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Target enrichment and high-throughput sequencing of 80 ribosomal protein genes to identify mutations associated with Diamond-Blackfan anaemia.
Diamond-Blackfan anaemia (DBA) is caused by inactivating mutations in ribosomal protein (RP) genes, with mutations in 13 of the 80 RP genes accounting for 50-60% of cases. The remaining 40-50% cases may harbour mutations in one of the remaining RP genes, but the very low frequencies render conventional genetic screening as challenging. We, therefore, applied custom enrichment technology combined with high-throughput sequencing to screen all 80 RP genes. Using this approach, we identified and validated inactivating mutations in 15/17 (88%) DBA patients. Target enrichment combined with high-throughput sequencing is a robust and improved methodology for the genetic diagnosis of DBA.
Neonatal thrombocytopenia.
Thrombocytopenia (platelets <150 x 10(9)/L) is one of the most common haematological problems in neonates, particularly those who are preterm and sick. In those preterm neonates with early thrombocytopenia who present within 72 h of birth, the most common cause is reduced platelet production secondary to intrauterine growth restriction and/or maternal hypertension. By contrast, the most common causes of thrombocytopenia arising after the first 72 h of life, both in preterm and term infants, are sepsis and necrotizing enterocolitis. The most important cause of severe thrombocytopenia (platelets <50 x 10(9)/L) is neonatal alloimmune thrombocytopenia (NAIT), as diagnosis can be delayed and death or long-term disability due to intracranial haemorrhage may occur. Platelet transfusion is the mainstay of treatment for severe thrombocytopenia. However, the correlation between thrombocytopenia and bleeding is unclear and no studies have yet shown clinical benefit for platelet transfusion in neonates. Studies to identify optimal platelet transfusion practice for neonatal thrombocytopenia are urgently required.
Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis.
Strategies for genetic prenatal diagnosis on fetal cells in the maternal circulation have been limited by lack of a cell type present only in fetal blood. However, the recent identification of mesenchymal stem cells (MSC) in first trimester fetal blood offers the prospect of targeting MSC for non-invasive prenatal diagnosis. We developed protocols for fetal MSC enrichment from maternal blood and determined sensitivity and specificity in mixing experiments of male fetal MSC added to female blood, in dilutions from 1 in 10(5) to 10(8). We then used the optimal protocol to isolate fetal MSC from maternal blood in the first trimester, using blood taken after surgical termination of pregnancy as a model of increased feto-maternal haemorrhage. In model mixtures, we could amplify one male fetal MSC in 2.5 x 10(7) adult female nucleated cells, yielding a 100% pure population of fetal cells, but not one fetal MSC in 10(8) nucleated cells. Fetal MSC were identified in one of 20 post-termination maternal blood samples and confirmed as fetal MSC by XY fluorescence in-situ hybridization (FISH), immunophenotyping and osteogenic and adipogenic differentiation. We report the isolation of fetal MSC from maternal blood; however, their rarity in post-termination blood suggests they are unlikely to have a role in non-invasive prenatal diagnosis. Failure to locate these cells routinely may be attributed to their low frequency in maternal blood, to sensitivity limitations of enrichment technology, and/or to their engraftment in maternal tissues soon after transplacental passage. We speculate that gender microchimerism in post-reproductive maternal tissues might result from feto-maternal trafficking of MSC in early pregnancy.
Thrombocytopenia in the neonate.
Thrombocytopenia is one of the commonest haematological problems in neonates, affecting at least 25% of all admissions to neonatal intensive care units (NICUs) [Murray NA, Howarth LJ, McCloy MP et al. Platelet transfusion in the management of severe thrombocytopenia in neonatal intensive care unit patients. Transfus Med 2002;12:35-41; Garcia MG, Duenas E, Sola MC et al. Epidemiologic and outcome studies of patients who received platelet transfusions in the neonatal intensive care unit. J Perinatol 2001;21:415-20; Del Vecchio A, Sola MC, Theriaque DW et al. Platelet transfusions in the neonatal intensive care unit: factors predicting which patients will require multiple transfusions. Transfusion 2001;41:803-8]. Although a long list of disorders associated with neonatal thrombocytopenia can be found in many textbooks, newer classifications based on the timing of onset of thrombocytopenia (early vs. late) are more useful for planning diagnostic investigations and day-to-day management. The mainstay of treatment of neonatal thrombocytopenia remains platelet transfusion although it is important to note that no studies have yet shown clinical benefit of platelet transfusion in this setting. Indeed some reports even suggest that there may be significant adverse effects of platelet transfusion in neonates, including increased mortality, and that the effects of transfusion may differ in different groups of neonates with similar degrees of thrombocytopenia [Bonifacio L, Petrova A, Nanjundaswamy S, Mehta R. Thrombocytopenia related neonatal outcome in preterms. Indian J Pediatr 2007;74:269-74; Kenton AB, Hegemier S, Smith EO et al. Platelet transfusions in infants with necrotizing enterocolitis do not lower mortality but may increase morbidity. J Perinatol 2005;25:173-7]. There is also considerable variation in transfusion practice between different countries and between different neonatal units. Here we review recent progress in understanding the prevalence, causes and pathogenesis of thrombocytopenia in the newborn, the clinical consequences of thrombocytopenia and developments in neonatal platelet transfusion.
