[Developments of high-throughput sequencing-based diagnosis of congenital thrombocytopenia/platelet disorders in a registry study].

Congenital thrombocytopenia/platelet disorders are heterogeneous disorders of platelet number and/or function. Pathogenic variants in the genes implicated in megakaryocyte differentiation and platelet formation cause thrombocytopenia in these patients. Recent advances have elucidated several causative genes for these disorders, but identifying the underlying causative genes remains challenging. Patients with these disorders often receive inappropriate treatments, including glucocorticoids and splenectomy, for chronic immune thrombocytopenia (ITP). In Japan, we have developed a diagnostic system using high-throughput DNA sequencing with a multigene panel and established a registry. Between 2018 and 2023, 245 patients were enrolled and analyzed. Pathogenic variants in 17 genes (42 MYH9, 19 ANKRD26, 17 ITGA2B/ITGB3, 8 ACTN1, 8 WAS, 6 ETV6, 6 VWF, 5 CYCS, and 14 others) were identified in 125 patients (51.0%). An additional 29 patients (11.8%) had suspected pathogenic variants under investigation. We also found that immature platelet fraction (IPF%) is useful in the differential diagnosis because the median IPF% in MYH9 disorders, 48.7%, was significantly higher than in all other groups (chronic ITP, 13.4%; controls, 2.6%). The results of this study provide new insight into congenital thrombocytopenia/platelet disorders.

Severe thrombocytopenia and intracranial hemorrhage in a newborn with Noonan syndrome and neonatal alloimmune thrombocytopenia.

Noonan syndrome (NS) is a genetic disorder with distinctive physical features and often multiple organ involvement. Bleeding disorders are reported in over half of patients with NS, including thrombocytopenia and platelet dysfunction. Neonatal alloimmune thrombocytopenia (NAIT) is an alloantigenic thrombocytopenia that can present with severe bleeding. Here, we present a case of intracranial hemorrhage and severe thrombocytopenia in a neonate found to have both NAIT and a de novo heterozygous pathogenic variant in PTPN11, consistent with Noonan syndrome.

Novel phenotypes observed in patients with ETV6-linked leukaemia/familial thrombocytopenia syndrome and a biallelic ARID5B risk allele as leukaemogenic cofactor.

Background. The phenotypes of patients with the recently discovered, dominant, ETV6-linked leukaemia predisposition and familial thrombocytopenia syndrome are variable, and the exact mechanism of leukaemogenesis remains unclear. Patients and Methods. Here, we present novel clinical and laboratory phenotypes of seven individuals from three families with ETV6 germline mutations and a refined genetic analysis of one child with additional high-hyperdiploid acute lymphoblastic leukaemia (HD-ALL), aiming to elucidate second oncogenic hits. Results. Four individuals from two pedigrees harboured one novel or one previously described variant in the central domain of ETV6 (c.592C>T, p.Gln198* or c.641C>T, p.Pro241Leu, respectively). Neutropenia was an accompanying feature in one of these families that also harboured a variant in RUNX1 (c.1098_1103dup, p.Ile366_Gly367dup), while in the other, an autism-spectrum disorder was observed. In the third family, the index patient suffered from HD-ALL and life-threatening pulmonary mucor mycosis, and had a positive family history of 'immune' thrombocytopenia. Genetic analyses revealed a novel heterozygous mutation in the ETS domain of ETV6 (c.1136T>C, p.Leu379Pro) along with absence of heterozygosity of chromosome (10)(q21.2q21.3), yielding a biallelic leukaemia risk allele in ARID5B (rs7090445-C). The neutrophil function was normal in all individuals tested, and the platelet immune histochemistry of all three pedigrees showed delta-storage-pool defect-like features and cytoskeletal defects. Conclusions. Our clinical observations and results of high-resolution genetic analyses extend the spectrum of possible phenotypes cosegregating with ETV6 germline mutations. Further, we propose ARID5B as potential leukaemogenic cofactor in patients with ETV6-linked leukaemia predisposition and familial thrombocytopenia syndrome.

