scholarly journals Mitochondrial DNA Variation in Individuals with Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-11
Author(s):  
Maliha Maryam Ahmad ◽  
Laxminath Tumburu ◽  
Chunyu Liu ◽  
Mehdi Pirooznia ◽  
Swee Lay Thein
Keyword(s):  
Mitochondrial Dna ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Mutant Allele ◽  
African Ancestry ◽  
Organ Damage ◽  
Median Number ◽  
Cell Disease ◽  
Mtdna Sequences ◽  
Mitochondrial Heteroplasmy

Background: Sickle cell disease (SCD) is a complex multi-system disorder that predominantly affects individuals of African heritage. While the sickle pathology is initiated by polymerization of HbS, multiple end-organ damage is inflicted by years of on-going inflammation and vasculopathy. An emerging marker of inflammation is the accumulation of mutations in mitochondrial DNA (mtDNA), the phenotypic effect of which will depend on the nature of the gene that harbors the mutation, the mutant allele fraction, and the pathogenicity of the mutant allele. A mutation in mtDNA is heteroplasmic when it is present in only a proportion of mtDNA, and homoplasmic, when it is present in all mtDNA molecules. Mitochondrial heteroplasmy can also occur at different tissue or cell levels, even within the same individual. Given the underlying chronic inflammation in SCD, we hypothesize that SCD patients display increased rates of mtDNA mutations. Methods: We analyzed and compared whole genome sequencing (WGS) data from the cohort of 683 SCD patients (SCD cohort) of African ancestry with that of 621 individuals of African ancestry from the 1000 Genomes Project (1KG). The SCD cohort included 561 HbSS & HbSβ0 thalassemia (combined), 90 HbSC, and 25 HbSβ+ thalassemia. The 1KG cohort included 516 HbAA and 105 sickle carriers (HbAS). mtDNA sequences of SCD and 1KG cohorts were initially aligned to the revised Cambridge Reference Sequence (rCRS NC_012920), and subsequently base recalibrated and deduplicated. Mitochondrial sequences extracted from the cleaned WGS data of both cohorts were analyzed for heteroplasmic and homoplasmic variants using mitoCaller from the package mitoAnalyzer. Results: The average depth per locus is ~6,828X for the SCD cohort and ~2,879X for the 1KG cohort. We performed a locus by locus comparison between the mtDNA sequences of both cohorts. No homoplasmic variants unique to the SCD cohort were found when compared to the 1KG cohort. In contrast, there were several "hotspots" of heteroplasmic variants that were unique to the SCD cohort, and largely shared amongst the SCD patient population (Figure 1A). To identify these unique variants, we used MITOMASTER and Ensembl VEP to annotate the heteroplasmic variants that had above 40% population frequency within the SCD cohort and below 10% population frequency in the 1KG cohort. Several heteroplasmic variants were non-synonymous and the selected variants originated from the Control Region (D-loop), RNR1, RNR2, ND1, ND4, and ND5. One of the heteroplasmic variants, 2623 A>G, was found to be age linked for HbSS & HbSβ0 (Figure 1B), with its minor allele frequency (MAF) increasing with age. Further analysis needs to be done in order to determine if more variants unique to the SCD cohort are age-linked. We then compared the quantity of heteroplasmic variants across the different SCD genotypic groups with the 1KG HbAA and the 1KG HbAS groups. The median number of heteroplasmic variants per individual increased progressively from HbAA, HbAS, HbSβ+ thalassemia, and HbSC with the highest median number of 119 in HbSS & HbSβ0 (Figure 1C). Mitochondrial heteroplasmy for 1KG HbAA and 1KG HbAS were statistically significant when tested against each other and against every SCD sub-group; however, the difference was not statistically significant between the different SCD genotypes (Table insert in Fig 1C). It is important to note that we did not apply a MAF threshold, thus many of the heteroplasmic variants may be present at very low levels. Conclusion: Our findings suggest that there is an increased prevalence of heteroplasmic variants in SCD compared to ethnic-matched healthy populations. Within the SCD genotypes, the heteroplasmic burden increased progressively (HbAS < HbSβ+ thalassemia < HbSC < HbSS & HbSβ0) with genotypic groups that are associated with increasing phenotypic severity. mtDNA heteroplasmic burden for one variant also increased with age in HbSS & HbSβ0 individuals, but further studies are needed to explore if mtDNA heteroplasmic burden correlates with the degree of organ damage and disease severity within the same genotypic group. Although it is not clear if the variants are a cause or effect of the sickle inflammatory pathology, our data suggest that mtDNA heteroplasmic burden is a potential biomarker of SCD severity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cell-Free Mitochondrial DNA Is Elevated in Sickle Cell Disease Patients, and Serve As a Potential Proinflammatory DAMP

