THROMKIDplus Patient Registry and Biomaterial Banking for Children with Inherited Platelet Disorders.

Inherited platelet disorders (IPDs) represent a heterogeneous group of disorders that include both quantitative (thrombocytopenia or thrombocytosis) and qualitative (thrombocytopathy) defects. To gain better knowledge about the prevalence, pathogenesis, and clinical consequences of specific diseases, to improve diagnosis and treatment of patients with IPD, and to support translational research on a genetic, molecular, and physiological basis, the THROMKIDplus study group currently comprising 24 sites in Germany, Austria, and Switzerland decided to establish a patient registry with associated biomaterial banking for children. This registry is designed as a retrospective-prospective, multicenter observational study and supposed to launch in the second half of 2023. Blood smears, plasma, platelet pellets, and DNA of patients will be stored in certified biomaterial banks for future translational research projects. The main inclusion criteria are (1) diagnosis of or highly suspected IPD after assessment of a THROMKIDplus competence center and (2) patients aged 0 to 17 years. Initial and follow-up data on patient history, laboratory parameters, standardized documentation of bleeding tendency, and congenital defects are collected according to good clinical practice and current data protection acts by using the MARVIN platform, a broadly used data management system supported by the German Society for Pediatric Oncology Hematology (GPOH). The THROMKIDplus study group intends to enroll ∼200 patients retrospectively and an annual amount of ∼50 patients prospectively.

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.

CAMT-MPL: congenital amegakaryocytic thrombocytopenia caused by MPL mutations - heterogeneity of a monogenic disorder - a comprehensive analysis of 56 patients.

Congenital amegakaryocytic thrombocytopenia caused by deleterious homozygous or compound heterozygous mutations in MPL (CAMT-MPL) is a rare inherited bone marrow failure syndrome presenting as an isolated thrombocytopenia at birth progressing to pancytopenia due to exhaustion of hematopoietic progenitors. The analysis of samples and clinical data from a large cohort of 56 patients with CAMT-MPL resulted in a detailed description of the clinical picture and reliable genotype-phenotype correlations for this rare disease. We extended the spectrum of CAMT causing MPL mutations regarding number (17 novel mutations) and impact. The clinical courses showed a great variability with respect to the severity of thrombocytopenia, the development of pancytopenia and the consequences from bleedings. The most severe clinical problems were (1) intracranial bleedings pre- and perinatally and the resulting long-term consequences, and (2) the development of aplastic anemia in the later course of the disease. An important and new finding was that thrombocytopenia was not detected at birth in a quarter of the patients. The rate of non-hematological abnormalities in CAMT-MPL was higher than described so far. Most of the anomalies were related to the head region (brain anomalies, ocular and orbital anomalies) and consequences of intracranial bleedings. The present study demonstrates a higher variability of clinical courses than described so far and has important implications on diagnosis and therapy. The diagnosis CAMT-MPL has to be considered even for those patients who are inconspicuous in the first months of life or show somatic anomalies typical for other inherited bone marrow failure syndromes.

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

Heterozygous 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.

Clinic, pathogenic mechanisms and drug testing of two inherited thrombocytopenias, ANKRD26-related Thrombocytopenia and MYH9-related diseases.

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count resulting in impaired hemostasis. Patients can have spontaneous hemorrhages and/or excessive bleedings provoked by hemostatic challenges as trauma or surgery. To date, ITs encompass 32 different rare monogenic disorders caused by mutations of 30 genes. This review will focus on the major discoveries that have been made in the last years on the diagnosis, treatment and molecular mechanisms of ANKRD26-Related Thrombocytopenia and MYH9-Related Diseases. Furthermore, we will discuss the use a Thrombopoietin mimetic as a novel approach to treat the thrombocytopenia in these patients. We will propose the use of a new 3D bone marrow model to study the mechanisms of action of these drugs and to test their efficacy and safety in patients. The overall purpose of this review is to point out that important progresses have been made in understanding the pathogenesis of ANKRD26-Related Thrombocytopenia and MYH9-Related Diseases and new therapeutic approaches have been proposed and tested. Future advancement in this research will rely in the development of more physiological models to study the regulation of human platelet biogenesis, disease mechanisms and specific pharmacologic targets.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

A novel G6PC3 gene mutation in severe congenital neutropenia: pancytopenia and variable bone marrow phenotype can also be part of this syndrome.

Glucose-6-phosphatase catalytic subunit 3 (G6PC3) deficiency is a newly described syndrome characterized by severe congenital neutropenia associated with multiple organ abnormalities including cardiac and urogenital malformations. The underlying pathophysiology of increased apoptosis of myeloid cells and of neutrophil dysfunction in G6PC3 deficiency involves disturbed glucose metabolism, increased endoplasmic reticulum stress and deficient protein folding. Here, we report a new case of G6PC3 deficiency caused by a novel homozygous G6PC3 gene mutation p.Trp59Arg. The patient showed pancytopenia and a variable bone marrow phenotype with maturation arrest and vacuolization in myeloid lineage cells and a normocellular marrow, respectively. She also showed persistent lymphopenia with low CD4 T- and CD19 B-cell counts. Lymphopenia and even pancytopenia as well as a variable bone marrow phenotype can be part of this syndrome. These clinical findings in a patient with chronic neutropenia should alert the clinician to consider a diagnosis of G6PC3 deficiency.

