Newborn screening for aromatic l-amino acid decarboxylase deficiency - Strategies, results, and implication for prevalence calculations.

BACKGROUND: Aromatic l-amino acid decarboxylase deficiency (AADCD) is a rare, autosomal-recessive neurometabolic disorder caused by variants in dopa decarboxylase (DDC) gene, resulting in a severe combined deficiency of serotonin, dopamine, norepinephrine, and epinephrine. Birth prevalence of AADCD varies by population. In pilot studies, 3-O-methyldopa (3-OMD) was shown to be a reliable biomarker for AADCD in high-throughput newborn screening (NBS) allowing an early diagnosis and access to gene therapy. To evaluate the usefulness of this method for routine NBS, 3-OMD screening results from the largest three German NBS centers were analyzed. METHODS: A prospective, multicenter (n = 3) NBS pilot study evaluated screening for AADCD by quantifying 3-OMD in dried blood spots (DBS) using tandem mass spectrometry (MS/MS). RESULTS: In total, 766,660 neonates were screened from January 2021 until June 2023 with 766,647 with unremarkable AADCD NBS (766,443 by 1st-tier analysis and 204 by 2nd-tier analysis) and 13 with positive NBS result recalled for confirmatory diagnostics (recall-rate about 1:59,000). Molecular genetic analysis confirmed AADCD (c.79C > T p.[Arg27Cys] in Exon 2 und c.215 A > C p.[His72Pro] in Exon 3) in one infant. Another individual was highly suspected with AADCD but died before confirmation (overall positive predictive value 0.15). False-positive results were caused by maternal L-Dopa use (n = 2) and prematurity (30th and 36th week of gestation, n = 2). However, in 63% (n = 7) the underlying etiology for false positive results remained unexplained. Estimated birth prevalence (95% confidence interval) was 1:766,660 (95% CI 1:775,194; 1:769,231) to 1:383,330 (95% CI 1:384,615; 1:383,142). The identified child remained asymptomatic until last follow up at the age of 9 months. CONCLUSIONS: The proposed screening strategy with 3-OMD detection in DBS is feasible and effective to identify individuals with AADCD. The estimated birth prevalence supports earlier estimations and confirms AADCD as a very rare disorder. Pre-symptomatic identification by NBS allows a disease severity adapted drug support to diminish clinical complications until individuals are old enough for the application of the gene therapy.

IgD shapes the pre-immune naïve B cell compartment in humans.

B cell maturation and immunoglobulin (Ig) repertoire selection are governed by expression of a functional B cell receptor (BCR). Naïve B cells co-express their BCR as IgM and IgD isotype. However, the role of the additionally expressed IgD on naïve B cells is not known. Here we assessed the impact of IgD on naïve B cell maturation and Ig repertoire selection in 8 individuals from 3 different families with heterozygous loss-of-function or loss-of expression mutations in IGHD. Although naïve B cells from these individuals expressed IgM on their surface, the IGHD variant in heterozygous state entailed a chimeric situation by allelic exclusion with almost half of the naïve B cell population lacking surface IgD expression. Flow cytometric analyses revealed a distinct phenotype of IgD-negative naïve B cells with decreased expression of CD19, CD20 and CD21 as well as lower BAFF-R and integrin-ß7 expression. IgD-negative B cells were less responsive in vitro after engaging the IgM-BCR, TLR7/9 or CD40 pathway. Additionally, a selective disadvantage of IgD-negative B cells within the T2 transitional and mature naïve B cell compartment as well as reduced frequencies of IgMlo/- B cells within the mature naïve B cell compartment lacking IgD were evident. RNA-Ig-seq of bulk sorted B cell populations showed an altered selection of distinct VH segments in the IgD-negative mature naïve B cell population. We conclude that IgD expression on human naïve B cells is redundant for generation of naïve B cells in general, but further shapes the naive B cell compartment starting from T2 transitional B cells. Our observations suggest an unexpected role of IgD expression to be critical for selection of distinct Ig VH segments into the pre-immune Ig repertoire and for the survival of IgMlo/- naïve B cells known to be enriched in poly-/autoreactive B cell clones.

Uncoupling of platelet granule release and integrin activation suggests GPIIb/IIIa as a therapeutic target in COVID-19.

