Symptoms Of Sickle Cell Disease Research Articles

Incidence of Vaso-Occlusive Crisis Does Not Increase with Achieving Higher Hemoglobin Levels on Voxelotor Treatment or after Discontinuation: Analyses of the HOPE Study

Incidence of Vaso-Occlusive Crisis Does Not Increase with Achieving Higher Hemoglobin Levels on Voxelotor Treatment or after Discontinuation: Analyses of the HOPE Study

Impact of Sickle Cell Disease Symptoms on Patients' Daily Lives: Interim Results from the International Sickle Cell World Assessment Survey (SWAY)

Impact of Sickle Cell Disease Symptoms on Patients' Daily Lives: Interim Results from the International Sickle Cell World Assessment Survey (SWAY)

Hydroxyurea Exposure throughout Pregnancy in Patients with Sickle-Cell Disease : 4 Case Reports from European Non-Interventional, Multicentric, Prospective Escort-HU Study

Over the last decades, the number of sickle-cell disease (SCD) women reaching childbearing age and planning to become pregnant is increasing due to improvements in the management of the disease.[1] Physiological changes of pregnancy, such as increased metabolic demand, increased blood viscosity and hyper-coagulability, lead to an increased incidence of complications, such as vaso-occlusive crisis (VOC), acute chest syndrome (ACS), osteonecrosis, hepatic necrosis, leg ulcers, and thromboembolic events. [2] Vaso-occlusion may also involve the placenta, with villi fibrosis, necrosis, and infarction leading to impaired uteroplacental circulation, with consequent chronic fetal hypoxia and adverse fetal outcomes. Therefore, prenatal care is important to evaluate if intensification of treatment as blood transfusion or Hydroxyurea (HU) may be required. HU is approved in EU and USA for VOC including ACS in patients with SCD aged 2 years and over. HU is not recommended for use during pregnancy, primarily because animal studies have suggested potential teratogenic effects on the fetus. In some rare cases, discontinuation of HU treatment to avoid fetal exposure is not possible. A few cases of exposure to HU throughout the pregnancy are published in the literature,[3] but none in SCD patients. We hereby present four cases in which HU was taken during all pregnancy collected in the ESCORT-HU study (European Sickle Cell Disease COhoRT - HydroxyUrea), a multicentric, prospective, non-interventional European study which collected long-term safety information on HU when used in current practice. Patient #1 (GxP2, SS) had a history of VOC, osteonecrosis and leg ulcers and was started on HU at age 18. She had two previous pregnancies without HU exposure. No oral contraception was taken and she spontaneously conceived at age 37. HU was continued at the same dose. Patient #2 (G5P1, Sβ+) had a history of VOC, chronic anemia and heart disease, and was started on HU at age 19. At age 34, she started to receive treatment for fertility (gonadotropin and follitropin). HU which had been started 17 years earlier was continued. Patient #3 (G5P1, Sβ+) had a history of chronic severe anemia and was started on HU at age 37. She had a gynecological history of 1 spontaneous abortion, 2 elective abortions and 1 live birth (pregnancy complicated with preeclampsia). She had been on oral contraception before becoming spontaneously pregnant when aged 38. HU, which had been initiated 11 months earlier, was continued at same dose. Patient #4 (SS, G3P0) female with history of stroke with sequelae and VOC, initiated on HU at the age of 25 following post-transfusion alloimmunization. Gynecological history included one spontaneous abortion and one elective abortion when aged 21 and 23 years (before initiation of HU). Following removal of contraceptive device, she conceived spontaneously at age 27. These four women suffered from severe SCD symptoms which required special management during pregnancies. HU exposure during pregnancy is not recommended but these case reports highlight the difficulty for medical teams to deal with both strong desire to become pregnant and maternal and fetal morbidity and mortality in SCD. Similarly to literature data in non-SCD patients, all four pregnancies in SCD women with HU exposure throughout pregnancy had a favorable outcome and no malformation was reported among the neonates. Details of pregnancy course and outcome are provided in Table 1. Although it is highly recommended to avoid any fetal exposure to HU, it can be difficult in some SCD patients with severe symptoms and a wish to become pregnant, to stop the treatment during pregnancy. In these cases, a strict monitoring of the mother and fetus is necessary. [1] Silva F.A.C., Ferreira A.L.C.G., Hazin-Costa M.F., Dias M.L.G., Araújo A.S., Souza A.I., Adverse clinical and obstetric outcomes among pregnant women with different sickle cell disease genotypes, International Federation of Gynecology and Obstetrics, 2018 [2] Jain D., Atmapoojya P., Colah R., Lodha P. Sickle cell disease and pregnancy. Mediterr J Hematol Infect Dis 2019, 11(1): e2019040 [3] C Thauvin-Robinet, Exposure to hydroxyurea during pregnancy: a case series, Correspondence 2001 Disclosures No relevant conflicts of interest to declare.

The Safety of Chelators for Iron Overload in Sickle Cell Disease: A Brief Systematic Review

Sickle cell disease (SCD) is a group of disorders that affects hemoglobin due to a mutation of the hemoglobin beta gene (HBB) on chromosome 11. Patients with SCD have atypical hemoglobin molecules called hemoglobinS (HbS), which distort erythrocytes into a “sickle-shape”. Typical symptoms of SCD include periodic episodes of pain, repeated infections, and anemia. This disorder is abundant in sub-Saharan African countries, the Mediterranean region, and also appears in some southern provinces in Turkey. Because of the high concentration of HbS in patients, a high risk of chronic anemia and vaso-occlusive events, such as stroke may deteriorate suddenly. In these conditions, transfusion of blood, especially erythrocytes, can be life-saving. However, chronic blood transfusions may lead to iron overload in SCD patients. Erythrocyte transfusion is associated with a higher risk in most patients with SCD than in the general population. Therefore, chelation therapy has become an important component of the transfusion program to prevent complications of iron accumulation in organs such as liver and heart. In this study, we sought to conduct a systematic review to assess the safety of iron chelating agents used by SCD patients with iron overload mainly due to necessary blood transfusion regime. Our evaluation revealed that in general iron chelation therapy, either deferasirox, deferoxamine or deferiprone, remains the most effective and safest available method to treat iron overload in SCD. Furthermore, current reports do not reflect any significant safety concerns against the use of available chelators.

