Diagnosis, treatment, and surveillance of Diamond-Blackfan anaemia syndrome: international consensus statement.

Diamond-Blackfan anaemia (DBA), first described over 80 years ago, is a congenital disorder of erythropoiesis with a predilection for birth defects and cancer. Despite scientific advances, this chronic, debilitating, and life-limiting disorder continues to cause a substantial physical, psychological, and financial toll on patients and their families. The highly complex medical needs of affected patients require specialised expertise and multidisciplinary care. However, gaps remain in effectively bridging scientific discoveries to clinical practice and disseminating the latest knowledge and best practices to providers. Following the publication of the first international consensus in 2008, advances in our understanding of the genetics, natural history, and clinical management of DBA have strongly supported the need for new consensus recommendations. In 2014 in Freiburg, Germany, a panel of 53 experts including clinicians, diagnosticians, and researchers from 27 countries convened. With support from patient advocates, the panel met repeatedly over subsequent years, engaging in ongoing discussions. These meetings led to the development of new consensus recommendations in 2024, replacing the previous guidelines. To account for the diverse phenotypes including presentation without anaemia, the panel agreed to adopt the term DBA syndrome. We propose new simplified diagnostic criteria, describe the genetics of DBA syndrome and its phenocopies, and introduce major changes in therapeutic standards. These changes include lowering the prednisone maintenance dose to maximum 0·3 mg/kg per day, raising the pre-transfusion haemoglobin to 9-10 g/dL independent of age, recommending early aggressive chelation, broadening indications for haematopoietic stem-cell transplantation, and recommending systematic clinical surveillance including early colorectal cancer screening. In summary, the current practice guidelines standardise the diagnostics, treatment, and long-term surveillance of patients with DBA syndrome of all ages worldwide.

Cell senescence and malignant transformation in the inherited bone marrow failure syndromes: Overlapping pathophysiology with therapeutic implications.

Fanconi anemia, telomeropathies and ribosomopathies are members of the inherited bone marrow failure syndromes, rare genetic disorders that lead to failure of hematopoiesis, developmental abnormalities, and cancer predisposition. While each disorder is caused by different genetic defects in seemingly disparate processes of DNA repair, telomere maintenance, or ribosome biogenesis, they appear to lead to a common pathway characterized by premature senescence of hematopoietic stem cells. Here we review the experimental data on senescence and inflammation underlying marrow failure and malignant transformation. We conclude with a critical assessment of current and future therapies targeting these pathways in inherited bone marrow failure syndromes patients.

Is COVID-19-associated cytokine storm distinct from non-COVID-19 secondary hemophagocytic lymphohistiocytosis?

Cytokine storm is an umbrella term that describes an inflammatory syndrome characterized by elevated levels of circulating cytokines and hyperactivation of innate and/or adaptive immune cells. One type of cytokine storm is hemophagocytic lymphohistiocytosis (HLH), which can be either primary or secondary. Severe COVID-19-associated pneumonia and acute respiratory distress syndrome (ARDS) can also lead to cytokine storm/cytokine release syndrome (CS/CRS) and, more rarely, meet criteria for the diagnosis of secondary HLH. Here, we review the immunobiology of primary and secondary HLH and examine whether COVID-19-associated CS/CRS can be discriminated from non-COVID-19 secondary HLH. Finally, we review differences in immunobiology between these different entities, which may inform both clinical diagnosis and treatment of patients.

Secondary hemophagocytic lymphohistiocytosis versus cytokine release syndrome in severe COVID-19 patients.

IMPACT STATEMENT: Severe COVID-19 associated pneumonia and acute respiratory distress syndrome has recently been described with life-threatening features of cytokine storm and loosely referred to as hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS). Although a recent report indicated favorable responses to the interleukin-1 receptor antagonist, anakinra in eight patients with COVID-19 secondary HLH diagnosed using the HScore calculation, others have suggested that the diagnosis of secondary HLH is uncommon and that the use of the HScore has limited value in guiding immunomodulatory therapy for COVID-19. Here, we provide additional perspective on this important controversy based upon comparisons between 14 COVID-19 cytokine storm patients and 10 secondary HLH patients seen immediately prior to the pandemic. We hypothesize that identification of HLH may relate to the severity or timing of cytokine release and suggest distinguishing between cytokine release syndrome and secondary HLH, reserving the latter term for cases fulfilling diagnostic criteria.

Large vessel stroke as initial presentation of thrombotic thrombocytopenic purpura.