Thromboembolic events are frequent and life-threating complications of COVID-19 but are also observed in patients with sepsis. Disseminated thrombosis can occur despite anticoagulation, suggesting that platelets play a direct but incompletely understood role. Several studies demonstrated altered platelet function in COVID-19 with some controversial findings, while underlying disease-specific mechanisms remain ill defined. We performed a comprehensive cohort study with 111 patients, comprising 37 with COVID-19, 46 with sepsis, and 28 with infection, compared with control participants. Platelet phenotype and function were assessed under static and flow conditions, revealing unexpected disease-specific differences. From hospital admission onward, platelets in COVID-19 failed to activate the integrin glycoprotein IIb/IIa (GPIIb/IIIa) in response to multiple agonists. Dense granule release was markedly impaired due to virtually missing granules, also demonstrated by whole-mount electron microscopy. By contrast, α-granule marker CD62P exposure was only mildly affected, revealing a subpopulation of PAC-1-/CD62P+ platelets, independently confirmed by automated clustering. This uncoupling of α-granule release was not observed in patients with sepsis, despite a similar disease severity. We found overall unaltered thrombus formation in COVID-19 and sepsis samples under venous shear rates, which was dependent on the presence of tissue factor. Unexpectedly, under arterial shear rates, thrombus formation was virtually abrogated in sepsis, whereas we detected overall normal-sized and stable thrombi in blood from patients with COVID-19. These thrombi were susceptible to subthreshold levels of GPIIb/IIIa blockers, eptifibatide, or tirofiban that had only a minor effect in control participants' blood. We provide evidence that low-dose GPIIb/IIIa blockade could be a therapeutic approach in COVID-19.

G6b-B regulates an essential step in megakaryocyte maturation.

G6b-B is a megakaryocyte lineage-specific immunoreceptor tyrosine-based inhibition motif-containing receptor, essential for platelet homeostasis. Mice with a genomic deletion of the entire Mpig6b locus develop severe macrothrombocytopenia and myelofibrosis, which is reflected in humans with null mutations in MPIG6B. The current model proposes that megakaryocytes lacking G6b-B develop normally, whereas proplatelet release is hampered, but the underlying molecular mechanism remains unclear. We report on a spontaneous recessive single nucleotide mutation in C57BL/6 mice, localized within the intronic region of the Mpig6b locus that abolishes G6b-B expression and reproduces macrothrombocytopenia, myelofibrosis, and osteosclerosis. As the mutation is based on a single-nucleotide exchange, Mpig6bmut mice represent an ideal model to study the role of G6b-B. Megakaryocytes from these mice were smaller, displayed a less-developed demarcation membrane system, and had a reduced expression of receptors. RNA sequencing revealed a striking global reduction in the level of megakaryocyte-specific transcripts, in conjunction with decreased protein levels of the transcription factor GATA-1 and impaired thrombopoietin signaling. The reduced number of mature MKs in the bone marrow was corroborated on a newly developed Mpig6b-null mouse strain. Our findings highlight an unexpected essential role of G6b-B in the early differentiation within the megakaryocytic lineage.

Effects of Cocoa Genotypes on Coat Color, Platelets and Coagulation Parameters in French Bulldogs.

A nonsense variant in HPS3, c.2420G>A or p.Trp807*, was recently discovered as the cause for a brown coat color termed cocoa in French Bulldogs. Here, we studied the genotype-phenotype correlation regarding coat color in HPS3 mutant dogs that carried various combinations of mutant alleles at other coat color genes. Different combinations of HPS3, MLPH and TYRP1 genotypes resulted in subtly different shades of brown coat colors. As HPS3 variants in humans cause the Hermansky-Pudlak syndrome type 3, which in addition to oculocutaneous albinism is characterized by a storage pool deficiency leading to bleeding tendency, we also investigated the phenotypic consequences of the HPS3 variant in French Bulldogs on hematological parameters. HPS3 mutant dogs had a significantly lowered platelet dense granules abundance. However, no increased bleeding tendencies in daily routine were reported by dog owners. We therefore conclude that in dogs, the phenotypic effect of the HPS3 variant is largely restricted to pigmentation. While an effect on platelet morphology is evident, we did not obtain any indications for major health problems associated with the cocoa coat color in French Bulldogs. Further studies will be necessary to definitely rule out very subtle effects on visual acuity or a clinically relevant bleeding disorder.

RhoA/Cdc42 signaling drives cytoplasmic maturation but not endomitosis in megakaryocytes.