Congenital amegakaryocytic thrombocytopenia (CAMT) presenting as severe pancytopenia in the first month of life.

Congenital amegakaryocytic thrombocytopenia (CAMT) is characterised by neonatal thrombocytopenia, with reduced or absent bone marrow megakaryocytes, leading eventually to pancytopenia. The mean age for progression to bone marrow failure is four years, with the earliest reported being six months. We describe a CAMT patient with compound heterozygous mutations of the causative MPL gene (one being a previously unreported splice site mutation in intron 11) who developed pancytopenia within the first month of life. This report emphasises the importance of considering CAMT in the differential diagnosis of congenital aplastic anaemia or idiopathic aplastic anaemia in babies.

The spectrum of ELANE mutations and their implications in severe congenital and cyclic neutropenia.

Neutrophil elastase gene (ELANE) mutations are responsible for the majority of cases of severe congenital neutropenia (CN) and cyclic neutropenia (CyN). We screened CN (n = 395) or CyN (n = 92) patients for ELANE mutations and investigated the impact of mutations on mRNA expression, protein expression, and activity. We found 116 different mutations in 162 (41%) CN patients and 26 in 51 (55%) CyN patients, 69 of them were novel. CyN-associated mutations were predicted to be more benign than CN-associated mutations, but the mutation severity largely overlapped. The frequency of acquired CSF3R mutations, malignant transformation, and the need for hematopoietic stem cell transplantation was significantly higher in CN patients with ELANE mutation than in ELANE mutation negative patients. Cellular elastase activity was reduced in neutrophils from CN/CyN patients, irrespective of the mutation status. In CN, enzymatic activity was significantly lower in patients with ELANE mutations compared with those with wild-type ELANE. Despite differences in the spectrum of mutations in CN or CyN, type or localization of mutation only partially determine the clinical phenotype. Specific ELANE mutations have limited predictive value for leukemogenesis; the risk for leukemia was correlated with disease severity rather than with occurrence of an ELANE mutation.

A novel G6PC3 gene mutation in a patient with severe congenital neutropenia.

Glucose-6-phosphatase catalytic subunit 3 (G6PC3) deficiency is a newly described syndromic type of severe congenital neutropenia, associated with multiple organ abnormalities including facial, cardiac, and urogenital abnormalities, and increased visibility of superficial veins. The molecular pathophysiology of G6PC3 deficiency is associated with the disturbed glucose homeostasis, increased endoplasmic reticulum stress, and apoptosis in neutrophils. We report a new case of G6PC3 deficiency caused by a novel homozygous G6PC3 gene mutation (p.Leu154Pro). Most remarkable is that the chronic neutropenia that originated from this novel G6PC3 genetic defect is also accompanied by some other unusual manifestations in this patient: myelokathexis and hypercholesterolemia.

Congenital amegakaryocytic thrombocytopenia: clinical presentation, diagnosis, and treatment.

Congenital amegakaryocytic thrombocytopenia (CAMT, MIM #604498) is a rare inherited bone marrow failure syndrome presenting as isolated hypomegakaryocytic thrombocytopenia at birth without other characteristic physical anomalies. Most of the patients develop a severe aplastic anemia and trilineage cytopenia during the first years of life and hematopoietic stem cell transplantation is the only curative treatment. In most of the cases the disease is caused by homozygous or compound heterozygous mutations in the gene MPL encoding the receptor for the hematopoietic growth factor thrombopoietin. The present review summarizes clinical and laboratory data for 96 patients with CAMT, reported since 1990.

Granulocyte colony-stimulating factor (G-CSF) treatment of childhood acute myeloid leukemias that overexpress the differentiation-defective G-CSF receptor isoform IV is associated with a higher incidence of relapse.

PURPOSE: This prospective, multicenter Acute Myeloid Leukemia Berlin-Frankfurt-Muenster (AML-BFM) 98 study randomly tested the ability of granulocyte colony-stimulating factor (G-CSF) to reduce infectious complications and to improve outcomes in children and adolescents with acute myeloid leukemia (AML). However, a trend toward an increased incidence of relapses in the standard-risk (SR) group after G-CSF treatment was observed. PATIENTS AND METHODS: Of 154 SR patients in the AML-BFM 98 cohort, 50 patients were tested for G-CSF receptor (G-CSFR) RNA isoform I and IV expression, G-CSFR cell surface expression, and acquired mutations in the G-CSFR gene. RESULTS: In patients randomly assigned to receive G-CSF after induction, 16 patients overexpressing the G-CSFR isoform IV showed an increased 5-year cumulative incidence of relapse (50% +/- 13%) compared with 14 patients with low-level isoform IV expression (14% +/- 10%; log-rank P = .04). The level of G-CSFR isoform IV had no significant effect in patients not receiving G-CSF (P = .19). Multivariate analyses of the G-CSF-treated subgroup, including the parameters G-CSFR isoform IV overexpression, sex, and favorable cytogenetics as covariables, revealed the prognostic relevance of G-CSFR isoform IV overexpression for 5-year event-free survival (P = .031) and the 5-year cumulative incidence of relapse (P = .049). CONCLUSION: Our results demonstrate that children and adolescents with AMLs that overexpress the differentiation-defective G-CSFR isoform IV respond to G-CSF administration after induction, but with a significantly higher incidence of relapse.