Hemoglobin F or: How I Learned to Stop Wondering and Love the Flow Cytometer

Abstract Sickle cell disease (SCD) is the most common inherited blood disorder in the United States. It is a hemoglobinopathy that leads to red blood cell (RBC) sickling and a broad range of disease complications including vaso-occlusive crisis, acute chest syndrome, and retinopathy. Hydroxyurea, a drug used to treat SCD, is known to increase expression of hemoglobin F (HbF), a type of hemoglobin normally expressed in infancy; HbF levels between 10% and 20% are associated with decreased vaso-occlusive episodes and improved survival. Hereditary persistent hemoglobin F (HPHF), a typically asymptomatic hemoglobinopathy associated with sustained hemoglobin F (HbF) expression into adulthood (HbF >10%), in combination with SCD is associated with decreased complications. Laboratories typically determine the HbF level via high-performance liquid chromatography (HPLC). HbF levels approaching 30% on HPLC are thought to be protective against SCD complications. However, HbF may be found within a majority or minority of RBCs, pancellular (deletional HPHF) or heterocellular distribution (nondeletional HPHF), respectively. Additionally, the quantity of HbF within cells can range from low (<10 picograms/cell) to high (>35 picograms). We sought to determine the quantity and distribution of HbF required to protect against sickle cell disease symptoms both via traditional HPLC as well as flow cytometry. This retrospective study was conducted at a large academic medical center over a period of 2 months (January-February 2019). We collected blood from sickle cell patients that had a detectable HbF level on hemoglobin electrophoresis. We then stained RBCs from 16 of the patients for HbF and performed flow cytometry to examine the HbF distribution. We calculated the cellular concentration of HbF within each HbF+ cell using the formula (MHC × %HbF)/%F-cells. We performed a chart review to determine the native hemoglobin type, exposure to hydroxyurea, and clinical symptoms of sickle cell disease. We identified four patients over the age of 20 with HbS/HPHP and no exposure to hydroxyurea. Two of these patients experienced no sickle cell disease complications; the protected patients had heterocellular distribution of HbF, but had a high concentration of HbF per HbF+ cell (>35 picograms/cell). Notably, these asymptomatic patients both had HbF level by HPLC less than 30. One of the symptomatic HbS/HPHF patients had heterocellular expression of HbF with low cellular concentration (28 picograms/cell) while the other patient had pancellular HbF expression with very low cellular concentration (6.4 picograms/cell). Our study demonstrates that HPHF alone does not prevent sickle cell disease complications. Our study highlights the importance of quantifying the cellular concentration of HbF, which can provide useful information beyond that of HPLC. In addition, our study raises the potential of the clinical use of hydroxyurea in patients with sickle cell disease even in the presence of HPHF.

δ-Aminolevulinate induces fetal hemoglobin expression by enhancing cellular heme biosynthesis.

Sickle cell disease (SCD) and β-thalassemia are inherited blood disorders caused by genetic defects in the β-globin gene on chromosome 11, producing severe disease in people worldwide. Induction of fetal hemoglobin consisting of two α-globin and two γ-globin chains ameliorates the clinical symptoms of both disorders. In the present study, we investigated the ability of δ-aminolevulinate (ALA), the heme precursor, to activate γ-globin gene expression as well as its effects on cellular functions in erythroid cell systems. We demonstrated that ALA induced γ-globin expression at both the transcriptional and protein levels in the KU812 erythroid cell line. Using inhibitors targeting two enzymes in the heme biosynthesis pathway, we showed that cellular heme biosynthesis was involved in ALA-mediated γ-globin activation. Moreover, the transcription factor NRF2 (nuclear factor [erythroid-derived 2]-like 2), a critical regulator of the cellular antioxidant response, was activated by ALA and contributed to mechanisms of γ-globin activation; ALA did not affect cell proliferation and was not toxic to cells. Subsequent studies demonstrated ALA-induced γ-globin activation in erythroid progenitors generated from normal human CD34+ stem cells. These data support future study to explore the potential of stimulating intracellular heme biosynthesis by ALA or similar compounds as a novel therapeutic strategy for treating SCD and β-thalassemia. Impact statement Inherited mutations in the β-globin-like genes result in the most common forms of genetic blood disease including sickle cell disease (SCD) and β-thalassemia worldwide. Therefore, effective inexpensive therapies that can be distributed widely are highly desirable. Currently, drug-mediated fetal hemoglobin (HbF) induction can ameliorate clinical symptoms of SCD and β-thalassemia and is the most effective strategy for developing new therapeutic options. In the current study, we confirmed that δ-Aminolevulinate (ALA), the precursor of heme, induces γ-globin expression at both the transcriptional and translational levels in primary human erythroid progenitors. Moreover, the results indicate activation of the transcription factor NRF2 (nuclear factor (erythroid-derived 2)-like 2) by ALA to enhance HbF expression. These data support future study to explore the potential of stimulating intracellular heme biosynthesis by ALA or similar compounds as a novel therapeutic strategy for treating SCD and β-thalassemia.