Hypomorphic promoter mutation in PIGM causes inherited glycosylphosphatidylinositol deficiency.
Attachment to the plasma membrane by linkage to a glycosylphosphatidylinositol (GPI) anchor is a mode of protein expression highly conserved from protozoa to mammals. As a clinical entity, deficiency of GPI has been recognized as paroxysmal nocturnal hemoglobinuria, an acquired clonal disorder associated with somatic mutations of the X-linked PIGA gene in hematopoietic cells. We have identified a novel disease characterized by a propensity to venous thrombosis and seizures in which deficiency of GPI is inherited in an autosomal recessive manner. In two unrelated kindreds, a point mutation (c --> g) at position -270 from the start codon of PIGM, a mannosyltransferase-encoding gene, disrupts binding of the transcription factor Sp1 to its cognate promoter motif. This mutation substantially reduces transcription of PIGM and blocks mannosylation of GPI, leading to partial but severe deficiency of GPI. These findings indicate that biosynthesis of GPI is essential to maintain homeostasis of blood coagulation and neurological function.
Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease.
Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell-derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.
Cord blood stem cell transplantation for haemoglobinopathies.
Despite improvements in supportive care, patients with beta-thalassaemia major or sickle cell disease (SCD) may benefit from haematopoietic stem cell transplantation at some point during their lives. Human leucocyte antigen (HLA)-matched sibling bone marrow donors are not always available and alternative sources of stem cells have been sought, including related and unrelated donor cord blood transplants (CBT). The outcome of CBT from related donors for the treatment of both thalassaemia major and SCD is now approaching that for bone marrow transplantation, with around 90% of patients surviving disease-free. The main complication is graft rejection, which may be reduced by increasing pretransplant immune suppression. Transplant-related mortality following HLA-identical matched related donor CBT is extremely low but is significant in the small series of unrelated and/or mis-matched donor CBT. The principal limitation to extending the use of CB stem cells for the cure of haemoglobinopathies is the need to better understand the mechanisms of action and optimal conditioning regimens used to secure long-term engraftment while minimizing morbidity and mortality. Further biological studies and clinical trials are needed to address this aim.
The role of mesenchymal stem cells in haemopoiesis.
The ontogeny of haemopoiesis during fetal life and the differentiation of blood cells in adult life depend upon a fully competent microenvironment to provide appropriate signals via production of soluble factors and cell contact interactions. The cellular constituents of the microenvironment, also defined as the haemopoietic niche, largely derive from a common progenitor of mesenchymal origin. Mesenchymal stem cells (MSC), initially identified in adult bone marrow, have also been described in fetal haemopoietic tissues where they accompany the migration of haemopoietic development. Their precise identity remains ill-defined because of the lack of specific markers. Their ability to self-renew and differentiate into tissues of mesodermal origin (osteocytes, adipocytes, chondrocytes) and their lack of expression of haemopoietic molecules are currently the main criteria for isolation. In the bone marrow the most important elements of the niche appear to be osteoblasts, whilst a less defined population of fibroblasts regulates the maturation of immature T cells in the thymus. Recently, MSC have been shown to exert a profound immunosuppressive effect on polyclonal as well as antigen-specific T cell responses by inducing a state of division arrest anergy. Thus, the multipotent capacity of MSC, their role in supporting haemopoiesis, and their immunoregulatory activity make MSC particularly attractive for therapeutic exploitation.
Bone involvement in sickle cell disease.
Bone involvement is the commonest clinical manifestation of sickle cell disease both in the acute setting such as painful vaso-occlusive crises, and as a source of chronic, progressive disability such as avascular necrosis. Management of these problems is often difficult because of the diagnostic imprecision of most laboratory and imaging investigations and because of the lack of evidence for most surgical procedures in sickle cell disease. This review first discusses the acute problems related to bone involvement in sickle cell disease, with particular reference to differentiating infection from infarction, and then describes the long-term effects of sickle cell disease on bone mineral density, growth, and chronic bone and joint damage.