Digenic mutations in severe congenital neutropenia.

Severe congenital neutropenia a clinically and genetically heterogeneous disorder. Mutations in different genes have been described as causative for severe neutropenia, e.g. ELANE, HAX1 and G6PC3. Although congenital neutropenia is considered to be a group of monogenic disorders, the phenotypic heterogeneity even within the yet defined genetic subtypes points to additional genetic and/or epigenetic influences on the disease phenotype. We describe congenital neutropenia patients with mutations in two candidate genes each, including 6 novel mutations. Two of them had a heterozygous ELANE mutation combined with a homozygous mutation in G6PC3 or HAX1, respectively. The other 2 patients combined homozygous or compound heterozygous mutations in G6PC3 or HAX1 with a heterozygous mutation in the respective other gene. Our results suggest that digenicity may underlie this disorder of myelopoiesis at least in some congenital neutropenia patients.

Eponym. Kostmann disease.

Rolf Kostmann (1909-1982) was a Swedish pediatrician and army doctor. He was the first to describe an inherited form of chronic neutropenia in childhood. In 1956, Kostmann published his article "Infantile genetic agranulocytosis" in Acta Paediatrica. "Infantile agranulocytosis," as Rolf Kostmann named this hereditary syndrome, has been known for more than half a century, yet the underlying genetic mutations have remained unknown for many decades. Fifty years later, homozygous mutations in the gene encoding the mitochondrial protein HCLS1-associated X1 were found in affected members of the original Kostmann pedigree. Therefore, the eponym "Kostmann disease" best fits this specific mutation and mode of inheritance. The identification of genetic cause now allows the analysis of genotype-phenotype correlations. After the development of recombinant human granulocyte colony-stimulating factor (G-CSF), the prognosis and quality of life improved dramatically. Hematopoietic stem cell transplantation remains the only currently available treatment for refractory cases to G-CSF and patients who have transformed into leukemia.

Kostmann disease with developmental delay in three patients.

Kostmann disease is a rare autosomal recessive form of severe congenital neutropenia characterized by maturation arrest at the stage of promyelocytes/myelocytes in bone marrow with peripheral blood absolute neutrophil counts below 0.5 x 10(9)/L and severe recurrent bacterial infections from early infancy. Kostmann disease is caused by homozygous mutations in the gene encoding the mitochondrial protein HCLS1-associated X1. Here, we report three patients with Kostmann disease who, besides recurrent infections, have developmental delay.

Advances in the understanding of congenital amegakaryocytic thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia (MIM #604498) is an extremely rare inherited bone marrow failure syndrome, usually presenting as a severe thrombocytopenia at birth due to ineffective megakaryocytopoiesis and no characteristic physical anomalies. Usually the isolated thrombocytopenia progresses to pancytopenia during the first years of life. The only curative therapy to date is haematopoietic stem cell transplantation. Most of the cases of congenital amegakaryocytic thrombocytopenia are caused by defective expression or function of the thrombopoietin receptor due to homozygous or compound heterozygous mutations in the gene MPL. The essential roles of thrombopoietin as a lineage specific regulator of platelet production and as a regulator of haematopoietic stem cell function are reflected in the haematological defects seen in affected individuals.

Acute lymphoblastic leukemia following severe congenital neutropenia or de novo ALL?

Acute lymphoblastic leukemia (ALL) presenting with neutropenia alone is very rare. We describe a newborn with an early life-threatening infection, severe neutropenia and bone marrow findings compatible with severe congenital neutropenia (SCN). She was treated with granulocyte colony-stimulating factor (G-CSF) with complete neutrophil recovery. Three months later she developed a pro-B ALL. We identified a rare loss of 5'-MLL present at the diagnosis of SCN and ALL by FISH analysis using two different MLL (11q23) probes. Molecular analyses for SCN causing mutations (ELA-2, HAX-1 and G6PC3) and for somatic mutations of the CSF3R gene were negative. The early presence of 5'-MLL loss in bone marrow samples may favor the diagnosis of de novo ALL. Nevertheless, the genetic background for SCN is heterogeneous and a non-described mutation for SCN followed by a secondary ALL cannot be excluded. Further genetic investigation may be useful to gain insight into this rare condition in children.