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1068-1068
Author(s):  
Laxminath Tumburu ◽  
Shohini Ghosh-Choudhary ◽  
Emilia Alina Barbu ◽  
Simon Yang ◽  
Lauren D Harrison Ware ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Acute Pain ◽  
Organ Damage ◽  
Healthy Controls ◽  
Cell Disease ◽  
Cross Sectional ◽  
Healthy Donors

Abstract Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by hemolysis and intermittent acute pain with multi-organ damage. Previously, we showed that acute pain in SCD was associated with >10-fold increases in cell-free DNA (cfDNA) when compared to steady state, that were significantly reduced during hydroxyurea therapy. Apoptosis, necrotic cell death and lysis of intact cells in the blood stream have been proposed as sources of plasma cfDNA. Here, we explored if the cfDNA increases could have a role in inflammation, a constant pathological feature of SCD. cfDNA was extracted using QIAamp MinElute ccfDNA Kit (Qiagen), from the platelet-poor plasma processed within 30 minutes from the blood drawn in EDTA tubes, and analyzed using whole genome sequencing (WGS) and targeted quantitative PCR (qPCR). SCD patients are defined as in acute pain if there is no evident cause other than SCD, for which the patient needs hospitalization, either as in- or outpatient, and is treated with parenteral narcotics. Steady state was defined as the period from at any time 8 weeks prior to or after a crisis. A cross-sectional study of 8 healthy controls and 34 SCD patients (18 steady-state; 16 crisis) mapped WGS reads showed significantly higher proportion of cell-free mitochondrial DNA (cf-mtDNA) compared to nuclear cfDNA (cf-nDNA) in SCD patients compared with healthy controls (Fig 1A: steady-state: 14 fold; crisis: 11 fold; p = 0.0001). We used targeted qPCR to quantify both cf-nDNA and cf-mtDNA in another cross-sectional cohort of 13 healthy controls and 92 patients (72 steady-state, 20 crisis) as well as 18 paired HbSS patients (steady-state and crisis) samples with 10 healthy controls. The nuclear reference genes used were GAPDH and TERT and mitochondrial genes were MT-ND1 and MT-ND6. While cf-nDNA (TERT) was significantly increased (> 3.5 fold, p = 0.0251; Fig 1B) in SCD patients compared with healthy controls only during crises, significantly higher levels of cf-mtDNA over cf-nDNA were observed in SCD patients compared with healthy volunteers in both steady-state and crises (Fig 1C: MT-ND1/GAPDH: steady-state >19 fold, crisis > 8 fold; MT-ND1/TERT: steady-state > 8 fold, crisis > 7 fold; MT-ND6/GAPDH: steady-state > 7 fold, crisis > 3 fold; MT-ND6/TERT: steady-state > 4 fold; crisis > 4 fold; p < 0.05). In the paired samples, cf-nDNA (GAPDH andTERT) was significantly increased (> 3 fold; Fig 1D-E) in crisis compared to steady-state (p < 0.05). The differential increase in cf-mtDNA (cf-mtDNA:cf-nDNA ratio) levels in these patients during crises, were significantly higher compared with healthy controls (Fig 1F: MT-ND1/GAPDH: steady-state >9 fold, crisis > 8 fold; MT-ND1/TERT: steady-state > 8 fold, crisis > 9 fold; MT-ND6/GAPDH: steady-state > 8 fold, crisis > 8 fold; MT-ND6/TERT: steady-state > 8 fold; crisis > 7 fold; p < 0.005). Using confocal microscopy and mitochondrial-specific dyes (MitoTracker Green and TMRM), we show that substantial numbers of red blood cells from SCD patients retain their mitochondria in the circulation. We next explored if the elevated cf-mtDNA in SCD could contribute to its pathophysiology, via activating neutrophils to form neutrophil extracellular traps (NETs), a recognized immunological response in inflammation. Initially, we confirmed that mtDNA can induce NETosis by treating neutrophils from healthy donors with mtDNA isolated from human platelets. mtDNA consistently induced a robust NETs response (N=8) while genomic nuclear DNA did not cause any NETosis. SCD plasma containing high levels of cf-mtDNA also caused a strong NETosis response while plasma from healthy donors did not (N=11). Cytosolic adaptor STING has a central role in sensing of cytosolic double stranded DNA. We sought to determine if the downstream STING-TBK1-IRF3 pathway is associated with the mtDNA-mediated formation of NETs. We inhibited the catalytic activity of the STING downstream effector TBK1 with BX795 prior to treating neutrophils with cf-mtDNA-containing plasma (N=5). The TBK1 inhibition consistently reduced the NETs response by at least 70% confirming that cytosolic DNA sensors are involved in promoting mtDNA-mediated formation of NETs. Our findings suggest that cf-mtDNA induces NETosis contributing to the pathological sterile inflammation in SCD patients. Continual release of these mitochondrial DAMPs in hemolysis may serve as key link between inflammation and organ damage in SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Individuals with Sickle Cell Disease Have a Higher Burden of Mitochondrial DNA Heteroplasmy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 954-954
Author(s):  
Laxminath Tumburu ◽  
Maliha Maryam Ahmad ◽  
Chunyu Liu ◽  
Clifton L. Dalgard ◽  
Mehdi Pirooznia ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Mitochondrial Dna ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Median Number ◽  
Sequencing Depth ◽  
Cumulative Distribution ◽  
Cell Disease ◽  
Mtdna Mutations ◽  
Hemoglobin C