Megakaryocytes (MKs), the precursors of blood platelets, are large, polyploid cells residing mainly in the bone marrow. We have previously shown that balanced signaling of the Rho GTPases RhoA and Cdc42 is critical for correct MK localization at bone marrow sinusoids in vivo. Using conditional RhoA/Cdc42 double-knockout (DKO) mice, we reveal here that RhoA/Cdc42 signaling is dispensable for the process of polyploidization in MKs but essential for cytoplasmic MK maturation. Proplatelet formation is virtually abrogated in the absence of RhoA/Cdc42 and leads to severe macrothrombocytopenia in DKO animals. The MK maturation defect is associated with downregulation of myosin light chain 2 (MLC2) and ß1-tubulin, as well as an upregulation of LIM kinase 1 and cofilin-1 at both the mRNA and protein level and can be linked to impaired MKL1/SRF signaling. Our findings demonstrate that MK endomitosis and cytoplasmic maturation are separately regulated processes, and the latter is critically controlled by RhoA/Cdc42.

Acquired platelet GPVI receptor dysfunction in critically ill patients with sepsis.

Glycoprotein VI (GPVI), the platelet immunoreceptor tyrosine activating motif (ITAM) receptor for collagen, plays a striking role on vascular integrity in animal models of inflammation and sepsis. Understanding ITAM-receptor signaling defects in humans suffering from sepsis may improve our understanding of the pathophysiology, especially during disease onset. In a pilot study, platelets from 15 patients with sepsis were assessed consecutively at day of admission, day 5 to 7, and the day of intensive care unit (ICU) discharge and subjected to comprehensive analyses by flow cytometry, aggregometry, and immunoblotting. Platelet function was markedly reduced in all patients. The defect was most prominent after GPVI stimulation with collagen-related peptide. In 14 of 15 patients, GPVI dysfunction was already present at time of ICU admission, considerably before the critical drop in platelet counts. Sepsis platelets failed to transduce the GPVI-mediated signal to trigger tyrosine phosphorylation of Syk kinase or LAT. GPVI deficiency was partially inducible in platelets of healthy donors through coincubation in whole blood, but not in plasma from patients with sepsis. Platelet aggregation upon GPVI stimulation increased only in those patients whose condition ameliorated. As blunted GPVI signaling occurred early at sepsis onset, this defect could be exploited as an indicator for early sepsis diagnosis, which needs to be confirmed in prospective studies.

Forming megakaryocytes from murine-induced pluripotent stem cells by the inducible overexpression of supporting factors.

BACKGROUND: Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low. OBJECTIVES: To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA-binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre-B-cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). METHODS: To avoid off-target effects, we generated iPSCs carrying the reverse tetracycline-responsive transactivator M2 (rtTA-M2) in the Rosa26 locus and expressed the factors from Tet-inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. RESULTS: Overexpression of GATA1 and Pbx1 increased MK output 2- to 2.5-fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro-generated platelets were functional in spreading on fibrinogen or collagen-related peptide. CONCLUSION: We demonstrate that the use of rtTA-M2 transgenic iPSCs transduced with Tet-inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.

Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1.

Rearrangements of the microtubule (MT) and actin cytoskeleton are pivotal for platelet biogenesis. Hence, defects in actin- or MT-regulatory proteins are associated with platelet disorders in humans and mice. Previous studies in mice revealed that loss of the actin-depolymerizing factor homology (ADF-H) protein Cofilin1 (Cof1) in megakaryocytes (MKs) results in a moderate macrothrombocytopenia but normal MK numbers, whereas deficiency in another ADF-H protein, Twinfilin1 (Twf1), does not affect platelet production or function. However, recent studies in yeast have indicated a critical synergism between Twf1 and Cof1 in the regulation of actin dynamics. We therefore investigated platelet biogenesis and function in mice lacking both Twf1 and Cof1 in the MK lineage. In contrast to single deficiency in either protein, Twf1/Cof1 double deficiency (DKO) resulted in a severe macrothrombocytopenia and dramatically increased MK numbers in bone marrow and spleen. DKO MKs exhibited defective proplatelet formation in vitro and in vivo as well as impaired spreading and altered assembly of podosome-like structures on collagen and fibrinogen in vitro. These defects were associated with aberrant F-actin accumulation and, remarkably, the formation of hyperstable MT, which appears to be caused by dysregulation of the actin- and MT-binding proteins mDia1 and adenomatous polyposis coli. Surprisingly, the mild functional defects described for Cof1-deficient platelets were only slightly aggravated in DKO platelets suggesting that both proteins are largely dispensable for platelet function in the peripheral blood. In summary, these findings reveal critical redundant functions of Cof1 and Twf1 in ensuring balanced actin/microtubule crosstalk during thrombopoiesis in mice and possibly humans.

