The Genetic Landscape of Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.

The Pathophysiology of Acquired Aplastic Anemia: Current Concepts Revisited.

Idiopathic acquired aplastic anemia is a rare, life-threatening bone marrow failure syndrome characterized by cytopenias and hypocellular bone marrow. The pathophysiology is unknown; the most favored model is of a dysregulated immune system leading to autoreactive T-cell destruction of hematopoietic stem and progenitor cells in a genetically susceptible host. The authors review the literature and propose that the major driver of acquired aplastic anemia is a combination of hematopoietic stem and progenitor cells intrinsic defects and an inappropriately activated immune response in the setting of a viral infection. Alterations in bone marrow microenvironment may also contribute to the disease process.

Cholesterol: the debate should be terminated.

Here, I offer personal perspectives on cholesterol homeostasis that reflect my belief that certain aspects of the debate have been overstated.-Nathan, D. G. Cholesterol: the debate should be terminated.

NNKTT120, an anti-iNKT cell monoclonal antibody, produces rapid and sustained iNKT cell depletion in adults with sickle cell disease.

Invariant NKT (iNKT) cells can be activated to stimulate a broad inflammatory response. In murine models of sickle cell disease (SCD), interruption of iNKT cell activity prevents tissue injury from vaso-occlusion. NKTT120 is an anti-iNKT cell monoclonal antibody that has the potential to rapidly and specifically deplete iNKT cells and, potentially, prevent vaso-occlusion. We conducted an open-label, multi-center, single-ascending-dose study of NKTT120 to determine its pharmacokinetics, pharmacodynamics and safety in steady-state patients with SCD. Doses were escalated in a 3+3 study design over a range from 0.001 mg/kg to 1.0 mg/kg. Twenty-one adults with SCD were administered NKTT120 as part of 7 dose cohorts. Plasma levels of NKTT120 predictably increased with higher doses. Median half-life of NKTT120 was 263 hours. All subjects in the higher dose cohorts (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg) demonstrated decreased iNKT cells below the lower limit of quantification within 6 hours after infusion, the earliest time point at which they were measured. In those subjects who received the two highest doses of NKTT120 (0.3, 1 mg/kg), iNKT cells were not detectable in the peripheral blood for a range of 2 to 5 months. There were no serious adverse events in the study deemed to be related to NKTT120. In adults with SCD, NKTT120 produced rapid, specific and sustained iNKT cell depletion without any infusional toxicity or attributed serious adverse events. The next step is a trial to determine NKTT120's ability to decrease rate of vaso-occlusive pain episodes. TRIAL REGISTRATION: clinicaltrials.gov NCT01783691.

Randomized phase 2 trial of regadenoson for treatment of acute vaso-occlusive crises in sickle cell disease.

Adenosine A2A receptor (A2AR) agonists have been shown to decrease tissue inflammation induced by hypoxia/reoxygenation in mice with sickle cell disease (SCD). The key mediator of the A2AR agonist's anti-inflammatory effects is a minor lymphocyte subset, invariant natural killer T (iNKT) cells. We tested the hypothesis that administration of an A2AR agonist in patients with SCD would decrease iNKT cell activation and dampen the severity of vaso-occlusive (VO) crises. In a phase 2, randomized, placebo-controlled trial, we administered a 48-hour infusion of the A2AR agonist regadenoson (1.44 µg/kg per hour) to patients with SCD during VO crises to produce a plasma concentration of ∼5 nM, a concentration known from prior studies to suppress iNKT cell activation in SCD. The primary outcome measure was a >30% reduction in the percentage of activated iNKT cells. Ninety-two patients with SCD were randomized to receive a 48-hour infusion of regadenoson or placebo, in addition to standard-of-care treatment, during hospital admission for a VO crisis and had analyzable iNKT cell samples. The proportion of subjects who demonstrated a reduction of >30% in activated iNKT cells was not significantly different between the regadenoson and placebo arms (43% vs 23%; P = .07). There were also no differences between regadenoson and placebo groups in length of hospital stay, mean total opioid use, or pain scores. These data demonstrate that a low-dose infusion of regadenoson intended to reduce the activity of iNKT cells is not sufficient to produce a statistically significant reduction in such activation or in measures of clinical efficacy. This trial was registered at www.clinicaltrials.gov as #NCT01788631.