Reliability and validity of PROMIS® pediatric family relationships short form in children 8-17years of age with chronic disease.

Families play a key role in managing pediatric chronic illness. The PROMIS® pediatric family relationships measure was developed primarily within the general pediatric population. We evaluated the Family Relationships short form in the context of pediatric chronic diseases. Children aged 8-17years with asthma (n = 73), type 1 diabetes (n = 122), or sickle cell disease (n = 80) completed the Family Relationships 8a short form and the PROMIS Pediatric Profile-25's six domains representing physical, mental, and social health. Parents (N = 275) of these children completed the parent versions of the same measures. We evaluated reliability of the Family Relationships measure using Cronbach's alpha and IRT-based marginal reliability, and the standard error of measurement (SEM). Convergent/discriminant validity were assessed from correlations between the Family Relationships domain and the PROMIS-25 domains. SEM increased for scores above the normative mean of 50. Cronbach's alpha and IRT-estimated marginal reliabilities exceeded 0.80 for children and parents across diseases, except in asthma, where marginal reliability was 0.75 for parents. Scores displayed small to large correlations in the expected directions with social and mental health domains. The largest correlations occurred with parents' proxy reports of children's depressive symptoms in sickle cell disease and asthma, r = - 0.60 (95% CI - 0.74, - 0.48) and r = - 0.58 (95% CI - 0.68, - 0.48) respectively. The Family Relationships 8-item short form demonstrated adequate reliability and convergent/discriminant validity for use in pediatric chronic conditions, though scores above the mean displayed greater uncertainty. Evidence of the measure's reliability and validity in multiple contexts furthers the case for its use.

Dietary supplementation with sulforaphane attenuates liver damage and heme overload in a sickle cell disease murine model

Sickle cell disease (SCD) is a recessively inherited blood disorder caused by abnormal β-globin production. The β-globin mutation changes erythrocyte morphology into a sickle shape and increases erythrocyte vulnerability to hemolysis. Oxidative stress and concomitant inflammation eventually result in damage to multiple organs. Nrf2 is a master regulator of the oxidative stress response, homeostasis, and metabolism. Keap1 modulates Nrf2 protein levels; Nrf2 inducers alter nuclear Nrf2 levels by interacting with Keap1. Genetic modification of Keap1 helps to reduce inflammation and tissue damage in SCD model mice through Nrf2 induction. Here, we investigated the benefits of a mild and safe Nrf2 agonist, sulforaphane (SFN), in ameliorating SCD pathology in a murine model. SFN is a phytochemical and is found in cruciferous vegetables as its inert precursor, glucoraphanin. We found that dietary SFN administration for 14 days or 2 months increased the expression of Nrf2-dependent cytoprotective genes, but SFN uptake did not have deleterious effects on the food consumption and growth of SCD model mice. SFN ameliorated the liver damage of SCD mice, which could be validated by the rescue of liver function and the significantly reduced liver necrotic area. SFN administration also helped to eliminate heme released from lysed sickle cells. These results indicate that dietary supplementation with SFN relieves SCD symptoms by inducing Nrf2 and support our contention that SFN is a potential drug for the long-term treatment of children with SCD.

PF731 A STRATEGY FOR THE MEASUREMENT OF SICKLE CELL DISEASE SYMPTOMS FROM THE PATIENT PERSPECTIVE

Please indicate where the abstract has been published before: Blood 2018Background:Sickle cell disease (SCD), an inherited blood disorder, impacts multiple organs leading to variable clinical presentation. In addition to severe pain episodes, patients may experience chronic symptoms (eg, pain, tiredness). Assessing treatment benefit using a patient‐reported outcome (PRO) measurement strategy is important in SCD research.Aims:To understand the SCD patient experience and inform the selection or development of fit‐for‐purpose PRO instrument(s) to assess daily chronic symptoms in clinical trials for adolescents and adults with SCD ≥16 years.Methods:A literature review and interviews with clinical experts (N = 5) and adolescents and adults with SCD (N = 20) were conducted to develop an SCD conceptual model summarizing important SCD signs, symptoms, and impacts. An instrument review identified and assessed the existing PRO measures used in SCD research and documented their development history and measurement properties. Concepts in the SCD conceptual model were compared to those assessed in reviewed PRO measures. Considerations for concept selection (eg, suitability of measuring symptoms via PRO measure) and item construction (eg, selection of recall period) in developing PRO measurement strategies were summarized.Results:Patients reported 22 symptoms that they experienced regularly and/or during pain crises, which were largely endorsed by the literature and/or clinical experts. Pain was reported by all patients, and tiredness was reported by 80%; both were reported as the most important to improve with treatment. Patients reported impacts of SCD on their daily lives across 9 domains (activities of daily living, cognitive, emotional, financial, leisure, physical, sleep, social, and work/school impacts).Five PRO measures used in SCD research were reviewed: The Adult Sickle Cell Quality of Life Measurement Information System (ASCQ‐MeSM), Pediatric Quality of Life Inventory (PedsQL™) sickle cell disease module, the Patient‐Reported Outcomes Measurement Information System (PROMIS) Fatigue and Pediatric Pain Interference measures, and Scribe Sickle Cell Record of Daily Symptom Burden™ (SSCRDSB). None of the measures assessed >5 of the symptoms included in the SCD conceptual model, with the SSCRDSB assessing the greatest number (5/22 concepts). Pain was the most frequently measured concept (assessed by 3/5 PRO measures). The most frequently measured impact domain was physical impacts, assessed to varying degrees by all 5 measures. The PedsQL™ SCD module assessed the greatest number of impact domains in the SCD conceptual model (7/9 domains). The reviewed measures were determined to have limitations (eg, inadequate concept coverage and recall period) in assessing SCD symptoms daily as part of regulated clinical trials.Summary/Conclusion:These results suggest that 1) patients experience several SCD symptoms regularly and intermittently, 2) existing instruments, while perhaps suitable for other contexts of use or in conjunction with other tools, may be inadequate for evaluating important SCD symptoms in a way that can demonstrate treatment benefit when measured daily in regulated clinical research. Instruments developed for this purpose should have documented evidence of content validity (ie, relevant instrument measures and understandable concepts) and should include clear instructions, a short recall period, items that measure dimensions of importance to patients (eg, symptom intensity), and well‐defined response options that are sufficiently sensitive to detect change.