Novel variants in FERMT3 and RASGRP2-Genetic linkage in Glanzmann-like bleeding disorders.

Defects of platelet intracellular signaling can result in severe platelet dysfunction. Several mutations in each of the linked genes FERMT3 and RASGRP2 on chromosome 11 causing a Glanzmann-like bleeding phenotype have been identified so far. We report on novel variants in two unrelated pediatric patients with severe bleeding diathesis-one with leukocyte adhesion deficiency type III due to a homozygous frameshift in FERMT3 and the other with homozygous variants in both, FERMT3 and RASGRP2. We focus on the challenging genetic and functional variant assessment and aim to accentuate the risk of obtaining misleading results due to the phenomenon of genetic linkage.

Pivotal role of PDK1 in megakaryocyte cytoskeletal dynamics and polarization during platelet biogenesis.

During thrombopoiesis, megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Phosphoinositide-dependent protein kinase-1 (PDK1) is required for Ca2+-dependent platelet activation, but its role in MK development and regulation of platelet production remained elusive. The present study explored the role of PDK1 in the regulation of MK maturation and polarization during thrombopoiesis using a MK/platelet-specific knockout approach. Pdk1-deficient mice (Pdk1-/-) developed a significant macrothrombocytopenia as compared with wild-type mice (Pdk1fl/fl). Pdk1 deficiency further dramatically increased the number of MKs without sinusoidal contact within the BM hematopoietic compartment, resulting in a pronounced MK hyperplasia and a significantly increased extramedullary thrombopoiesis. Cultured Pdk1-/- BM-MKs showed impaired spreading on collagen, associated with an altered actin cytoskeleton structure with less filamentous actin (F-actin) and diminished podosome formation, whereas the tubulin cytoskeleton remained unaffected. This phenotype was associated with abrogated phosphorylation of p21-activated kinase (PAK) as well as its substrates LIM domain kinase and cofilin, supporting the hypothesis that the defective F-actin assembly results from increased cofilin activity in Pdk1-deficient MKs. Pdk1-/- BM-MKs developed increased ploidy and exhibited an abnormal ultrastructure with disrupted demarcation membrane system (DMS). Strikingly, Pdk1-/- BM-MKs displayed a pronounced defect in DMS polarization and produced significantly less proplatelets, indicating that PDK1 is critically required for proplatelet formation. In human MKs, genetic PDK1 knockdown resulted in increased maturity but reduced platelet-like particles formation. The present observations reveal a pivotal role of PDK1 in the regulation of MK cytoskeletal dynamics and polarization, proplatelet formation, and thrombopoiesis.

Impact of Itga2-Gp6-double collagen receptor deficient mice for bone marrow megakaryocytes and platelets.

The two main collagen receptors on platelets, GPVI and integrin α2ß1, play an important role for the recognition of exposed collagen at sites of vessel injury, which leads to platelet activation and subsequently stable thrombus formation. Both receptors are already expressed on megakaryocytes, the platelet forming cells within the bone marrow. Megakaryocytes are in permanent contact with collagen filaments in the marrow cavity and at the basal lamina of sinusoids without obvious preactivation. The role of both collagen receptors for megakaryocyte maturation and thrombopoiesis is still poorly understood. To investigate the function of both collagen receptors, we generated mice that are double deficient for Gp6 and Itga2. Flow cytometric analyses revealed that the deficiency of both receptors had no impact on platelet number and led to the expected lack in GPVI responsiveness. Integrin activation and degranulation ability was comparable to wildtype mice. By immunofluorescence microscopy, we could demonstrate that both wildtype and double-deficient megakaryocytes were overall normally distributed within the bone marrow. We found megakaryocyte count and size to be normal, the localization within the bone marrow, the degree of maturation, as well as their association to sinusoids were also unaltered. However, the contact of megakaryocytes to collagen type I filaments was decreased at sinusoids compared to wildtype mice, while the interaction to type IV collagen was unaffected. Our results imply that GPVI and α2ß1 have no influence on the localization of megakaryocytes within the bone marrow, their association to the sinusoids or their maturation. The decreased contact of megakaryocytes to collagen type I might at least partially explain the unaltered platelet phenotype in these mice, since proplatelet formation is mediated by these receptors and their interaction to collagen. It is rather likely that other compensatory signaling pathways and receptors play a role that needs to be elucidated.

