Case report: Refractory Evans syndrome in two patients with spondyloenchondrodysplasia with immune dysregulation treated successfully with JAK1/JAK2 inhibition.

Biallelic mutations in the ACP5 gene cause spondyloenchondrodysplasia with immune dysregulation (SPENCDI). SPENCDI is characterized by the phenotypic triad of skeletal dysplasia, innate and adaptive immune dysfunction, and variable neurologic findings ranging from asymptomatic brain calcifications to severe developmental delay with spasticity. Immune dysregulation in SPENCDI is often refractory to standard immunosuppressive treatments. Here, we present the cases of two patients with SPENCDI and recalcitrant autoimmune cytopenias who demonstrated a favorable clinical response to targeted JAK inhibition over a period of more than 3 years. One of the patients exhibited steadily rising IgG levels and a bone marrow biopsy revealed smoldering multiple myeloma. A review of the literature uncovered that approximately half of the SPENCDI patients reported to date exhibited increased IgG levels. Screening for multiple myeloma in SPENCDI patients with rising IgG levels should therefore be considered.

Congenital anemia reveals distinct targeting mechanisms for master transcription factor GATA1.

Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.

The active component of ginseng, ginsenoside Rb1, improves erythropoiesis in models of Diamond-Blackfan anemia by targeting Nemo-like kinase.

Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond-Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1-mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3'-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1-mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3'-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase.

SLC25A38 congenital sideroblastic anemia: Phenotypes and genotypes of 31 individuals from 24 families, including 11 novel mutations, and a review of the literature.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited disorders of erythropoiesis characterized by pathologic deposits of iron in the mitochondria of developing erythroblasts. Mutations in the mitochondrial glycine carrier SLC25A38 cause the most common recessive form of CSA. Nonetheless, the disease is still rare, there being fewer than 70 reported families. Here we describe the clinical phenotype and genotypes of 31 individuals from 24 families, including 11 novel mutations. We also review the spectrum of reported mutations and genotypes associated with the disease, describe the unique localization of missense mutations in transmembrane domains and account for the presence of several alleles in different populations.

Pyruvate kinase deficiency in children.

BACKGROUND: Pyruvate kinase deficiency (PKD) is a rare, autosomal recessive red blood cell enzyme disorder, which leads to lifelong hemolytic anemia and associated complications from the disease and its management. METHODS: An international, multicenter registry enrolled 124 individuals younger than 18 years old with molecularly confirmed PKD from 29 centers. Retrospective and prospective clinical data were collected. RESULTS: There was a wide range in the age at diagnosis from 0 to 16 years. Presentation in the newborn period ranged from asymptomatic to neonatal jaundice to fulminant presentations of fetal distress, myocardial depression, and/or liver failure. Children <5 years old were significantly more likely to be transfused than children >12 to <18 years (53% vs. 14%, p = .0006), which correlated with the timing of splenectomy. Regular transfusions were most common in children with two severe PKLR variants. In regularly transfused children, the nadir hemoglobin goal varied considerably. Impact on quality of life was a common reason for treatment with regular blood transfusions and splenectomy. Splenectomy increased the hemoglobin and decreased transfusion burden in most children but was associated with infection or sepsis (12%) and thrombosis (1.3%) even during childhood. Complication rates were high, including iron overload (48%), perinatal complications (31%), and gallstones (20%). CONCLUSIONS: There is a high burden of disease in children with PKD, with wide practice variation in monitoring and treatment. Clinicians must recognize the spectrum of the manifestations of PKD for early diagnostic testing, close monitoring, and management to avoid serious complications in childhood.

L-leucine improves anemia and growth in patients with transfusion-dependent Diamond-Blackfan anemia: Results from a multicenter pilot phase I/II study from the Diamond-Blackfan Anemia Registry.