Sickle Cell Disease-Genetics, Pathophysiology, Clinical Presentation and Treatment.

Sickle cell disease (SCD) is a monogenetic disorder due to a single base-pair point mutation in the β-globin gene resulting in the substitution of the amino acid valine for glutamic acid in the β-globin chain. Phenotypic variation in the clinical presentation and disease outcome is a characteristic feature of the disorder. Understanding the pathogenesis and pathophysiology of the disorder is central to the choice of therapeutic development and intervention. In this special edition for newborn screening for haemoglobin disorders, it is pertinent to describe the genetic, pathologic and clinical presentation of sickle cell disease as a prelude to the justification for screening. Through a systematic review of the literature using search terms relating to SCD up till 2019, we identified relevant descriptive publications for inclusion. The scope of this review is mainly an overview of the clinical features of pain, the cardinal symptom in SCD, which present following the drop in foetal haemoglobin as young as five to six months after birth. The relative impact of haemolysis and small-vessel occlusive pathology remains controversial, a combination of features probably contribute to the different pathologies. We also provide an overview of emerging therapies in SCD.

Targeted deletion of BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta thalassemia disease

Hemoglobinopathies, such as β-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of β-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous β-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of β-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating β-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.

Protein kinase C‐beta isoform as a mechanism promoting chronic pain in sickle cell disease

Pain is a hallmark symptom in sickle cell disease (SCD). Besides acute painful vaso‐occlusive crises, SCD is also accompanied by intractable chronic pain. This persistent, and often unrelieved, pain starts early in childhood and continues throughout life. The neurobiological mechanisms of chronic pain in SCD remain unclear, which limits effective pain management and the quality of life in patients with SCD. Taking advantage of a humanized mouse model of SCD, this study aimed to investigate protein kinase C (PKC)‐beta isoform mechanism for chronic pain in SCD. We characterized pain phenotypes in the Townes' mouse model (TOW) of SCD that exclusively expresses human alleles encoding normal α‐ and sickle β‐globin. TOW mice exhibited ongoing spontaneous pain as well as hypersensitivity to evoked pain stimuli compared with littermate control mice. We then examined spinal PKCbeta‐mediated signaling and behavioral consequences. Prominent activation of PKCbeta was identified in the superficial laminae of the spinal cord in TOW sickle mice. Functional inhibition and genetic silencing of the PKCbeta attenuated sickle cell pain phenotypes, abolishing PKCbeta activation, spontaneous pain, mechanical allodynia, and heat hyperalgesia in the sickle cell mice. Taken together, these data suggest PKCbeta may be one of the mechanisms for the generation and maintenance of ongoing and evoked pain in SCD. These findings offer new insights into sickle cell pain mechanisms, which may offer new pharmacological interventions.Support or Funding InformationR35 HL140031 and K99 HL133590This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of HBG Promoter DNA Methylation

Pharmacological or genetic induction of fetal hemoglobin (HbF, α2γ2) in adult red blood cells is a proven strategy to ameliorate the clinical symptoms of sickle cell disease (SCD) and β-thalassemia. Therefore, efforts are underway to better understand mechanisms that mediate the perinatal switch from HbF to adult hemoglobin (HbA, α2β2). We performed a CRISPR-Cas9/guide (g) RNA screen to identify novel proteins that regulate HbF production in HUDEP-2 cells, a human erythroid line that normally expresses HbA. We identified UHRF1 (ubiquitin-like with PHD and RING finger domains 1) as a repressor of HbF production. UHRF1 binds hemi-methylated DNA and recruit DNA methyltransferase 1 (DNMT1) to ensure faithful maintenance of DNA methylation during DNA replication. Numerous UHRF1-interacting proteins, including DNMT1, EHMT1/2 and HDAC2 are associated with γ-globin repression. We used CRISPR/Cas9 and RNA interference to validate UHRF1 as a HbF regulator. Compared to non-targeting gRNA UHRF1 disruption using Cas9 + 2 separate gRNAs increased the γ-globin/γ+β-globin RNA ratio from 1.9 to 25.8/27.1% (P<0.01), increased the fraction of HbF immunostaining cells (“F-cells”) from 7.5 to 25.1/35.4% and increased HbF protein from 2.10 to 16.3/15.0% (P<0.01) in HUDEP-2 cells. Compared to a control luciferase shRNA, 2 different UHRF1 shRNAs increased theγ-globin/γ+β-globin RNA ratio from 9.68% to 21.59/28.93% (P<0.01), increased the F-cell fraction from 37.9 to 49.8/55.6% and increased HbF protein from 9.1 to 16.18/18.5% (P<0.05) in erythroid cells derived from normal adult peripheral blood CD34+ cells. UHRF1 deficiency did not alter erythroid maturation or expression of key transcription factor genes that regulate HbF expression in HUDEP-2 or CD34+ cells (BCL11A, ZBTB7A, MYB and KLF1). UHRF1 mutant proteins defective in recognizing H3K9me2 (FW237/238AA), binding to hemi-methylated DNA (R491A) or ubiquitination of H3K23 to enhance DNMT1 recruitment (C741A), were unable to repress HBG1/HBG2. These mutations have the most profound effects on maintaining DNA methylation, indicating that UHRF1 represses HBG1/HBG2 in HUDEP-2 cells through this mechanism.UHRF1 knockout induced genome-wide demethylation including 6 CpG sites located at positions -162, -53, -50, +6, +17, +50 positions relative to the γ-globin (HBG1 and HBG2) transcription start site. Demethylation of these sites is thought to be required for γ-globin de-repression. However, forced demethylation of these cytosines in HUDEP-2 cells using specific gRNAs + dead (d) Cas9-TET1 was not sufficient to activate γ-globin expression when UHRF1 was present. Additionally, dCas9-DNMT3a-mediated methylation of the HBG promoter CpG residues in UHRF1 knockdown HUDEP-2 cells did not inhibit γ-globin expression in UHRF1 knockout HUDEP-2 cells. Based on these studies, we conclude that: 1) UHRF1 regulates γ-globin transcription; 2) demethylation of CpG sites at the HBG gene promoters is neither necessary or sufficient for γ-globin induction; 3) UHRF1 regulates γ-to-β globin gene switching either by methylating DNA regions other than those present around the HBG promoter or through non-canonical activities. Distinguishing these mechanisms will elucidate further our understanding of globin gene switching and could identify new pathways for pharmacological induction of HbF. DisclosuresNo relevant conflicts of interest to declare.

