsequent lines are TPO receptor agonists used outside the approved indications in research institutions, based on published clinical trial data. Splenectomy rate before and during the study was only 7.5% (7 pts) with chronic ITP; in 1 child (14.3%) splenectomy was ineffective. Low acceptance of splenectomy suggests TPO-mimetics as potential second-line therapy. In total, good disease control is achievable in the majority of pediatric pts with ITP. DisclosuresOff Label Use: use of TPO-mimetics in children.

Zygosity Diagnosis: When Physicians and DNA Disagree.

Physicians and other medical professionals do not always provide new parents with an accurate diagnosis of their twins' zygosity. An overview of this problem is presented, supplemented by an interview with a mother who recently learned that her 2-year-old 'dizygotic (DZ)' twin girls are actually 'monozygotic (MZ)'. Reviews of two case studies, one of twins with sex-discordance and chimerism and the other of twins with congenital amegakaryotic thrombocytopenia, follow. Two additional studies, one a twin analysis of attractiveness to mosquitoes and the other a study of twins coping with crisis, are also described. Several articles and letters from the popular media, concerning less favored twins, paternity issues surrounding superfecundation, twins with late-onset Tay-Sachs disease, and triplets admitted to MIT are informative and insightful.

A family having type 2B von Willebrand disease with a novel VWF p.R1308S mutation: Detection of characteristic platelet aggregates on peripheral blood smears as the key aspect of diagnosis

Type 2B von Willebrand disease (VWD) is frequently associated with distinct platelet morphology. Here we present a familial case of type 2B VWD with a novel VWF mutation (p.R1308S), which caused neonatal thrombocytopenia. The mother had been treated for refractory immune thrombocytopenia (ITP) for more than 20years. The most important hematological features of this case were large platelets and platelet aggregates detected on peripheral blood smears. Hemostatic tests showed enhanced ristocetin-induced platelet agglutination at low-ristocetin concentrations, absence of high-molecular weight von Willebrand factor (VWF) multimers, and low VWF cofactor activity/antigen ratio. In patients with intractable ITP, family history of ITP and consecutive neonatal thrombocytopenia, the differential diagnosis of congenital thrombocytopenia is mandatory. For this purpose, the identification of large platelets and platelet aggregates on peripheral blood smears is the key aspect of type 2B VWD diagnosis.