[Congenital thrombocytopenia].

Congenital thrombocytopenia is a group of heterogeneous disorders caused by mutations in the responsible genes that play crucial roles in normal megakaryopoiesis and subsequent platelet production. The diagnosis of congenital thrombocytopenia is clinically necessary to distinguish it from immune thrombocytopenia and select the appropriate therapeutic modalities. The number of responsible genes reported so far is up to 56, and data on their targeted sequencing and subsequent exome sequencing analysis are available in Japan. Here, we report the disease outlines, disease classification based on platelet sizes (small, normal, large, and giant platelets), disease descriptions, consultation system, list of responsible genes, therapeutic options, and follow-up system for congenital thrombocytopenia.

Congenital thrombocytopenia associated with GNE mutations in twin sisters: a case report and literature review.

BACKGROUND: Neonatal thrombocytopenia is common in preterm and term neonates admitted to neonatal intensive care units. The etiology behind neonatal thrombocytopenia is complex. Inherited thrombocytopenia is rare and usually results from genetic mutations. CASE PRESENTATION: Here we report a case of twins with severe inherited thrombocytopenia presented in the neonatal period who were shown to be compound heterozygotes for 2 UDP-N-acetylglucosamine 2-epimerase (GNE) gene mutations, c.1351C > T and c.1330G > T, of which c.1330G > T is a novel mutation. CONCLUSION: These two GNE mutations may help in the diagnosis and management of thrombocytopenia diagnosed in neonates.

[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.

Congenital thrombocytopenia in a neonate with an interstitial microdeletion of 3q26.2q26.31.

Interstitial deletions encompassing the 3q26.2 region are rare. Only one case-report was published this far describing a patient with an interstitial deletion of 3q26.2 (involving the MDS1-EVI1 complex (MECOM)) and congenital thrombocytopenia. In this report we describe a case of a neonate with congenital thrombocytopenia and a constitutional 4.52 Mb deletion of 3q26.2q26.31 including TERC and the first 2 exons of MECOM, involving MDS1 but not EVI1. The deletion was demonstrated by array-CGH on lymphocytes. Our report confirms that congenital thrombocytopenia can be due to a constitutional deletion of 3q26.2 involving MECOM. We suggest that in case of unexplained neonatal thrombocytopenia, with even just slight facial dysmorphism, DNA microarray on peripheral blood should be considered early in the diagnostic work-up.

"CHildren with Inherited Platelet disorders Surveillance" (CHIPS) retrospective and prospective observational cohort study by Italian Association of Pediatric Hematology and Oncology (AIEOP).

Background: Inherited thrombocytopenias (ITs) are rare congenital bleeding disorders characterized by different clinical expression and variable prognosis. ITs are poorly known by clinicians and often misdiagnosed with most common forms of thrombocytopenia. Material and methods: "CHildren with Inherited Platelet disorders Surveillance" study (CHIPS) is a retrospective - prospective observational cohort study conducted between January 2003 and January 2022 in 17 centers affiliated to the Italian Association of Pediatric Hematology and Oncology (AIEOP). The primary objective of this study was to collect clinical and laboratory data on Italian pediatric patients with inherited thrombocytopenias. Secondary objectives were to calculate prevalence of ITs in Italian pediatric population and to assess frequency and genotype-phenotype correlation of different types of mutations in our study cohort. Results: A total of 139 children, with ITs (82 male - 57 female) were enrolled. ITs prevalence in Italy ranged from 0.7 per 100,000 children during 2010 to 2 per 100,000 children during 2022. The median time between the onset of thrombocytopenia and the diagnosis of ITs was 1 years (range 0 - 18 years). A family history of thrombocytopenia has been reported in 90 patients (65%). Among 139 children with ITs, in 73 (53%) children almost one defective gene has been identified. In 61 patients a pathogenic mutation has been identified. Among them, 2 patients also carry a variant of uncertain significance (VUS), and 4 others harbour 2 VUS variants. VUS variants were identified in further 8 patients (6%), 4 of which carry more than one variant VUS. Three patients (2%) had a likely pathogenic variant while in 1 patient (1%) a variant was identified that was initially given an uncertain significance but was later classified as benign. In addition, in 17 patients the genetic diagnosis is not available, but their family history and clinical/laboratory features strongly suggest the presence of a specific genetic cause. In 49 children (35%) no genetic defect were identified. In ninetyseven patients (70%), thrombocytopenia was not associated with other clinically apparent disorders. However, 42 children (30%) had one or more additional clinical alterations. Conclusion: Our study provides a descriptive collection of ITs in the pediatric Italian population.