CD34+ cells from first-trimester fetal blood are enriched in primitive hemopoietic progenitors.
OBJECTIVE: The purpose of this study was to investigate whether purified CD34(+) cells from first-trimester fetal blood are a source of primitive and committed hemopoietic progenitors. STUDY DESIGN: CD34(+) cells from first-trimester fetal blood and term cord blood were assayed for committed hemopoietic progenitor cells, high proliferative potential colony-forming cells, and long-term culture-initiating cells. RESULTS: First-trimester CD34(+) cells that were compared with cells at term generated fewer hemopoietic progenitor cells and fewer high proliferative potential colony-forming cells with lower recloning efficiency(P <.001). First-trimester CD34(+) cells tended to contain more long-term culture-initiating cells, both in bulk cultures and by limiting dilution analysis. The ratio between committed and primitive progenitors was 3 in the first-trimester and 20 in the term cord blood, respectively. CONCLUSION: First-trimester fetal blood is enriched in primitive (compared with committed) hemopoietic progenitors and may be an advantageous source of stem cells for prenatal therapy.
Characterization of first trimester fetal erythroblasts for non-invasive prenatal diagnosis.
Isolating fetal erythroblasts from first trimester maternal blood offers a promising non-invasive alternative for prenatal diagnosis. The aim of this study was to characterize the biological properties of first trimester primitive erythroblasts to facilitate their enrichment from first trimester maternal blood. Primitive erythroblasts were the predominant cell type until 12 weeks gestation, after which time their numbers declined steeply; 100% were epsilon-globin-positive versus <0.06% definitive erythroblasts. Buoyant densities of first trimester fetal erythroblasts ranged from 1.077 to 1.130 g/ml, and optimal recoveries were obtained with Percoll 1118. Although primitive erythroblasts carried a negative surface charge and were resistant to NH(4)Cl lysis, these properties had only a limited role in fetal cell enrichment. Immunophenotyping showed that primitive, like definitive, erythroblasts were GPA+, CD47+, CD45- and CD35-, whereas CD71 expression was weak/undetectable on primitive erythroblasts but strongly positive on 100% of definitive erythroblasts; primitive erythroblasts were also CD36- whereas definitive erythroblasts were CD36+. We therefore used CD45/GPA selection of Percoll 1118-separated cells to demonstrate successful enrichment of male epsilon-globin-positive fetal erythroblasts from model mixtures, and as proof of principle from some first trimester maternal blood samples. Fetal cell enrichment protocols based on first trimester epsilon-globin-positive primitive erythroblasts may allow reliable enrichment of fetal cells from maternal blood for early non-invasive prenatal diagnosis of genetic disorders.
Correction of severe anaemia using immuno-regulated gene therapy is achieved by restoring the early erythroblast compartment.
Patients with chronic renal failure usually require exogenous erythropoietin (epo) to alleviate anaemia resulting from inadequate epo production by the kidneys. We have recently shown that severe anaemia in genetically manipulated epo-deficient mice (EpoTAg) can be corrected by adoptively transferred epo-producing lymphocytes. The aim of this study was to investigate the precise effects of human epo administration by this route on erythropoietic development in epo-deficient mice. The erythroblast compartments of untreated and treated EpoTAg mice were analysed in comparison with wild-type mice. The early erythroblast population was reduced in the bone marrow of epo-deficient mice, whilst the number of erythroid colony-forming units (CFU-E) was not significantly compromised. This paucity in marrow early erythroblasts was restored to normal values in treated mutant mice. In addition, the early erythroblast population was expanded in the spleens of treated animals. These findings show that the early erythroblasts are important targets of epo and that epo corrects anaemia of epo-deficient mice by restoring marrow function and splenic erythropoiesis.
Recommendations regarding splenectomy in hereditary hemolytic anemias.
Hereditary hemolytic anemias are a group of disorders with a variety of causes, including red cell membrane defects, red blood cell enzyme disorders, congenital dyserythropoietic anemias, thalassemia syndromes and hemoglobinopathies. As damaged red blood cells passing through the red pulp of the spleen are removed by splenic macrophages, splenectomy is one possible therapeutic approach to the management of severely affected patients. However, except for hereditary spherocytosis for which the effectiveness of splenectomy has been well documented, the efficacy of splenectomy in other anemias within this group has yet to be determined and there are concerns regarding short- and long-term infectious and thrombotic complications. In light of the priorities identified by the European Hematology Association Roadmap we generated specific recommendations for each disorder, except thalassemia syndromes for which there are other, recent guidelines. Our recommendations are intended to enable clinicians to achieve better informed decisions on disease management by splenectomy, on the type of splenectomy and the possible consequences. As no randomized clinical trials, case control or cohort studies regarding splenectomy in these disorders were found in the literature, recommendations for each disease were based on expert opinion and were subsequently critically revised and modified by the Splenectomy in Rare Anemias Study Group, which includes hematologists caring for both adults and children.