Protein Phosphorylation Mechanisms Underlying Chronic Pain in Sickle Cell Disease

Pain is one of the most dreadful symptoms in sickle cell disease (SCD) and is often refractory to currently available analgesics. Besides acute painful vaso-occlusive crises, SCD is also accompanied by intractable chronic pain. This persistent, and often unrelieved, pain starts early in childhood and continues throughout life. The neurobiological mechanisms of chronic pain in SCD remain unclear, which markedly limits effective pain management and the quality of life in patients with SCD. Taking advantage of two humanized mouse models of SCD, this study aimed to investigate protein phosphorylation mechanisms for chronic pain in SCD.We characterized pain in two transgenic mice models of SCD that exclusively express human alleles encoding normal α- and sickle β-globin. Berkeley SCD mice (BERK mice) and Townes' SCD mice (TOW mice) exhibited ongoing spontaneous pain behavior and increased sensitivity to evoked pain stimuli compared with littermate control mice expressing normal human hemoglobins. To investigate the underlying protein phosphorylation mechanisms of chronic pain in SCD, we examined PKC isoform mediated nociceptive signaling. Prominent activation of multiple PKC isoforms were observed in the superficial laminae of the spinal cord dorsal horn in BERK and TOW mice. Functional inhibition and silencing of specific PKC isoforms attenuated spontaneous pain, mechanical allodynia, and heat hyperalgesia in both transgenic SCD mice. Furthermore, employing hematopoietic stem cell transplantation approach, we were able to generate a sickle cell anemia model in PKC-null mice, allowing us to specifically target neuronal PKC in SCD. Neither spontaneous pain nor evoked pain was detected in the mice lacking specific PKC isoform despite the full establishment of SCD phenotypes.In summary, this study is the first to identify the presence of ongoing spontaneous pain in preclinical sickle cell models, which closely mimic the most prevalent manifestation in patients with SCD. Moreover, we found that spinal PKC is a critical mechanism for the generation and maintenance of ongoing and evoked pain in SCD. These findings offer insights into sickle cell pain mechanisms, which may become a potential target for pharmacological interventions. DisclosuresNo relevant conflicts of interest to declare.

Soluble Guanylate Cyclase Modulators Effect Fetal Hemoglobin Levels through a Non-cGMP-Protein Kinase G Signaling Pathway