A 67-year-old right-handed woman presented with dysarthria, left upper extremity weakness and right-sided neglect of 3 hours duration. Imaging of the brain revealed acute right middle cerebral artery stroke; however, tissue plasminogen activator could not be administered due to severe thrombocytopenia. A peripheral smear revealed schistocytes and the patient was treated empirically for thrombotic thrombocytopenic purpura (TTP) with therapeutic plasma exchange. An extensive workup revealed no embolic source or other cause for stroke, and a diagnosis of large vessel infarct secondary to TTP was made. After a prolonged hospital course, the patient had partial neurological recovery and was discharged to a rehabilitation facility. Although transient neurologic deficits due to small vessel occlusions are well described in TTP, large vessel infarct can occur as well. This diagnosis should be considered in patients presenting with concomitant stroke and thrombocytopenia, as untreated TTP is nearly always fatal.

Toward RNA Repair of Diamond Blackfan Anemia Hematopoietic Stem Cells.

Diamond blackfan anemia (DBA) is a well-known inherited bone marrow failure syndrome mostly caused by mutations in ribosomal protein (RP) genes but also rarely in the hematopoietic transcription factor gene, GATA1, or TSR2, a ribosomal protein (Rps26) chaperone gene. About 25% of patients have heterozygous mutations in the RPS19 gene, which leads to haploinsufficiency of Rps19 protein in most cases. However, some RPS19 missense mutations appear to act in a dominant negative fashion. DBA typically leads to a hypoplastic anemia that becomes apparent during the first year of life, and standard treatment includes steroids or red blood cell transfusions, each modality having attendant side effects. The only curative therapy is allogeneic stem-cell transplantation, but this option is limited to patients with a histocompatible donor. DBA-mutant embryonic, induced pluripotent, and hematopoietic stem cells all exhibit growth abnormalities that can be corrected by DNA gene transfer, suggesting the possibility of ex vivo autologous gene therapy. The authors have been interested in the application of spliceosome-mediated mRNA trans-splicing (SMaRT) technology to RNA repair of DBA stem cells. Compared with gene replacement or other RNA re-programming approaches, SMaRT has several potential advantages. First, delivery of the entire normal cDNA is unnecessary, thus minimizing the overall size of the construct for packaging into a viral delivery vector. Second, RNA transcription of the corrected gene relies on the cell's endogenous transcriptional, processing, and regulatory machinery, thereby ensuring faithful and contextual expression. Third, RNA trans-splicing employs the endogenous spliceosome enzymatic machinery present in nearly all cells. Fourth, RNA trans-splicing converts mutant transcripts into therapeutically useful mRNA, and thus may be capable of treating disorders caused by dominant negative mutations. This review critically assesses prospects for both gene and RNA repair in DBA stem cells.

Pomalidomide reverses γ-globin silencing through the transcriptional reprogramming of adult hematopoietic progenitors.

Current therapeutic strategies for sickle cell anemia are aimed at reactivating fetal hemoglobin. Pomalidomide, a third-generation immunomodulatory drug, was proposed to induce fetal hemoglobin production by an unknown mechanism. Here, we report that pomalidomide induced a fetal-like erythroid differentiation program, leading to a reversion of γ-globin silencing in adult human erythroblasts. Pomalidomide acted early by transiently delaying erythropoiesis at the burst-forming unit-erythroid/colony-forming unit-erythroid transition, but without affecting terminal differentiation. Further, the transcription networks involved in γ-globin repression were selectively and differentially affected by pomalidomide including BCL11A, SOX6, IKZF1, KLF1, and LSD1. IKAROS (IKZF1), a known target of pomalidomide, was degraded by the proteasome, but was not the key effector of this program, because genetic ablation of IKZF1 did not phenocopy pomalidomide treatment. Notably, the pomalidomide-induced reprogramming was conserved in hematopoietic progenitors from individuals with sickle cell anemia. Moreover, multiple myeloma patients treated with pomalidomide demonstrated increased in vivo γ-globin levels in their erythrocytes. Together, these data reveal the molecular mechanisms by which pomalidomide reactivates fetal hemoglobin, reinforcing its potential as a treatment for patients with ß-hemoglobinopathies.

p53-Independent cell cycle and erythroid differentiation defects in murine embryonic stem cells haploinsufficient for Diamond Blackfan anemia-proteins: RPS19 versus RPL5.

Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome caused by ribosomal protein haploinsufficiency. DBA exhibits marked phenotypic variability, commonly presenting with erythroid hypoplasia, less consistently with non-erythroid features. The p53 pathway, activated by abortive ribosome assembly, is hypothesized to contribute to the erythroid failure of DBA. We studied murine embryonic stem (ES) cell lines harboring a gene trap mutation in a ribosomal protein gene, either Rps19 or Rpl5. Both mutants exhibited ribosomal protein haploinsufficiency and polysome defects. Rps19 mutant ES cells showed significant increase in p53 protein expression, however, there was no similar increase in the Rpl5 mutant cells. Embryoid body formation was diminished in both mutants but nonspecifically rescued by knockdown of p53. When embryoid bodies were further differentiated to primitive erythroid colonies, both mutants exhibited a marked reduction in colony formation, which was again nonspecifically rescued by p53 inhibition. Cell cycle analyses were normal in Rps19 mutant ES cells, but there was a significant delay in the G2/M phase in the Rpl5 mutant cells, which was unaffected by p53 knockdown. Concordantly, Rpl5 mutant ES cells had a more pronounced growth defect in liquid culture compared to the Rps19 mutant cells. We conclude that the defects in our RPS19 and RPL5 haploinsufficient mouse ES cells are not adequately explained by p53 stabilization, as p53 knockdown appears to increase the growth and differentiation potential of both parental and mutant cells. Our studies demonstrate that gene trap mouse ES cells are useful tools to study the pathogenesis of DBA.

Diminutive somatic deletions in the 5q region lead to a phenotype atypical of classical 5q- syndrome.

Classical 5q- syndrome is an acquired macrocytic anemia of the elderly. Similar to Diamond Blackfan anemia (DBA), an inherited red cell aplasia, the bone marrow is characterized by a paucity of erythroid precursors. RPS14 deletions in combination with other deletions in the region have been implicated as causative of the 5q- syndrome phenotype. We asked whether smaller, less easily detectable deletions could account for a syndrome with a modified phenotype. We employed single-nucleotide polymorphism array genotyping to identify small deletions in patients diagnosed with DBA and other anemias lacking molecular diagnoses. Diminutive mosaic deletions involving RPS14 were identified in a 5-year-old patient with nonclassical DBA and in a 17-year-old patient with myelodysplastic syndrome. Patients with nonclassical DBA and other hypoproliferative anemias may have somatically acquired 5q deletions with RPS14 haploinsufficiency not identified by fluorescence in situ hybridization or cytogenetic testing, thus refining the spectrum of disorders with 5q- deletions.

Mitochondrial function is impaired in yeast and human cellular models of Shwachman Diamond syndrome.

Shwachman Diamond syndrome (SDS) is an inherited bone marrow failure syndrome typically characterized by neutropenia, exocrine pancreas dysfunction, metaphyseal chondrodysplasia, and predisposition to myelodysplastic syndrome and leukemia. SBDS, the gene affected in most cases of SDS, encodes a protein known to influence many cellular processes including ribosome biogenesis, mitotic spindle assembly, chemotaxis, and the regulation of reactive oxygen species production. The best characterized role for the SBDS protein is in the production of functional 60S ribosomal subunits. Given that a reduction in functional 60S subunits could impact on the translational output of cells depleted of SBDS we analyzed protein synthesis in yeast cells lacking SDO1, the ortholog of SBDS. Cells lacking SDO1 selectively increased the synthesis of POR1, the ortholog of mammalian VDAC1 a major anion channel of the mitochondrial outer membrane. Further studies revealed the cells lacking SDO1 were compromised in growth on non-fermentable carbon sources suggesting mitochondrial function was impaired. These observations prompted us to examine mitochondrial function in human cells where SBDS expression was reduced. Our studies indicate that reduced expression of SBDS decreases mitochondrial membrane potential and oxygen consumption and increases the production of reactive oxygen species. These studies indicate that mitochondrial function is also perturbed in cells expressing reduced amounts of SBDS and indicate that disruption of mitochondrial function may also contribute to SDS pathophysiology.

Impaired growth, hematopoietic colony formation, and ribosome maturation in human cells depleted of Shwachman-Diamond syndrome protein SBDS.