Abstract Background: The simple point mutation that causes sickle cell disease (SCD) belies the extensive systemic damage it can cause. While the sickle pathology is initiated by polymerization of HbS, the multiple end-organ damage is inflicted by years of on-going inflammation and vasculopathy. An emerging marker of inflammation is the accumulation of acquired heteroplasmy mutations in mitochondrial DNA (mtDNA). Given the underlying chronic inflammation in SCD, we hypothesized that SCD patients display increased rates of mtDNA mutations, and previously confirmed (1). Here, we further performed indepth analyses in an ethnically matched normal (HbAA) as well as sickle trait (HbAS) subjects from another independent cohort, the Jackson Heart Study (JHS). Methods: We analyzed and compared whole genome sequencing (WGS) data from the from NIH cohort of 676 SCD patients of African ancestry with that of 621 ethnic-matched indviduals from the 1000 Genomes Project (1KG), and 3,580 individuals from the JHS cohort. The NIH SCD cohort included 561 HbSS & HbSβ 0thalassemia (combined), 90 HbSC, and 25 HbSβ + thalassemia genotypes, the 1KG cohort - 516 HbAA and 105 HbAS and JHS cohort - 3,200 HbAA, 89 HbAC (hemoglobin C trait), and 291 HbAS. Additionally, to further understand any potential sequencing depth bias, as well as to compare between two patient cohorts (NIH SCD & JHS cohorts) with underlying conditons that may influence the heteroplasmy bias, we downsampled 300 NIH cohort HbSS samples to a sequencing depth similar to JHS cohort, and compared their heteroplasmy burden. Mitochondrial sequences extracted from the cleaned WGS data of these 3 cohorts were analyzed for heteroplasmic and homoplasmic variants using mitoCaller from the package mitoAnalyzer. Results: The average depth per locus was ~6,671X for the NIH SCD cohort , ~2,879X for the 1KG cohort, and ~2169X for JHS cohort. We compared the quantity of heteroplasmic variants across the different NIH SCD genotype with 1KG (HbAA & HbAS), and JHS (HbAA, HbAC and HbAS) genotypic groups. The median number of heteroplasmic variants per individual increased progressively from HbAA, HbAS, HbSβ +thalassemia, and HbSC with the highest median number of 118 in HbSS & HbSβ 0 (Fig 1A) in NIH SCD cohort. It is noteworthy that the median mtDNA heteroplasmy in HbAA individuals in 1KG cohort was significantly lower than those in JHS cohort (Table insert in Fig 1A) which may be related to the underlying cardiovascular disease in the JHS cohort; whereas similar heteroplasmy burden in HbAS individuals between these 2 cohorts may underscore the genotype (HbAS) as the driver of heteroplasmy in these cohorts. We compared the heteroplasmy burden of a downsampled subset (n=300) NIH HbSS with that of JHS HbAA, HbAC and HbAS genotypes (Fig 1B). Although, the 70% reduction in sequencing depth resulted in the slight reduction in heteroplasmy burden, we noticed higher heteroplasmic variability (standard deviation) in this subset of NIH HbSS patients. This variability may be attributable to extreme variation in SCD phenotypic severity. We then applied cumulative distribution function to this downsampled subset and compared with JHS genotypes. We found the NIH HbSS patients have disproportionately higher proportion of heteroplasmy variants (Fig 1D) when compared to the JHS genotypes (HbAA, HbAC, and HbAS). Conclusion: We conclude that there is an increased prevalence of heteroplasmic mtDNA variants in SCD compared to ethnic-matched normal (HbAA) populations. Normal individuals with HbAA in JHS cohort have significantly higher heteroplasmic burden compared to those in 1KG cohort, suggesting an underlying cardiovascular disease in JHS cohort as a driving factor. Within each 1KG and JHS cohorts, individuals with sickle cell trait (HbAS) have similar heteroplasmy burden and also higher than those with HbAA, highlighting the potential significance of this genotype. Reducing the sequencing depth by &gt; 70% (downsampling) led to the filtering out of heteroplasmy variants that would have been discovered with the original deeper sequencing depth of ~7300X. Nonetheless, downsampled HbSS samples still retained disproportionately higher heteroplasmy burden compared to non-SCD subjects. We are currently investigating if there is any correlation between mtDNA heteroplasmy burden and severity of clinical phenotypes among the SCD patients. 1. Ahmad, MM et al, Blood 136 (1):11-11 (2020) Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sickle Cell Disease: Role of Oxidative Stress and Antioxidant Therapy