Individualized Bleeding Risk Assessment through Thromboelastography: A Case Report of May-Hegglin Anomaly in Preterm Twin Neonates.

May-Hegglin anomaly (MHA) is a rare autosomal dominant disorder in the spectrum of myosin heavy chain-related disorders (MYH9-RD), characterized by congenital macrothrombocytopenia and white blood cell inclusions. MHA carries a potential risk of hemorrhagic complications. Bleeding diathesis is usually mild, but sporadic, life-threatening events have been reported. Data regarding the clinical course and outcomes of neonatal MYH9-RD are limited, and specific guidelines on platelet transfusion in asymptomatic patients are lacking. We present monochorionic twins born preterm at 32 weeks of gestation to an MHA mother; both presented with severe thrombocytopenia at birth. Peripheral blood smear demonstrated the presence of macrothrombocytes, and immunofluorescence confirmed the diagnosis of MHA. Close clinical monitoring excluded bleeding complications, and serial hemostatic assessments through a viscoelastic system demonstrated functionally normal primary hemostasis in both patients. Therefore, prophylactic platelet transfusions were avoided. Whole DNA sequencing confirmed the pathogenetic variant of MHA of maternal origin in both twins. Thromboelastography allowed real-time bedside bleeding risk assessment and supported individualized transfusion management in preterm newborns at risk of hemostatic impairment. This report suggests that dynamic and appropriate clotting monitoring may contribute to the more rational use of platelets' transfusions while preserving patients with hemorrhagic complications and potential transfusion-related side effects.

Congenital amegakaryocytic thrombocytopenia - Not a single disease.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare inherited bone marrow failure syndrome (IBMFS) that is characterized by severe thrombocytopenia at birth due to ineffective megakaryopoiesis and development towards aplastic anemia during the first years of life. CAMT is not a single monogenetic disorder; rather, many descriptions of CAMT include different entities with different etiologies. CAMT in a narrow sense, which is primarily restricted to the hematopoietic system, is caused mainly by mutations in the gene for the thrombopoietin receptor (MPL), sometimes in the gene for its ligand (THPO). CAMT in association with radio-ulnar synostosis, which is not always clinically apparent, is mostly caused by mutations in MECOM, rarely in HOXA11. Patients affected by other IBMFS - especially Fanconi anemia or dyskeratosis congenita - may be misdiagnosed as having CAMT when they lack typical disease features of these syndromes or have only mild symptoms. This article reviews scientific and clinical aspects of the various disorders associated with the term "CAMT" with a main focus on the disease caused by mutations in the MPL gene.

1q21.1 deletion and a rare functional polymorphism in siblings with thrombocytopenia-absent radius-like phenotypes.