BACKGROUND: Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, short stature, congenital anomalies, and cancer predisposition. Most cases are due to mutations in genes encoding ribosomal proteins (RP) leading to RP haploinsufficiency. Effective treatments for the anemia of DBA include chronic red cell transfusions, long-term corticosteroid therapy, or hematopoietic stem cell transplantation. In a small patient series and in animal models, there have been hematologic responses to L-leucine with amelioration of anemia. The study objectives of this clinical trial were to determine feasibility, safety, and efficacy of L-leucine in transfusion-dependent patients with DBA. PROCEDURE: Patients ≥2 years of age received L-leucine 700 mg/m2 orally three times daily for nine months to determine a hematologic response and any improvement in growth (NCT01362595). RESULTS: This multicenter, phase I/II study enrolled 55 subjects; 43 were evaluable. There were 21 males; the median age at enrollment was 10.4 years (range, 2.5-46.1 years). No significant adverse events were attributable to L-leucine. Two subjects had a complete erythroid response and five had a partial response. Nine of 25, and 11 of 25, subjects experienced a positive weight and height percentile change, respectively, at the end of therapy. CONCLUSIONS: L-leucine is safe, resulted in an erythroid response in 16% of subjects with DBA, and led to an increase in weight and linear growth velocity in 36% and 44% of evaluable subjects, respectively. Further studies will be critical to understand the role of L-leucine in the management of patients with DBA.

The variable manifestations of disease in pyruvate kinase deficiency and their management.

Pyruvate kinase deficiency (PKD) is the most common cause of chronic hereditary non-spherocytic hemolytic anemia and results in a broad spectrum of disease. The diagnosis of PKD requires a high index of suspicion and judicious use of laboratory tests that may not always be informative, including pyruvate kinase enzyme assay and genetic analysis of the PKLR gene. A significant minority of patients with PKD have occult mutations in non-coding regions of PKLR which are missed on standard genetic tests. The biochemical consequences of PKD result in hemolytic anemia due to red cell pyruvate and ATP deficiency while simultaneously causing increased red cell 2,3-diphosphoglycerate, which facilitates oxygen unloading. This phenomenon, in addition to numerous other factors such as genetic background and differences in splenic function result in a poor correlation between symptoms and degree of anemia from patient to patient. Red cell transfusions should, therefore, be symptom-directed and not based on a hemoglobin threshold. Patients may experience specific complications, such as paravertebral extramedullary hematopoiesis and chronic debilitating icterus, which require personalized treatment. The decision to perform splenectomy or hematopoietic stem cell transplantation is nuanced and depends on disease burden and long-term outlook given that targeted therapeutics are in development. In recognition of the complicated nature of the disease and its management and the limitations of the PKD literature, an international working group of ten PKD experts convened to better define the disease burden and manifestations. This article summarizes the conclusions of this working group and is a guide for clinicians and investigators caring for patients with PKD.

MMP9 inhibition increases erythropoiesis in RPS14-deficient del(5q) MDS models through suppression of TGF-ß pathways.

The del(5q) myelodysplastic syndrome (MDS) is a distinct subtype of MDS, associated with deletion of the ribosomal protein S14 (RPS14) gene that results in macrocytic anemia. This study sought to identify novel targets for the treatment of patients with del(5q) MDS by performing an in vivo drug screen using an rps14-deficient zebrafish model. From this, we identified the secreted gelatinase matrix metalloproteinase 9 (MMP9). MMP9 inhibitors significantly improved the erythroid defect in rps14-deficient zebrafish. Similarly, treatment with MMP9 inhibitors increased the number of colony forming unit-erythroid colonies and the CD71+ erythroid population from RPS14 knockdown human BMCD34+ cells. Importantly, we found that MMP9 expression is upregulated in RPS14-deficient cells by monocyte chemoattractant protein 1. Double knockdown of MMP9 and RPS14 increased the CD71+ population compared with RPS14 single knockdown, suggesting that increased expression of MMP9 contributes to the erythroid defect observed in RPS14-deficient cells. In addition, transforming growth factor ß (TGF-ß) signaling is activated in RPS14 knockdown cells, and treatment with SB431542, a TGF-ß inhibitor, improved the defective erythroid development of RPS14-deficient models. We found that recombinant MMP9 treatment decreases the CD71+ population through increased SMAD2/3 phosphorylation, suggesting that MMP9 directly activates TGF-ß signaling in RPS14-deficient cells. Finally, we confirmed that MMP9 inhibitors reduce SMAD2/3 phosphorylation in RPS14-deficient cells to rescue the erythroid defect. In summary, these study results support a novel role for MMP9 in the pathogenesis of del(5q) MDS and the potential for the clinical use of MMP9 inhibitors in the treatment of patients with del(5q) MDS.

Immunosuppressive therapy for pediatric aplastic anemia: a North American Pediatric Aplastic Anemia Consortium study.