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 296
Author(s):  
Rosa Vona ◽  
Nadia Maria Sposi ◽  
Lorenza Mattia ◽  
Lucrezia Gambardella ◽  
Elisabetta Straface ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Organ Damage ◽  
Cell Disease ◽  
Vessel Occlusion ◽  
Antioxidant Agents ◽  
Oxidant Status

Sickle cell disease (SCD) is the most common hereditary disorder of hemoglobin (Hb), which affects approximately a million people worldwide. It is characterized by a single nucleotide substitution in the β-globin gene, leading to the production of abnormal sickle hemoglobin (HbS) with multi-system consequences. HbS polymerization is the primary event in SCD. Repeated polymerization and depolymerization of Hb causes oxidative stress that plays a key role in the pathophysiology of hemolysis, vessel occlusion and the following organ damage in sickle cell patients. For this reason, reactive oxidizing species and the (end)-products of their oxidative reactions have been proposed as markers of both tissue pro-oxidant status and disease severity. Although more studies are needed to clarify their role, antioxidant agents have been shown to be effective in reducing pathological consequences of the disease by preventing oxidative damage in SCD, i.e., by decreasing the oxidant formation or repairing the induced damage. An improved understanding of oxidative stress will lead to targeted antioxidant therapies that should prevent or delay the development of organ complications in this patient population.

scholarly journals Targeting pain at its source in sickle cell disease

2018 ◽  
Vol 315 (1) ◽  
pp. R104-R112 ◽  
Author(s):  
Kanika Gupta ◽  
Om Jahagirdar ◽  
Kalpna Gupta
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
Genetic Disorder ◽  
Somatosensory System ◽  
Organ Damage ◽  
Mast Cell Activation ◽  
Cell Disease ◽  
Central Nervous Systems

Sickle cell disease (SCD) is a genetic disorder associated with hemolytic anemia, end-organ damage, reduced survival, and pain. One of the unique features of SCD is recurrent and unpredictable episodes of acute pain due to vasoocclusive crisis requiring hospitalization. Additionally, patients with SCD often develop chronic persistent pain. Currently, sickle cell pain is treated with opioids, an approach limited by adverse effects. Because pain can start at infancy and continue throughout life, preventing the genesis of pain may be relatively better than treating the pain once it has been evoked. Therefore, we provide insights into the cellular and molecular mechanisms of sickle cell pain that contribute to the activation of the somatosensory system in the peripheral and central nervous systems. These mechanisms include mast cell activation and neurogenic inflammation, peripheral nociceptor sensitization, maladaptation of spinal signals, central sensitization, and modulation of neural circuits in the brain. In this review, we describe potential preventive/therapeutic targets and their targeting with novel pharmacologic and/or integrative approaches to ameliorate sickle cell pain.