Living with Inflammatory Bowel Disease: A review of qualitative research studies.
BACKGROUND: Inflammatory Bowel Disease is a chronic, untreatable condition represented by two illnesses, Crohn's and Ulcerative Colitis. Despite high incidence in well-developed industrialised countries, and the significant impact of symptoms on patient's quality of life, little is known about living with Inflammatory Bowel Disease. AIM: To explore the patients' experiences of living with Inflammatory Bowel Disease. DESIGN: A qualitative systematic review. DATA SOURCES: CINAHL, Medline, British Nursing Index and PsycINFO were searched using the following keywords: Inflammatory Bowel Disease AND experiences. We have limited the search to studies published in English from 2000 to 2017. REVIEW METHOD: Thematic synthesis. RESULTS: Data from 23 studies, identified that fatigue, incontinence and uncertainty about future, body image, and lack of information from healthcare professionals dominated the experiences of those living with Inflammatory Bowel Disease. Also, patients living with Inflammatory Bowel Disease were reluctant to disclose their illness due to lack of public awareness and stigma surrounding symptoms. From these, an overarching theme has been identified: Living in isolation and exclusion. CONCLUSION: Patients with Inflammatory Bowel Disease face a variety of problems, often their priorities and those of healthcare professionals differ greatly. Healthcare professionals have little evidence needed to provide adequate, holistic care to this group. With a rise in the Inflammatory Bowel Disease population in newly industrialised countries it is estimated that the condition is turning into a global disease, potentially making long term care unsustainable. More evidence is needed to understand the concerns of this group.
Gremlin 1 - small protein, big impact: the multiorgan consequences of disrupted BMP antagonism†.
Highly conserved, complex and interacting morphogen signalling pathways regulate adult stem cells and control cell fate determination across numerous different organs. In homeostasis, the bone morphogenetic protein (BMP) pathway predominantly promotes cell differentiation. Localised expression of ligand sequestering BMP antagonists, such as Gremlin 1 (Grem1), necessarily restricts BMP activity within the stem cell niche and facilitate stemness and self-renewal. In a new paper, Rowan, Jahns et al show that acute deletion of Grem1 in adult mice, using a ubiquitous ROSA26-Cre recombinase, induced not only severe intestinal enteropathy but also hypocellular bone marrow failure suggestive of stem cell niche collapse in both tissues. Grem1 has an increasingly recognised pleiotrophic role in a number of organ systems and is implicated across a wide range of disease states. Although the importance of Grem1 in intestinal stem cell regulation has been well described, a putative function in haematopoietic niche maintenance is novel and requires further exploration. Moreover, the complex and context-specific regulation of Grem1, among a host of functionally convergent but structurally disparate BMP antagonists, warrants further research as we learn more about the pathogenic consequences of deranged expression of this small, but important, protein. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Double Relapsed and/or Refractory Multiple Myeloma: Clinical Outcomes and Real World Healthcare Costs.
Double relapsed and/or refractory multiple myeloma (DRMM), MM that is relapsed and/or refractory to bortezomib and lenalidomide, carries a poor prognosis. The healthcare costs of DRMM have not previously been reported. We analyzed detailed medical resource utilization (MRU) costs, drug costs and outcomes for 39 UK patients receiving standard DRMM therapy. Median OS in this cohort was 5.6 months. The mean cost of DRMM treatment plus MRU until death was £23,472 [range: £1,411-£90,262], split between drug costs £11,191 and other resource use costs £12,281. The cost per assumed quality-adjusted life year (QALY) during DRMM was £66,983. These data provide a standard of care comparison when evaluating the cost-effectiveness of new drugs in DRMM.
Gemcitabine-induced large vessel vasculitis demonstrated by PET CT: a rare, important side effect
Gemcitabine is a nucleoside analogue used widely across haemato-oncology. Side effects are generally predictable, and typically consist of cytopenia, nausea, and infection. As the present case clearly demonstrates, gemcitabine is in rare cases associated with life-threatening large vessel vasculitis, which can involve the aorta. It is important to consider gemcitabine-induced vasculitis in non-specifically unwell patients with raised inflammatory markers and fever of unknown origin, with or without signs of vascular compromise. Early recognition, cessation of gemcitabine therapy, and high-dose steroids are critical for a good outcome. PET CT is valuable to diagnose large vessel vasculitis and monitor treatment response. © 2014 The Japanese Society of Hematology.