Defective Zn2+ homeostasis in mouse and human platelets with α- and δ-storage pool diseases.

Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d-/- mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2-/- mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2-/- and Unc13d-/- mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.

Functional Classification of Paediatric Patients with Non-syndromic Delta-Storage Pool Deficiency.

Storage pool disease (SPD) covers a group of platelet defects in which α- and/or delta-granules are reduced or cannot be secreted adequately in response to agonists. The detection of delta-granule release defects is hampered by a lack of fast and feasible tests. We aimed to implement a flow cytometry-based kinetic mepacrine assay to better identify and subgroup childhood patients with a mild to moderate bleeding diathesis and compare our method to established laboratory tests. We analysed 50 children with suspected SPD whose initial parameters were re-assessed in a second site visit. Mepacrine uptake and release patterns were correlated with CD63 exposure, platelet ADP/ATP release and content, and the bleeding score ascertained by the ISTH-BAT. Mepacrine release was overall significantly reduced in investigated patients compared with controls. Summarizing, our time-resolved approach proved to be a quick and inexpensive tool that was additionally able to distinguish between mepacrine uptake, mepacrine release, and combined defects. Classification of patients using such a kinetic assay makes it feasible to sensitively detect frequently missed SPD and to group these patients for further analyses and clinical correlations.

Use of Targeted High-Throughput Sequencing for Genetic Classification of Patients with Bleeding Diathesis and Suspected Platelet Disorder.

Inherited platelet disorders (IPD) form a rare and heterogeneous disease entity that is present in about 8% of patients with non-acquired bleeding diathesis. Identification of the defective cellular pathway is an important criterion for stratifying the patient's individual risk profile and for choosing personalized therapeutic options. While costs of high-throughput sequencing technologies have rapidly declined over the last decade, molecular genetic diagnosis of bleeding and platelet disorders is getting more and more suitable within the diagnostic algorithms. In this study, we developed, verified, and evaluated a targeted, panel-based next-generation sequencing approach comprising 59 genes associated with IPD for a cohort of 38 patients with a history of recurrent bleeding episodes and functionally suspected, but so far genetically undefined IPD. DNA samples from five patients with genetically defined IPD with disease-causing variants in WAS , RBM8A , FERMT3 , P2YR12 , and MYH9 served as controls during the validation process. In 40% of 35 patients analyzed, we were able to finally detect 15 variants, eight of which were novel, in 11 genes, ACTN1 , AP3B1 , GFI1B , HPS1 , HPS4 , HPS6 , MPL , MYH9 , TBXA2R , TPM4 , and TUBB1 , and classified them according to current guidelines. Apart from seven variants of uncertain significance in 11% of patients, nine variants were classified as likely pathogenic or pathogenic providing a molecular diagnosis for 26% of patients. This report also emphasizes on potentials and pitfalls of this tool and prospectively proposes its rational implementation within the diagnostic algorithms of IPD.

Diagnosis of platelet function disorders: A standardized, rational, and modular flow cytometric approach.

A high proportion of patients with mucocutaneous bleeding diathesis and suspected inherited or acquired platelet disorder remain without diagnosis even after comprehensive laboratory testing. Since flow cytometry allows investigation of resting and activated platelets on the single cell level by requiring only minimal amounts of blood, this method has become an important assay within the diagnostic algorithm, especially in pediatrics. We therefore developed a standardized and modular flow cytometric approach that contributes to clarify impaired platelet function in a rational step-by-step manner. Due to simultaneous analysis of four fluorophores in a basic panel design, we are able to readily detect the most common and clinically significant platelet disorders: Glanzmann thrombasthenia or Glanzmann-like diseases (fibrinogen receptor GPIIb-IIIa), Bernard-Soulier syndrome (von Willebrand-factor receptor complex GPIb-IX-V) and less well characterized ß1-integrins that serve as the collagen, laminin or fibronectin receptor (CD29-CD49b, e and f, respectively). Platelet reactivity was investigated in response to the agonists adenosine diphosphate (ADP) and thrombin receptor activator peptide 6 (TRAP6) in suboptimal and optimal concentrations by quantifying surface expression of activation markers CD62P and CD63 as well as binding of PAC-1 antibody to the high affinity conformation of the fibrinogen receptor. For advanced diagnostic questions, several further modules were implemented: (i) calcium mobilization for evaluation of early signal transduction, (ii) a kinetically resolved mepacrine assay for estimation of delta-granule content and release, and (iii) a module to determine platelet reactivity upon additional agonists like the thromboxane A2-analogue U46619 or collagen. Blood withdrawn from a healthy control cohort allowed generating preliminary standard values for all parameters. The modules were validated by analysis of patients with known or suspected platelet defects (leukocyte-adhesion deficiency type III, Wiskott-Aldrich syndrome, acute myeloid leukemia, sickle cell disease and chronic immune thrombocytopenia).