Thrombocytopenia-absent radii (TAR) syndrome, characterized by neonatal thrombocytopenia and bilateral radial aplasia with thumbs present, is typically caused by the inheritance of a 1q21.1 deletion and a single-nucelotide polymorphism in RBM8A on the nondeleted allele. We evaluated two siblings with TAR-like dysmorphology but lacking thrombocytopenia in infancy. Family NCI-107 participated in an IRB-approved cohort study and underwent comprehensive clinical and genomic evaluations, including aCGH, whole-exome, whole-genome, and targeted sequencing. Gene expression assays and electromobility shift assays (EMSAs) were performed to evaluate the variant of interest. The previously identified TAR-associated 1q21.1 deletion was present in the affected siblings and one healthy parent. Multiple sequencing approaches did not identify previously described TAR-associated SNPs or mutations in relevant genes. We discovered rs61746197 A > G heterozygosity in the parent without the deletion and apparent hemizygosity in both siblings. rs61746197 A > G overlaps a RelA-p65 binding motif, and EMSAs indicate the A allele has higher transcription factor binding efficiency than the G allele. Stimulation of K562 cells to induce megakaryocyte differentiation abrogated the shift of both reference and alternative probes. The 1q21.1 TAR-associated deletion in combination with the G variant of rs61746197 on the nondeleted allele is associated with a TAR-like phenotype. rs61746197 G could be a functional enhancer/repressor element, but more studies are required to identify the specific factor(s) responsible. Overall, our findings suggest a role of rs61746197 A > G and human disease in the setting of a 1q21.1 deletion on the other chromosome.

Further evidence for the involvement of EFL1 in a Shwachman-Diamond-like syndrome and expansion of the phenotypic features.

Recent evidence has implicated EFL1 in a phenotype overlapping Shwachman-Diamond syndrome (SDS), with the functional interplay between EFL1 and the previously known causative gene SBDS accounting for the similarity in clinical features. Relatively little is known about the phenotypes associated with pathogenic variants in the EFL1 gene, but the initial indication was that phenotypes may be more severe, when compared with SDS. We report a pediatric patient who presented with a metaphyseal dysplasia and was found to have biallelic variants in EFL1 on reanalysis of trio whole-exome sequencing data. The variant had not been initially reported because of the research laboratory's focus on de novo variants. Subsequent phenotyping revealed variability in her manifestations. Although her metaphyseal abnormalities were more severe than in the original reported cohort with EFL1 variants, the bone marrow abnormalities were generally mild, and there was equivocal evidence for pancreatic insufficiency. Despite the limited number of reported patients, variants in EFL1 appear to cause a broader spectrum of symptoms that overlap with those seen in SDS. Our report adds to the evidence of EFL1 being associated with an SDS-like phenotype and provides information adding to our understanding of the phenotypic variability of this disorder. Our report also highlights the value of exome data reanalysis when a diagnosis is not initially apparent.

Congenital macrothrombocytopenia associated with a combination of functional polymorphisms in the TUBB1 gene.

Congenital thrombocytopenia in childhood and adolescence requires an extensive diagnostic workup to find the underlying reason. We report on a 13-year-old female patient who was incidentally found to have moderate thrombocytopenia which was also diagnosed in her father and brother. Within the microscopic evaluation of a peripheral blood smear macrothrombocytes were found. Immunofluorescence microscopy of the patient's platelets detected the lack of ß1-tubulin. Analysis of the TUBB1 gene revealed three known missense variants in heterozygous state which in combination might explain the ß1-tubulin defect.

Two novel RUNX1 mutations in a patient with congenital thrombocytopenia that evolved into a high grade myelodysplastic syndrome.

Here we report two new RUNX1 mutations in one patient with congenital thrombocytopenia that transformed into a high grade myelodysplastic syndrome with myelomonocytic features. The first mutation was a nucleotide base substitution from guanine to adenine within exon 8, resulting in a nonsense mutation in the DNA-binding inhibitory domain of the Runx1 protein. This nonsense mutation is suspected a de novo germline mutation since both parents are negative for the mutation. The second mutation identified was an in-frame six nucleotide base pair insertion in exon 5 of the RUNX1 gene, which is predicted to result in an insertion in the DNA-binding runt homology domain (RHD). This mutation is believed to be a somatic mutation as it was mosaic before allogeneic hematopoietic cell transplantation and disappeared after transplant. As no other genetic mutation was found using genetic screening, it is speculated that the combined effect of these two RUNX1 mutations may have exerted a stronger dominant negative effect than either RUNX1 mutation alone, thus leading to a myeloid malignancy.

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.