BACKGROUND: Shwachman-Diamond syndrome (SDS), associated with SBDS mutations, is characterized by pancreatic exocrine dysfunction and marrow failure. Sdo1, the yeast ortholog of SBDS, is implicated in maturation of the 60S ribosomal subunit, with delayed export of 60S-like particles from the nucleoplasm when depleted. Sdo1 is needed for release of the anti-subunit association factor Tif6 from 60S subunits, and Tif6 may not be recycled to the nucleus when Sdo1 is absent. METHODS: To clarify the role of SBDS in human ribosome function, TF-1 erythroleukemia and A549 lung carcinoma cells were transfected with vectors expressing RNAi against SBDS. RESULTS: Growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to controls. To analyze the effect of SBDS on 60S subunit maturation in A549 cells, subunit localization was assessed by transfection with a vector expressing a fusion between human RPL29 and GFP: we found a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits. Polysome analysis of TF-1 knockdown cells showed a decrease in free 60S and 80S subunits. We also analyzed the levels of eIF6 (human ortholog of Tif6) following near-complete knockdown of SBDS in TF-1 cells and found an approximately 20% increase in the amount of eIF6 associated with the 60S subunit. CONCLUSIONS: We conclude that knockdown of SBDS leads to growth inhibition and defects in ribosome maturation, suggesting a role for wild-type SBDS in nuclear export of pre-60S subunits. Furthermore, knockdown of SBDS may interfere with eIF6 recycling.

A clinical algorithm predicts hematological complications in Shwachman-Diamond syndrome?

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in the SBDS gene in approximately 90% of cases. SDS is characterized by exocrine pancreatic insufficiency and bone marrow failure, which predisposes to the development of myelodysplastic syndrome and/or acute myeloid leukemia. In a new report, the French national cohort studied 102 SDS patients with a median follow-up of 11.6 years, focusing on the natural history of severe cytopenias. The authors concluded that SDS patients with a young age (<3 months) at first symptomatic presentation or cytopenia at diagnosis were at a high risk of subsequent severe hematological complications (either malignant or nonmalignant). Their findings raise the possibility that a clinical algorithm may predict the subsequent development of hematological complications in SDS.

Sixth International Congress on Shwachman-Diamond syndrome: from patients to genes and back.

At the Sixth International Congress on Shwachman-Diamond syndrome, held at the New York Academy of Sciences on June 28-30, 2011, researchers from around the world met to discuss the latest clinical and basic science relating to this puzzling condition.

Draft consensus guidelines for diagnosis and treatment of Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine insufficiency and bone marrow failure, often associated with neurodevelopmental and skeletal abnormalities. Mutations in the SBDS gene have been shown to cause SDS. The purpose of this document is to provide draft guidelines for diagnosis, evaluation of organ and system abnormalities, and treatment of hematologic, pancreatic, dietary, dental, skeletal, and neurodevelopmental complications. New recommendations regarding diagnosis and management are presented, reflecting advances in understanding the genetic basis and clinical manifestations of the disease based on the consensus of experienced clinicians from Canada, Europe, and the United States. Whenever possible, evidence-based conclusions are made, but as with other rare diseases, the data on SDS are often anecdotal. The authors welcome comments from readers.

Non-Diamond Blackfan anemia disorders of ribosome function: Shwachman Diamond syndrome and 5q- syndrome.

A number of human disorders, dubbed ribosomopathies, are linked to impaired ribosome biogenesis or function. These include but are not limited to Diamond Blackfan anemia (DBA), Shwachman Diamond syndrome (SDS), and the 5q- myelodysplastic syndrome (MDS). This review focuses on the latter two non-DBA disorders of ribosome function. Both SDS and 5q- syndrome lead to impaired hematopoiesis and a predisposition to leukemia. SDS, due to bi-allelic mutations of the SBDS gene, is a multi-system disorder that also includes bony abnormalities, and pancreatic and neurocognitive dysfunction. SBDS associates with the 60S subunit in human cells and has a role in subunit joining and translational activation in yeast models. In contrast, 5q- syndrome is associated with acquired haplo-insufficiency of RPS14, a component of the small 40S subunit. RPS14 is critical for 40S assembly in yeast models, and depletion of RPS14 in human CD34(+) cells is sufficient to recapitulate the 5q- erythroid defect. Both SDS and the 5q- syndrome represent important models of ribosome function and may inform future treatment strategies for the ribosomopathies.

Ectopic human mesenchymal stem cell-coated scaffolds in NOD/SCID mice: an in vivo model of the leukemia niche.

Human mesenchymal stem cells form the supportive structure in which the functional cells of a differentiated tissue reside. We describe the creation of ectopic niches within polyurethane scaffolds coated with human mesenchymal stem cells. When implanted subcutaneously in NOD/SCID mice, these niches supported engraftment of primary human acute myeloid leukemia cells. The scaffolds showed vascularization and presence of osteoclasts and adipocytes, suggestive of an organizing human bone marrow microenvironment in the murine host. The chemokine stromal-derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 are critical for homing and migration of acute myeloid leukemia. We found that a CXCR4 antagonist could disrupt homing to the ectopic niches, possibly by modulation of the mesenchymal stroma. We believe that these scaffold niches provide a new and powerful tool to study the leukemia stem cell microenvironment and may be useful for identification of novel drug targets.