Impact of genetic variants on haematopoiesis in patients with thrombocytopenia absent radii (TAR) syndrome.

Thrombocytopenia absent radii (TAR) syndrome is clearly defined by the combination of radial aplasia and reduced platelet counts. The genetics of TAR syndrome has recently been resolved and comprises a microdeletion on Chromosome 1 including the RBM8A gene and a single nucleotide polymorphism (SNP) either at the 5' untranslated region (5'UTR) or within the first intron of RBM8A. Although phenotypically readily diagnosed after birth, the genetic determination of particular SNPs in TAR syndrome harbours valuable information to evaluate disease severity and treatment decisions. Here, we present clinical data in a cohort of 38 patients and observed that platelet counts in individuals with 5'UTR SNP are significantly lower compared to patients bearing the SNP in intron 1. Moreover, elevated haemoglobin values could only be assessed in patients with 5'UTR SNP whereas white blood cell count is unaffected, indicating that frequently observed anaemia in TAR patients could also be SNP-dependent whereas leucocytosis does not correlate with genetic background. However, this report on a large cohort provides an overview of important haematological characteristics in TAR patients, facilitating evaluation of the various traits in this disease and indicating the importance of genetic validation for TAR syndrome.

Establishment and Characterization of a Pair of Patient-derived Human Non-small Cell Lung Cancer Cell Lines from a Primary Tumor and Corresponding Lymph Node Metastasis.

BACKGROUND: Non-small lung cancer is the leading cause of cancer-related mortality worldwide. For a deeper understanding of tumor biology, we established a pair of cell lines derived from a primary tumor and a corresponding lymph node metastasis. MATERIAL AND METHODS: The cell line BC4323 from the primary tumor (PT) and a mediastinal lymph node metastasis (LN) were derived from an adenocarcinoma (pT2, pN2, G3, UICC stage IIIa) in a 47-year-old female patient. Comparative characterization was performed by in vitro analysis. A murine xenograft was established for analysis of in vivo behavior. RESULTS: Chromosomal aberrations were detected in multiple chromosomal sections throughout the entire genome, with only a few differences between PT and LN cells. High-level Kirsten ras oncogene homolog (KRAS) mutation and amplification were seen based on a chromosomal translocation and novel assembled chromosome. In contrast to the genomic level, at the mRNA and protein levels, multiple differences were detectable, in particular in markers for cell adhesion [e.g. epithelial cell adhesion molecule (EpCAM), CD44, P-selectin binding, epidermal growth factor receptor (EGFR) and integrin alphaV] and the epithelial-mesenchymal transition. Due to accelerated tumor growth in vivo by the PT cells, a shortened overall survival was seen (60 vs. 101 days, p=0.005). CONCLUSION: We provide detailed analysis of a cell line derived from a primary tumor and a corresponding LN metastasis. This unique feature allows further investigative analysis of the differences and regulatory processes underlying the metastatic process during tumor progression in non-small cell lung cancer.

GABP is necessary for stem/progenitor cell maintenance and myeloid differentiation in human hematopoiesis and chronic myeloid leukemia.

Maintenance of hematopoietic stem cells and their potential to give rise to progenitors of differentiated lymphoid and myeloid cells are accomplished by a network of regulatory processes. As a part of this network, the heteromeric transcription factor GA-binding protein (GABP) plays a crucial role in self-renewal of murine hematopoietic and leukemic stem cells. Here, we report the consequences of functional impairment of GABP in human hematopoietic and in leukemic stem/progenitor cells. Ectopic overexpression of a dominant-negative acting GABP mutant led to impaired myeloid differentiation of CD34(+) hematopoietic stem/progenitor cells obtained from healthy donors. Moreover, drastically reduced clonogenic capacity of leukemic stem/progenitor cells isolated from bone marrow aspirates of chronic myeloid leukemia (CML) patients underlines the importance of GABP on stem/progenitor cell maintenance and confirms the relevance of GABP for human myelopoiesis in healthy and diseased states.