Quality of response to immunosuppressive therapy and long-term outcomes for pediatric severe aplastic anemia remain incompletely characterized. Contemporary evidence to inform treatment of relapsed or refractory severe aplastic anemia for pediatric patients is also limited. The clinical features and outcomes for 314 children treated from 2002 to 2014 with immunosuppressive therapy for acquired severe aplastic anemia were analyzed retrospectively from 25 institutions in the North American Pediatric Aplastic Anemia Consortium. The majority of subjects (n=264) received horse anti-thymocyte globulin (hATG) plus cyclosporine (CyA) with a median 61 months follow up. Following hATG/CyA, 71.2% (95%CI: 65.3,76.6) achieved an objective response. In contrast to adult studies, the quality of response achieved in pediatric patients was high, with 59.8% (95%CI: 53.7,65.8) complete response and 68.2% (95%CI: 62.2,73.8) achieving at least a very good partial response with a platelet count ≥50×109L. At five years post-hATG/CyA, overall survival was 93% (95%CI: 89,96), but event-free survival without subsequent treatment was only 64% (95%CI: 57,69) without a plateau. Twelve of 171 evaluable patients (7%) acquired clonal abnormalities after diagnosis after a median 25.2 months (range: 4.3-71 months) post treatment. Myelodysplastic syndrome or leukemia developed in 6 of 314 (1.9%). For relapsed/refractory disease, treatment with a hematopoietic stem cell transplant had a superior event-free survival compared to second immunosuppressive therapy treatment in a multivariate analysis (HR=0.19, 95%CI: 0.08,0.47; P=0.0003). This study highlights the need for improved therapies to achieve sustained high-quality remission for children with severe aplastic anemia.

Hb Adana (HBA2 or HBA1: c.179G > A) and alpha thalassemia: Genotype-phenotype correlation.

Alpha thalassemia due to nondeletional mutations usually leads to more severe disease than that caused by deletional mutations. Devastating outcomes such as hydrops fetalis can occur with two nondeletional mutations, therefore warranting DNA-based workup for suspected carriers with subtle hematological abnormalities for family counseling purposes. We describe three cases with hemoglobin (Hb) Adana, a nondeletional alpha chain mutation, compounded with an alpha globin gene deletion resulting in thalassemia intermedia. We review the literature, draw genotype-phenotype correlations from published cases of Hb Adana, and propose that this correlation can be used by clinicians to help direct diagnostic studies and urge hematologists to thoroughly workup high-risk patients.

Loss of Forkhead box M1 promotes erythropoiesis through increased proliferation of erythroid progenitors.

Forkhead box M1 (FOXM1) belongs to the forkhead/winged-helix family of transcription factors and regulates a network of proliferation-associated genes. Its abnormal upregulation has been shown to be a key driver of cancer progression and an initiating factor in oncogenesis. FOXM1 is also highly expressed in stem/progenitor cells and inhibits their differentiation, suggesting that FOXM1 plays a role in the maintenance of multipotency. However, the exact molecular mechanisms by which FOXM1 regulates human stem/progenitor cells are still uncharacterized. To understand the role of FOXM1 in normal hematopoiesis, human cord blood CD34+ cells were transduced with FOXM1 short hairpin ribonucleic acid (shRNA) lentivirus. Knockdown of FOXM1 resulted in a 2-fold increase in erythroid cells compared to myeloid cells. Additionally, knockdown of FOXM1 increased bromodeoxyuridine (BrdU) incorporation in erythroid cells, suggesting greater proliferation of erythroid progenitors. We also observed that the defective phosphorylation of FOXM1 by checkpoint kinase 2 (CHK2) or cyclin-dependent kinases 1/2 (CDK1/2) increased the erythroid population in a manner similar to knockdown of FOXM1. Finally, we found that an inhibitor of FOXM1, forkhead domain inhibitor-6 (FDI-6), increased red blood cell numbers through increased proliferation of erythroid precursors. Overall, our data suggest a novel function of FOXM1 in normal human hematopoiesis.

Hematologic outcomes after total splenectomy and partial splenectomy for congenital hemolytic anemia.