scholarly journals The gut microbiome in sickle cell disease: Characterization and potential implications

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255956
Author(s):  
Hassan Brim ◽  
James Taylor ◽  
Muneer Abbas ◽  
Kimberly Vilmenay ◽  
Mohammad Daremipouran ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Gut Microbiome ◽  
Tissue Injury ◽  
Organ Damage ◽  
Composition Analysis ◽  
Cell Disease ◽  
Blood Disorder ◽  
Major Player ◽  
Next Generation Sequencing Ngs

Background Sickle Cell Disease (SCD) is an inherited blood disorder that leads to hemolytic anemia, pain, organ damage and early mortality. It is characterized by polymerized deoxygenated hemoglobin, rigid sickle red blood cells and vaso-occlusive crises (VOC). Recurrent hypoxia-reperfusion injury in the gut of SCD patients could increase tissue injury, permeability, and bacterial translocation. In this context, the gut microbiome, a major player in health and disease, might have significant impact. This study sought to characterize the gut microbiome in SCD. Methods Stool and saliva samples were collected from healthy controls (n = 14) and SCD subjects (n = 14). Stool samples were also collected from humanized SCD murine models including Berk, Townes and corresponding control mice. Amplified 16S rDNA was used for bacterial composition analysis using Next Generation Sequencing (NGS). Pairwise group analyses established differential bacterial groups at many taxonomy levels. Bacterial group abundance and differentials were established using DeSeq software. Results A major dysbiosis was observed in SCD patients. The Firmicutes/Bacteroidetes ratio was lower in these patients. The following bacterial families were more abundant in SCD patients: Acetobacteraceae, Acidaminococcaceae, Candidatus Saccharibacteria, Peptostreptococcaceae, Bifidobacteriaceae, Veillonellaceae, Actinomycetaceae, Clostridiales, Bacteroidacbactereae and Fusobacteriaceae. This dysbiosis translated into 420 different operational taxonomic units (OTUs). Townes SCD mice also displayed gut microbiome dysbiosis as seen in human SCD. Conclusion A major dysbiosis was observed in SCD patients for bacteria that are known strong pro-inflammatory triggers. The Townes mouse showed dysbiosis as well and might serve as a good model to study gut microbiome modulation and its impact on SCD pathophysiology.

scholarly journals Sickle Cell Disease Model Mice Lacking 2,3-Dpg Show Reduced RBC Sickling and Improvements in Markers of Hemolytic Anemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 27-28
Author(s):  
Kelly M. Knee ◽  
Amey Barakat ◽  
Lindsay Tomlinson ◽  
Lila Ramaiah ◽  
Zane Wenzel ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Hemolytic Anemia ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Organ Damage ◽  
Complete Elimination ◽  
Cell Disease ◽  
The Impact ◽  
2,3 Dpg