Sickle cell anemia results from a point mutation in both alleles of the β-globin gene. This homozygous mutation ultimately leads to a structural alteration of the hemoglobin protein that promotes polymerization of the mutant sickle hemoglobin tetramer (HbS) upon deoxygenation. HbS polymerization results in rigid, sickle-shaped RBCs with increased cell-to-cell adhesion properties. These sticky and rigid RBCs are prone to become trapped in small capillary networks leading to ischemia-reperfusion injury, endothelial damage and the hallmark pain crisis of sickle cell anemia. Neonates are protected from deoxy-HbS polymerization by high levels of fetal hemoglobin (HbF). HbF is composed of two α-globin and two γ-globin subunits(α2γ2), The γ-globin molecule cannot interact with deoxy-βS-globin polymers, which makes HbF an effective inhibitor of deoxy-HbS polymerization. The level of HbF required to reduce the symptoms of sickle cell anemia is 20-25%, but levels as low as 9% can prolong red cell survival. Treatment with hydroxyurea (HU) induces HbF and reduces the hematologic and clinical consequences of sickle cell anemia. Basal and inducible HbF levels are important in predicting the severity of sickle cell anemia and are highly phenotypically variable among patients, leading to varied responses to treatment. Patients are also variably susceptible to HU-induced cytopenias, which limits the use of HU in certain patients. HU is currently the only FDA-approved HbF-inducer for the treatment of sickle cell anemia and there is clearly a need for alternative HbF-inducers. Understanding the signaling pathways that regulate HbF induction will lead to novel therapeutic targets for sickle cell anemia. The soluble guanylate cyclase/cyclic guanosine monophosphate-dependent protein kinase (sGC/PKG) signaling pathway potentially links HU to the induction of HbF expression. In this study we investigated the direct role of sGC in HbF induction using novel pharmacologic modulators of sGC. Nitric oxide (NO) activates sGC by binding to the ferrous iron (Fe2+) in the active site heme moiety. Once activated, sGC converts GTP to cGMP, which in-turn activates PKG. Reactive oxygen species (ROS) oxidize the active site heme of sGC leading to NO-insensitivity. We tested the ability of a novel sGC activator, BAY 58-2667, to induce γ-globinin primary and immortalized (HUDEP-2) human erythroid progenitor cells. BAY 54-6544 binds to heme-free inactivated sGC to restore its guanylyl cyclase activity independent of NO. We also tested the ability of the sGC stimulator, BAY 41-2272, to induce γ-globinin primary and HUDEP-2 human erythroid progenitor cells. BAY 41-2272, binds to the ferrous iron at the active site of non-oxidized sGC to stimulate guanylyl cyclase activity in a synergistic manner with NO. We compared g-globin mRNA and protein expressionin the primary and immortalized human erythroid progenitors after treatment with different concentrations and combinations of BAY 54-6544, BAY 41-2667 and HU. We also evaluated g-globin induction in cellstreated with the pan-phosphodiesterase inhibitor IBMX and a synthetic cGMP analog. Although we see robust induction of cGMP and activation of PKG with all treatments, we only see significant induction of g-globin expression in the HU treated cells. This data suggests that the induction of HbF occurs through a non-sGC/PKG-dependent signaling pathway. These data demonstrate a very limited induction of γ-globin by BAY 54-6544 and BAY 41-2667 that appears to be disproportionate to, and independent of, cGMP/PKG signaling. These data also demonstrate, for the first time, that HU treatment of the immortalized HUDEP-2 cell line induces γ-globin expression more consistently than in primary erythroid progenitors. DisclosuresNo relevant conflicts of interest to declare.

Measurement of Sickle Cell Disease Symptoms from the Patient Perspective: Considerations for Clinical Trials

OBJECTIVES: Sickle cell disease (SCD), a group of inherited blood disorders, can impact multiple organs and can lead to variable clinical presentations. In addition to severe pain crises, patients may experience chronic symptoms (e.g. pain, tiredness). Assessing treatment benefit using a patient-reported outcome (PRO) measurement strategy can be critical for supporting clinical SCD research. We conducted qualitative research activities to inform considerations for PRO measurement strategies to assess daily chronic symptoms in regulated SCD clinical trials in patients 16 years and older.METHODS: Research activities aimed to identify the relevant and important signs/symptoms of SCD and determine their potential for measurement via existing PRO instruments. A literature review, meetings with clinical experts (N=5), and interviews with adolescent and adult SCD patients (N=20) were conducted. Trained researchers conducted semi-structured qualitative interviews. Results were analyzed to develop a synthesized SCD conceptual model describing the literature, expert, and patient perspectives of SCD.Five instruments used in SCD research were selected for review: Adult Sickle Cell Quality of Life Measurement Information System (ASCQ-MeSM), Pediatric Quality of Life Inventory (PedsQL™) sickle cell disease module, the Patient-Reported Outcomes Measurement Information System (PROMIS) Fatigue and Pediatric Pain Interference measures, and Scribe Sickle Cell Record of Daily Symptom Burden™ (SSCRDSB). Signs/symptoms in the SCD conceptual model were compared to those assessed in these instruments and measurement properties were reviewed. Considerations for concept selection (e.g. suitability of measuring a symptom via PRO instrument) and item development (e.g. selection of recall period) in developing a PRO measurement strategy were evaluated. Instruments developed to assess SCD concepts should have documented evidence of content validity (i.e., qualitative data confirming the instrument measures concepts important, relevant and understandable to the target patient population) and should include clear instructions, a short recall period, items that measure dimensions of importance to patients (e.g., symptom intensity), and well-defined response options that are sufficiently sensitive to detect change.RESULTS: Patients reported 22 signs/symptoms that they experienced generally with SCD and/or during pain episodes, which were largely endorsed by the literature and/or clinical experts. Pain was reported by all patients and tiredness was reported by 80.0% of patients; both were cited as the most important to improve with treatment. Patients reported 29 impacts of SCD across 9 domains.Across the 5 instruments, pain was the most frequently measured SCD symptom concept, measured in different ways by 3 instruments. None of the reviewed instruments assessed more than 5 of the signs/symptoms included in our SCD conceptual model with the SSCRDSB assessing the greatest number of signs/symptoms (n=5/22 concepts, 22.7%). The remaining 4 instruments assessed 3 or fewer of the signs/symptoms reported by patients. Physical impacts were the most frequently measured impact domain, assessed to varying degrees by all 5 instruments evaluated. The PedsQL™ SCD module assessed the greatest number of impact domains in our SCD conceptual model (n=7/9 domains, 77.8%). The reviewed instruments were determined to have limitations in the context of assessing SCD signs/symptoms daily as part of regulated clinical trials.CONCLUSIONS: The research activities conducted as part of this work suggest that (1) patients experience numerous SCD signs/symptoms regularly and intermittently and (2) existing instruments, while perhaps suitable for other contexts of use or in conjunction with other tools, may be inadequate for evaluating the most important symptoms in a way that can demonstrate treatment benefit when measured daily in regulated clinical research. A de novo instrument developed for these purposes may better assess treatment benefit from the patient perspective in the context of measuring variable SCD symptoms daily in a regulated clinical trial setting. DisclosuresOjo:Ironwood Pharmaceuticals: Employment. Litcher-Kelly:Ironwood Pharmaceuticals: Consultancy. Mazar:Ironwood Pharmaceuticals: Consultancy. Klooster:Ironwood Pharmaceuticals: Consultancy. Ollis:Ironwood Pharmaceuticals: Consultancy. Chaston:Ironwood Pharmaceuticals: Consultancy. Roeder:Ironwood Pharmaceuticals: Consultancy. Treadwell:Global Blood Therapeutics: Consultancy; Ironwood Pharmaceuticals: Consultancy; Pfizer: Consultancy. Reasner:Ironwood Pharmaceuticals: Employment, Equity Ownership.