PURPOSE: The purpose of this study was to define the hematologic response to total splenectomy (TS) or partial splenectomy (PS) in children with hereditary spherocytosis (HS) or sickle cell disease (SCD). METHODS: The Splenectomy in Congenital Hemolytic Anemia (SICHA) consortium registry collected hematologic outcomes of children with CHA undergoing TS or PS to 1 year after surgery. Using random effects mixed modeling, we evaluated the association of operative type with change in hemoglobin, reticulocyte counts, and bilirubin. We also compared laparoscopic to open splenectomy. RESULTS: The analysis included 130 children, with 62.3% (n=81) undergoing TS. For children with HS, all hematologic measures improved after TS, including a 4.1g/dl increase in hemoglobin. Hematologic parameters also improved after PS, although the response was less robust (hemoglobin increase 2.4 g/dl, p<0.001). For children with SCD, there was no change in hemoglobin. Laparoscopy was not associated with differences in hematologic outcomes compared to open. TS and laparoscopy were associated with shorter length of stay. CONCLUSION: Children with HS have an excellent hematologic response after TS or PS, although the hematologic response is more robust following TS. Children with SCD have smaller changes in their hematologic parameters. These data offer guidance to families and clinicians considering TS or PS.

Clinical outcomes of splenectomy in children: report of the splenectomy in congenital hemolytic anemia registry.

The outcomes of children with congenital hemolytic anemia (CHA) undergoing total splenectomy (TS) or partial splenectomy (PS) remain unclear. In this study, we collected data from 100 children with CHA who underwent TS or PS from 2005 to 2013 at 16 sites in the Splenectomy in Congenital Hemolytic Anemia (SICHA) consortium using a patient registry. We analyzed demographics and baseline clinical status, operative details, and outcomes at 4, 24, and 52 weeks after surgery. Results were summarized as hematologic outcomes, short-term adverse events (AEs) (≤30 days after surgery), and long-term AEs (31-365 days after surgery). For children with hereditary spherocytosis, after surgery there was an increase in hemoglobin (baseline 10.1 ± 1.8 g/dl, 52 week 12.8 ± 1.6 g/dl; mean ± SD), decrease in reticulocyte and bilirubin as well as control of symptoms. Children with sickle cell disease had control of clinical symptoms after surgery, but had no change in hematologic parameters. There was an 11% rate of short-term AEs and 11% rate of long-term AEs. As we accumulate more subjects and longer follow-up, use of a patient registry should enhance our capacity for clinical trials and engage all stakeholders in the decision-making process.

TNF-mediated inflammation represses GATA1 and activates p38 MAP kinase in RPS19-deficient hematopoietic progenitors.

Diamond-Blackfan anemia (DBA) is an inherited disorder characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of tumor necrosis factor-α (TNF-α) in nonerythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF-α or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF-α inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-α, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA.

Novel protocol including liver biopsy to identify and treat CD8+ T-cell predominant acute hepatitis and liver failure.

In the majority of children with ALF, the etiology is unknown and liver transplantation is often needed for survival. A patient case prompted us to consider that immune dysregulation may be the cause of indeterminate acute hepatitis and liver failure in children. Our study includes nine pediatric patients treated under a multidisciplinary clinical protocol to identify and treat immune-mediated acute liver injury. Patients with evidence of inflammation and no active infection on biopsy received treatment with intravenous immune globulin and methylprednisolone. Seven patients had at least one positive immune marker before or after treatment. All patients had a CD8+ T-cell predominant liver injury that completely or partially responded to immune therapy. Five of the nine patients recovered liver function and did not require liver transplantation. Three of these patients subsequently developed bone marrow failure and were treated with either immunosuppression or stem cell transplant. This series highlights the importance of this tissue-based approach to diagnosis and treatment that may improve transplant-free survival. Further research is necessary to better characterize the immune injury and to predict the subset of patients at risk for bone marrow failure who may benefit from earlier and stronger immunosuppressive therapy.

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia.

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis - a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.

Immune thrombocytopenia in children less than 1 year of age: a single-institution 10-year experience.

Immune thrombocytopenia (ITP) in children less than one year of age is less well characterized compared to ITP in toddlers and school-age children. We performed a 10-year retrospective review of ITP patients in this age-cohort at our institution. Diagnosis and classification were made according to the 2009 International Working Group criteria. Fourteen infants were identified. Their bleeding scores were Grades 1 to 2 (79%), Grade 3 (22%), Grades 4 to 5 (0%). Eight patients received treatment with a 75% response rate. Three patients (21%) developed chronic ITP. These observations suggest that ITP in very young patients is similar to typical childhood ITP.