Eulogy for the clinical research center.

The extramural General Clinical Research Center (GCRC) program has been funded for more than 50 years, first by the National Center for Research Resources, NIH, and more recently as part of the Clinical Translational Science Award (CTSA) program through the newly formed National Center for Advancing Translation Sciences (NCATS). The GCRCs represent the federally funded laboratories that employ a highly trained cadre of research nurses, dietitians, and other support staff and in which generations of clinical investigators trained and performed groundbreaking human studies that advanced medical science and improved clinical care. Without the opportunity for adequate discussion, NCATS has now stopped funding these Research Centers. In this "eulogy," we review the origins and history of the GCRCs, their contributions to the advancement of medicine, and the recent events that have essentially defunded them. We mourn their loss.

Thalassemia: a look to the future.

To assess the future of a clinical science one must first assess the rate of accrual of understanding in the past century. From the time of Cooley's description (1925) to 1964, the year of the first Symposium, progress was glacial because the molecular biology revolution in medicine was in its infancy and therapy other than transfusion was impossible. But between 1964 and 2015, progress has been huge on every front. Our patients ushered in the molecular biology revolution in medicine. They have benefited from far better understanding of molecular pathophysiology, substantial improvements in transfusion and chelation, more effective stem cell transplantation, the beginnings of gene therapy, and now major advances in our capacity to reinduce fetal hemoglobin. We have only lagged in the application of prevention technology in the less developed world that suffers the most from thalassemia and sickle cell disease. We must redouble our efforts to spare patients from these cruel diseases.

Amino acid uptake in erythropoiesis.

Erythroblasts produce huge amounts of hemoglobin, for which they must incorporate vast stores of iron and amino acids. In the 14 April issue of Science Signaling, Chung et al. describe the complex mechanism linking neutral amino acid uptake to hemoglobin production in these hungry cells.

Advances in sickle cell therapies in the hydroxyurea era.

In the hydroxyurea era, insights into mechanisms downstream of erythrocyte sickling have led to new therapeutic approaches for patients with sickle cell disease (SCD). Therapies have been developed that target vascular adhesion, inflammation and hemolysis, including innovative biologics directed against P-selectin and invariant natural killer T cells. Advances in hematopoietic stem cell transplant and gene therapy may also provide more opportunities for cures in the near future. Several clinical studies are underway to determine the safety and efficacy of these new treatments. Novel approaches to treat SCD are desperately needed, since current therapies are limited and rates of morbidity and mortality remain high.

The role of adenosine signaling in sickle cell therapeutics.

Data suggest a role for adenosine signaling in the pathogenesis of sickle cell disease (SCD). Signaling through the adenosine A2A receptor (A2AR) has demonstrated beneficial effects. Activation of A2ARs decreases inflammation with SCD by blocking activation of invariant natural killer T cells. Decreased inflammation may reduce the severity of vasoocclusive crises. Adenosine signaling through the adenosine A2B receptor (A2BR) may be detrimental in SCD. Whether adenosine signaling predominantly occurs through A2ARs or A2BRs may depend on differing levels of adenosine and disease state (steady state versus crisis). There may be opportunities to develop novel therapeutic approaches targeting A2ARs and/or A2BRs for patients with SCD.

NF-κB is activated in CD4+ iNKT cells by sickle cell disease and mediates rapid induction of adenosine A2A receptors.