Clinical Characteristics and Health Care Utilization Patterns of Sickle Cell Disease Patients Using Marijuana

Background:Opioid analgesics have served as the cornerstone of acute and chronic pain management in patients with sickle cell disease (SCD) in the United States. Despite significant opioid use, hospitalization for painful crises comprises the majority of healthcare costs for patients with SCD. Moreover, greater use of opioids may correlate with poorer response to treatment of acute painful crises. Insufficient pain management, the rise of opioid use disorder and opioid-related fatal overdose have opened the door for new strategies of pain management for these patients. Medical cannabis represents a potential new strategy for the management of chronic SCD pain. There is a substantial body of literature describing the analgesic properties of cannabis, but little research has explored its role in the management of SCD-related pain. We sought to evaluate the prevalence of marijuana use in an urban SCD patient population and to identify demographic and clinical characteristics that may predict marijuana use in this cohort.Methods:Adult patients with SCD seen at an urban outpatient SCD center completed an intake form with questions regarding individual demographics, cumulative disease complications, current or prior marijuana use, ED utilization, frequency of pain, and opioid use. Data from 78 patients seen between October 2017 and June 2018 were available for analysis. Additional data including information regarding disease genotype and race were collected from the electronic medical record. Data were analyzed using Microsoft Excel and Chi-Square analysis.Results:Among the 78 patients, mean age of the population was 35.7 years (range 20-69), and 50% were male. Genotype was 69% HbSS/SB0 and 31% HbSC/SB+. Twenty-three patients (29%) reported ever using marijuana, and 15 (19%) endorsed current use; most patients (80%) reported smoking. Marijuana users were more likely than non-users to be male (67% v 46%), to have HbSS/SB0 (80% v 67%), to have avascular necrosis (AVN) (47% v 17%, p=.025), to report daily pain (40% v 13%, p=.013), and to be prescribed opioids (93% v 65%, p=.031). Frequent ED visits (at least monthly) were more common in marijuana users (33% v 6% p=.002) compared to non-users. Age, leg ulcers, priapism, and hydroxyurea use were not different in users versus non-users.Discussion:In this cohort of patients with SCD seeking outpatient care, 19% reported current marijuana use for the management of SCD-related symptoms. Most patients reported smoking marijuana, and only one patient reported ingestion of edible marijuana. These rates are similar to marijuana use in other populations with chronic pain. SCD marijuana users were similar in age but not in sex distribution, as men more commonly reported marijuana use than women in our population. Marijuana users were enriched for the HbSS genotype, reported more daily pain, had more frequent ED visits and were more likely to be diagnosed with AVN of bones. There was a strong association between marijuana use and self-reported opioid use for pain control. These preliminary data suggest that patients with more severe manifestations of SCD, namely daily pain, frequent ED visits, AVN of bones, and significant utilization of opioids, are more likely to be using marijuana as adjunctive therapy for their chronic pain.As of July 2018, 31 US states and the District of Columbia have legalized the use of medical cannabis, with chronic pain an indication in most states and opioid replacement an indication in some. The expansion of access to medical cannabis highlights the need for careful evaluation of the potential benefits and harms of medical cannabis in the SCD population. These benefits might include better control of daily pain, reduced visits to the ED, and possibly decreased utilization of opioids. As medical cannabis is becoming a more available therapy, carefully controlled prospective studies are needed to assess its efficacy in the management of debilitating symptoms of SCD. [Display omitted] DisclosuresStarrels:Opioid Post-Marketing Requirement Consortium: Other: Research and travel support from the Opioid Post-Marketing Requirement Consortium for a FDA-mandated observational study of the risks of opioid medications. Minniti:Novartis: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Other: Adjudicating Committee; Global Blood Therapeutics: Research Funding; Teutona: Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding.