Thrombocytopenia: an update.

Thrombocytopenia is a common clinical problem with numerous potential causes including decreased bone marrow platelet production, increased peripheral platelet destruction, increased splenic sequestration, and dilution. Investigation of the etiology of thrombocytopenia requires careful consideration of clinical history and laboratory features. A complete blood count and peripheral smear review are essential components of the diagnostic work-up, and physicians should be knowledgeable about appropriate selection and interpretation of more specialized tests, including bone marrow examination, to assist with diagnosis. This review article aims to summarize and address appropriate work-up of the major and/or life-threatening causes of thrombocytopenia and some of the better-characterized congenital thrombocytopenias.

Trombocitopenia en el embarazo: gestacional, inmune y congénita / Thrombocytopenia in pregnancy: gestational, immune and congenital

La trombocitopenia afecta hasta el 10 por ciento de todos los embarazos y es un diagnóstico común y un problema en el manejo de las pacientes, ya que puede estar relacionado con condiciones prexistentes presentes en las mujeres en edad fértil, como la trombocitopenia inmune primaria y las trombocitopenias congénitas; o con trastornos intrínsecos del embarazo, como la trombocitopenia gestacional. Se recomienda que todas las mujeres embarazadas con recuento de plaquetas por debajo de 100 x 10(9)/L sean sometidas a una evaluación por el hematólogo y el obstetra. El análisis cuidadoso del momento del inicio de la trombocitopenia asociado a las manifestaciones clínicas y las pruebas de laboratorio específicas, es indispensable para proporcionar un diagnostico apropiado y una asistencia médica materna-fetal en el momento oportuno, en preparación para el desafío homeostático(AU)

Thrombocytopenia affects up to 10 percent of all pregnant women and is a common diagnosis and a problem in the management of patients as it may be related to preexisting conditions in women of childbearing age, such as primary immune thrombocytopenia and congenital thrombocytopenia or intrinsic disorders of pregnancy as gestational thrombocytopenia. It is recommended that all pregnant women with a platelet count below 100 x 10(9) / L should undergo an evaluation by the hematologist and the obstetrician. Careful analysis of the time of onset of thrombocytopenia associated to clinical manifestations and specific laboratory tests are essential to provide appropriate diagnosis and maternal - fetal medical care at the right time, when preparing for the homeostatic challenge(AU)

Trombocitopenia en el embarazo: gestacional, inmune y congénita / Thrombocytopenia in pregnancy: gestational, immune and congenital

La trombocitopenia afecta hasta el 10 por ciento de todos los embarazos y es un diagnóstico común y un problema en el manejo de las pacientes, ya que puede estar relacionado con condiciones prexistentes presentes en las mujeres en edad fértil, como la trombocitopenia inmune primaria y las trombocitopenias congénitas; o con trastornos intrínsecos del embarazo, como la trombocitopenia gestacional. Se recomienda que todas las mujeres embarazadas con recuento de plaquetas por debajo de 100 x 10(9)/L sean sometidas a una evaluación por el hematólogo y el obstetra. El análisis cuidadoso del momento del inicio de la trombocitopenia asociado a las manifestaciones clínicas y las pruebas de laboratorio específicas, es indispensable para proporcionar un diagnostico apropiado y una asistencia médica materna-fetal en el momento oportuno, en preparación para el desafío homeostático...

Thrombocytopenia affects up to 10 percent of all pregnant women and is a common diagnosis and a problem in the management of patients as it may be related to preexisting conditions in women of childbearing age, such as primary immune thrombocytopenia and congenital thrombocytopenia or intrinsic disorders of pregnancy as gestational thrombocytopenia. It is recommended that all pregnant women with a platelet count below 100 x 10(9) / L should undergo an evaluation by the hematologist and the obstetrician. Careful analysis of the time of onset of thrombocytopenia associated to clinical manifestations and specific laboratory tests are essential to provide appropriate diagnosis and maternal - fetal medical care at the right time, when preparing for the homeostatic challenge...