Distinct ribosome maturation defects in yeast models of Diamond-Blackfan anemia and Shwachman-Diamond syndrome.

BACKGROUND: Diamond-Blackfan anemia and Shwachman-Diamond syndrome are inherited bone marrow failure syndromes linked to defects in ribosome synthesis. The purpose of this study was to determine whether yeast models for Diamond-Blackfan anemia and Shwachman-Diamond syndrome differed in the mechanism by which ribosome synthesis was affected. DESIGN AND METHODS: Northern blotting, pulse-chase analysis, and polysome profiling were used to study ribosome synthesis in yeast models. Localization of 60S ribosomal subunits was assessed using RPL25eGFP. RESULTS: Relative to wild-type controls, each disease model showed defects in 60S subunit maturation, but with distinct underlying mechanisms. In the model of Diamond-Blackfan anemia, 60S subunit maturation was disrupted at a relatively early stage with abortive complexes subject to rapid degradation. 5S ribosomal RNA, unlike other large subunit ribosomal RNA in this model, accumulated as an extra-ribosomal species. In contrast, subunit maturation in the Shwachman-Diamond syndrome model was affected at a later step, giving rise to relatively stable pre-60S particles with associated 5S ribosomal RNA retained in the nucleus. Conclusions These differences between the yeast Diamond-Blackfan anemia and Shwachman-Diamond syndrome models have implications for signaling mechanisms linking abortive ribosome assembly to cell fate decisions and may contribute to the divergent clinical presentations of Diamond-Blackfan anemia and Shwachman-Diamond syndrome.

Depletion of the Shwachman-Diamond syndrome gene product, SBDS, leads to growth inhibition and increased expression of OPG and VEGF-A.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure and leukemia predisposition, pancreatic exocrine dysfunction, and skeletal abnormalities, manifesting as skeletal dysplasia and osteoporosis. Mutations in SBDS have been shown to cause SDS, but the function of the SBDS gene product is unclear. Accelerated angiogenesis has recently been described in bone marrow cells from SDS patients. To clarify the unknown function of SBDS, we performed experiments analyzing the cellular effects of depleting SBDS by RNA interference. The growth of HeLa cells constitutively depleted of SBDS was markedly hindered when compared to cells stably transfected with siRNA against an irrelevant control gene. Similarly, growth of HeLa cells induced to express siRNA against SBDS was specifically inhibited. Inducible SBDS knockdown was associated with modestly increased levels of apoptosis, suggesting a partial contribution of this process to growth inhibition. By microarray analysis of knockdown cells, we found marked differences in expression of genes in multiple pathways, and we chose to examine a selected subset more closely using quantitative PCR arrays. In constitutive and inducible SBDS-depleted HeLa cell clones, we found 3- to 6-fold elevated mRNA levels of osteoprotegerin (OPG or TNFRSF11B) and vascular endothelial growth factor-A (VEGF-A). We confirmed significant overexpression of both secreted proteins by ELISA from supernatants of SBDS-depleted HeLa cells. Osteoprotegerin and VEGF-A are known to have diverse effects on osteoclast differentiation, angiogenesis, and monocyte/macrophage migration, all processes that may be aberrant in SDS, and we propose that overexpression of these factors may contribute to its pathology.

Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference.

Diamond Blackfan anaemia (DBA) is a rare, genetically and clinically heterogeneous, inherited red cell aplasia. Classical DBA affects about seven per million live births and presents during the first year of life. However, as mutated genes have been discovered in DBA, non-classical cases with less distinct phenotypes are being described in adults as well as children. In caring for these patients it is often difficult to have a clear understanding of the treatment options and their outcomes because of the lack of complete information on the natural history of the disease. The purpose of this document is to review the criteria for diagnosis, evaluate the available treatment options, including corticosteroid and transfusion therapies and stem cell transplantation, and propose a plan for optimizing patient care. Congenital anomalies, mode of inheritance, cancer predisposition, and pregnancy in DBA are also reviewed. Evidence-based conclusions will be made when possible; however, as in many rare diseases, the data are often anecdotal and the recommendations are based upon the best judgment of experienced clinicians. The recommendations regarding the diagnosis and management described in this report are the result of deliberations and discussions at an international consensus conference.