Precision Medicine for Sickle Cell Disease through Whole Genome Sequencing

▪Although sickle cell disease (SCD) is a monogenic disorder, the severity and specific organ dysfunction and failure are strongly influenced by genetic modifiers. Rapid identification of all modifiers in patients and well-phenotyped cohorts will better define the impact of relevant variants on clinical status, inform disease biology, and identify new therapeutic strategies. We created the Sickle Genome Project (SGP), a whole genome sequencing (WGS) strategy, to define genomic variation and modifiers of SCD. We performed WGS on 871 African American SCD patients from St. Jude Children's Research Hospital who participated in the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer, 2018) and Texas Children's Hospital Hematology Center (TCHC). We developed robust pipelines for accurate detection of single nucleotide polymorphisms (SNPs), identification of structural variants and data retrieval/sharing via the St. Jude Cloud platform (to be described elsewhere). Notable findings include:1) Confirmed associations of common genetic modifiers with SCD phenotypes, including levels of fetal hemoglobin (BCL11A, HBS1L-MYB, HBB), bilirubin (UGT1A1), and microalbuminuria (APOL1). Additional associations approaching genome-wide significance require further investigation, including replication in independent samples.2) Improved determination of the SCD modifier α-thalassemia. The most common α-thalassemia mutations in SCD are 3.7 kb or 4.2 kb deletions (-α3.7 and -α4.2 alleles), which arose from recombination between homologous HBA1 and HBA2 genes and are difficult to map using standard WGS reads. Three independent crossover events are described for -α3.7 and one for -α4.2 in SCD cohorts. We developed a novel approach to identify α-globin gene deletions by local de novo assembly of WGS data and coverage depth analysis. We identified 5 -α3.7 alleles (frequencies 0.77-32.12%) and 7 -α4.2 alleles (frequencies 0.19-5.77%). Collectively, the frequency of all -α alleles was 57%, reflecting at least 12 distinct recombination events, greatly exceeding previously published counts. These findings better define the evolution of α-globin genes to allow improved understanding of their regulation and influence on SCD.3) Characterization of β0-thalassemia alleles. Mutations in the extended β-globin locus influence SCD phenotypes. Five SGP patients had large β-globin (HBB) deletions associated with elevated fetal hemoglobin, which ameliorates symptoms of SCD. Twenty-three patients had HbSβ0-thalassemia, which reduces the severity of some SCD phenotypes. Overall, 48.6% (18/37) of patients clinically designated as HbSβ0 -thalassemia had no identified β-thalassemia mutation. Moreover, 4/680 patients (0.6%) designated HbSS were identified to be β0-thalassemia heterozygotes. The MCV, RBC and %HbA2 distributions overlapped substantially in correct vs. incorrect genotype assignments. Improved discrimination of HbSβ0 vs HbSS genotypes by WGS will better define associated phenotype differences to impact clinical care.4) Determination of a genetic variant linked to vaso-occlusive crisis (VOC). Previously, a single GWAS study linked rs3115229, located 63.7 kb 5′ upstream of the KIAA1109 gene, with VOC at borderline significance (P = 5.63 × 10−8) (Chaturvedi et al, Blood 130, 2017). Using WGS data for 327 SGP participants (HbSS or HbSβ0-thalassemia) enrolled in the SCCRIPP study, we found strong association (p = 7 x 10-5) between the onset of VOC and a 4-SNP diplotype within an adjacent LD block of the KIAA1109-TENR-IL2-IL21 region (chr4: 122.8Mb - 123.8Mb) which has been previously associated with numerous inflammatory disorders. We validated this association using imputed genome-wide array data in an independent group of SCD patients (Sleep and Asthma Cohort, n= 181 patients, p = 0.05) (Cohen et al, Ann Am Thorac Soc, 2016). This works provides confirmation that the region surrounding KIAA1109 is associated with pain crisis in SCD.Our studies provide new information on the genomic architecture of SCD patients and delineate a consolidated approach for future applications of precision medicine. [Display omitted] DisclosuresHankins:Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy. Estepp:Global Blood Therapeutics: Consultancy, Research Funding; ASH Scholar: Research Funding; NHLBI: Research Funding; Daiichi Sankyo: Consultancy.

Elevated fetal haemoglobin levels are associated with decreased incidence of retinopathy in adults with sickle cell disease

Among the many vascular complications of sickle cell disease (SCD), retinopathy is the most prevalent and represents a leading cause of blindness. Hydroxycarbamide therapy ameliorates many symptoms of SCD, and high fetal haemoglobin (HbF) levels have been shown to protect against the development of retinopathy in children with HbSS. Its effect on adults with SCD, who are at a much higher risk of developing retinopathy, has not been studied. We aimed to investigate the effect of hydroxycarbamide use and HbF level on sickle cell retinopathy development in adults. We performed a retrospective cross-sectional study and collected demographics, comorbidities, and ocular and haematological data from 300 adult sickle cell subjects examined at the Henkind Eye Institute at Montefiore Medical Center during a 5-year period, from October 2012 to November 2017. The cohort was comprised mainly of Black and Hispanic subjects with all SCD genotypes, aged 18-71years. Results show that in HbSS patients treated with hydroxycarbamide, those with retinopathy had significantly lower HbF levels compared to patients without retinopathy (P=0·018). Our study identified the optimal HbF cut-off point for retinopathy protection to be 14·87%. Thus, a HbF level of 15% appears to be the threshold above which the odds for developing retinopathy in SS patients are reduced by 50%.

Bartonella henselae Infection in Sickle Cell Disease Mice Is Associated with Hyperalgesia.

Sickle cell disease (SCD) is the most prevalent hematologic genetic disorder. Acute vaso-occlusive painful crisis is the hallmark of the disease and may be related to subclinical infections. Bartonellosis, a rare and neglected infection, is caused by Bartonella spp., which can be found in donated blood. These bacteria cause intraerythrocytic and endothelial infection and pain, all of which occur in SCD. It is likely that this infection is transmitted to SCD patients during transfusion from donated blood, leading to pain. We, therefore, evaluated whether Bartonella henselae infection would cause hyperalgesia in mice with SCD. SCD mice were generated by transplantation of nucleated bone marrow cells harvested from transgenic Berkeley sickle mice into 2-month-old irradiated C57BL/6 mice. We infected four SCD mice by intraperitoneal inoculation with B. henselae, and inoculated four other mice with the same volume of saline. Mechanical hyperalgesia was determined using von Frey monofilaments by two blinded observers. Thereafter, the animals were anesthetized and euthanized to collect blood, liver, and spleen samples to seek B. henselae infection by PCR. We confirmed the experimental infection in all animals by PCR. Tremors and mechanical hypersensitivity were demonstrated by SCD mice infected with B. henselae infection but not in those receiving saline. B. henselae infection may be related to pain and other symptoms in SCD.