Reperfusion injury following tissue ischemia occurs as a consequence of vaso-occlusion that is initiated by activation of invariant natural killer T (iNKT) cells. Sickle cell disease (SDC) results in widely disseminated microvascular ischemia and reperfusion injury as a result of vaso-occlusion by rigid and adhesive sickle red blood cells. In mice, iNKT cell activation requires NF-κB signaling and can be inhibited by the activation of anti-inflammatory adenosine A2A receptors (A2ARs). Human iNKT cells are divided into subsets of CD4+ and CD4- cells. In this study we found that human CD4+ iNKT cells, but not CD4- cells undergo rapid NF-κB activation (phosphorylation of NF-κB on p65) and induction of A2ARs (detected with a monoclonal antibody 7F6-G5-A2) during SCD painful vaso-occlusive crises. These findings indicate that SCD primarily activates the CD4+ subset of iNKT cells. Activation of NF-κB and induction of A2ARs is concordant, i.e. only CD4+ iNKT cells with activated NF-κB expressed high levels of A2ARs. iNKT cells that are not activated during pVOC express low levels of A2AR immunoreactivity. These finding suggest that A2AR transcription may be induced in CD4+ iNKT cells as a result of NF-κB activation in SCD. In order to test this hypothesis further we examined cultured human iNKT cells. In cultured cells, blockade of NF-κB with Bay 11-7082 or IKK inhibitor VII prevented rapid induction of A2AR mRNA and protein upon iNKT activation. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells may serve as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. As activated iNKT cells express high levels of A2ARs following their activation, they may become highly sensitive to inhibition by A2AR agonists.

Hemoglobin disorders: a look to the future.

In this issue of Blood, Locatelli et al compare the results of histocompatible family donor bone marrow and cord blood transplants (BMT and CBT) for severe ß thalassemia (SBT) and sickle cell disease (SCD) as experienced by the Eurocord and European Blood and Marrow Transplantation group and collaborating centers in the United States, Hong Kong, and Israel between 1994 and 2005. Obviously, many changes in medical care and particularly MHC typing occurred over that decade, so this retrospective represents a moving target, but some firm points can be made for which we are indebted to this excellent group.

Sickle cell vaso-occlusion causes activation of iNKT cells that is decreased by the adenosine A2A receptor agonist regadenoson.

Adenosine A2A receptor (A2AR) agonists reduce invariant natural killer T (iNKT) cell activation and decrease inflammation in sickle cell disease (SCD) mice. We conducted a phase 1 trial of the A2AR agonist regadenoson in adults with SCD. The target dose was 1.44 µg/kg/h. iNKT cell activation was evaluated using antibodies targeting the p65 subunit of nuclear factor-κB (phospho-NF-κB p65), interferon-γ (IFN-γ), and A2AR. Regadenoson was administered to 27 adults with SCD. We examined 21 patients at steady state and 6 during painful vaso-occlusive crises (pVOC). iNKT cell activation was also measured in 14 African-American controls. During pVOC, the fraction of iNKT cells demonstrating increased phospho-NF-κB p65 and A2AR expression was significantly higher compared with controls (P < .01) and steady-state patients (P < .05). IFN-γ expression was also significantly higher compared with controls (P = .02). After a 24-hour infusion of regadenoson during pVOC, phospho-NF-κB p65 activation in iNKT cells decreased compared to baseline by a median of 48% (P = .03) to levels similar to controls and steady-state SCD. No toxicities were identified. Infusional regadenoson administered to adults with SCD at 1.44 µg/kg/h during pVOC decreases activation of iNKT cells without toxicity.

Pathophysiology and Clinical Manifestations of the ß-Thalassemias.

The ß-thalassemia syndromes reflect deficient or absent ß-globin synthesis usually owing to a mutation in the ß-globin locus. The relative excess of α-globin results in the formation of insoluble aggregates leading to ineffective erythropoiesis and shortened red cell survival. A relatively high capacity for fetal hemoglobin synthesis is a major genetic modifier of disease severity, with polymorphisms in other genes also having a significant role. Iron overload secondary to enhanced absorption and red cell transfusions causes an increase in liver iron and in various other tissues, leading to endocrine and cardiac dysfunction. Modern chelation regimens are effective in removing iron and preserving or restoring organ function.