Sickle cell disease (SCD) is a severe genetic disorder caused by a mutation in hemoglobin (b6Glu-Val), which allows the mutant hemoglobin to assemble into long polymers when deoxygenated. Over time, these polymers build up and deform red blood cells, leading to hemolytic anemia, vaso-occlusion, and end organ damage. A number of recent therapies for SCD have focused on modulating the mutant hemoglobin directly, however, reduction or elimination of 2,3-DPG to reduce Hb S polymerization and RBC sickling has recently been proposed as a therapeutic strategy for SCD. Current clinical studies focus on activation of pyruvate kinase to reduce 2,3-DPG, however, direct targeting of the enzyme which produces 2,3-DPG; Bisphosphoglycerate Mutase (BPGM) may also be possible. In this study we evaluate the impact of elimination of 2,3-DPG on SCD pathology by complete knockout of BPGM in Townes model mice. Animals with complete knockout of BPGM (BPGM -/-) have no detectable 2,3-DPG, while animals that are heterozygous for BPGM (BPGM -/+) have 2,3-DPG levels comparable to Townes mice. Western Blot analysis confirms that BPGM -/- animals completely lack BPGM, while BPGM -/+ animals have BPGM levels that are nearly equivalent to Townes mice. As expected from the lack of 2,3-DPG, BPGM -/- animals have increased oxygen affinity, observed as a 39% decrease in p50 relative to Townes mice. Complete elimination of 2,3-DPG has significant effects on markers of hemolytic anemia in BPGM -/- mice. Mice lacking 2,3-DPG have a 60% increase in hemoglobin (3.7 g/dL), a 53% increase in red blood cell count, and a 29% increase in hematocrit relative to Townes mice. The BPGM -/- mice also have a 57% decrease in reticulocytes, and a 61% decrease in spleen weight relative to Townes animals, consistent with decreased extramedullary hematopoiesis. Consistent with the reduction in hemolysis, BPGM -/- animals had a 59% reduction in red blood cell sickling under robust hypoxic conditions. BPGM -/+ animals had hemoglobin, RBC, and hematocrit levels that were similar to Townes animals, and a similar degree of RBC sickling to Townes mice. Liver phenotype was similar across all variants, with areas of random necrosis observed in BPGM -/-, BPGM -/+ and Townes mice. Higher percentages of microcytic and/or hyperchromic RBCs were observed in BPGM -/- animals relative to BPGM -/+ or Townes animals. These results suggest that modulation of 2,3-DPG has a positive effect on RBC sickling and hemolytic anemia, which may have therapeutic benefits for SCD patients. However, the lack of improvement in organ damage suggests that modulation of 2,3-DPG alone may not be sufficient for complete elimination of SCD phenotypes, and further investigation of this therapeutic avenue may be necessary. Disclosures No relevant conflicts of interest to declare.

Abstract P726: Cytochrome B5 Reductase 3 Modifies The Brain-Blood Axis Response To Ischemic Stroke In Mice With Sickle Cell Disease

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Katherine C Wood ◽  
Heidi M Schmidt ◽  
Scott Hahn ◽  
Mehdi Nouraie ◽  
Mara Carreno ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Ischemic Stroke ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
African Ancestry ◽  
Cytochrome B5 ◽  
Heme Iron ◽  
Wild Type ◽  
Cell Disease ◽  
Cytochrome B5 Reductase

Introduction: Stroke and silent infarcts are serious complications of sickle cell disease (SCD), occurring frequently in children. Decreased nitric oxide bioavailability and responsiveness contribute to neurovascular disease. Cytochrome b5 reductase 3 (Cyb5R3) is a heme iron reductase that reduces oxidized soluble guanylate cyclase heme iron (Fe 3+ --> Fe 2+ ) to preserve nitric oxide signaling. A loss-of-function Cyb5R3 missense variant (T117S) occurs with high frequency (0.23 minor allele) in persons of African ancestry. Hypothesis: We hypothesized that impaired reductase function of T117S Cyb5R3 exacerbates brain damage after ischemic stroke in SCD. Methods: Bone marrow transplant was used to create male SCD mice with wild type (SS/WT) or T117S (SS/T117S) Cyb5R3. Blood was sampled before and after middle cerebral artery occlusion (55 minutes occlusion, 48 hours reperfusion). Infarct volume (IV) was determined by 2,3,5-triphenyltetrazolium chloride. Intravascular hemolysis and correlation (Pearson’s R) of hematology changes with IV were determined. Baseline Walk-PHaSST (NCT00492531) data were analyzed for stroke occurrence. Results: Brain IV (63 vs 27 cm 3 , P=0.003) and mortality (3/6 vs 0/8) were greater in SS/T117S vs SS/WT. Red blood cells, hemoglobin and hematocrit declined as IV increased. Plasma oxyhemoglobin increased in parallel with IV (r = 0.74, P=0.09). There were different signatures to hematologic changes that occurred with IV in SCD. Relative to wild type, T117S contracted the erythroid compartment (red blood cell: -13% vs 13%, P=0.003; hematocrit: -20% vs 1%, P=0.008; hemoglobin: -18% vs 2%, P=0.007). Mean platelet volume correlated with IV in SS/T117S (r = 0.87, P=0.06), while the inverse occurred in SS/WT (r = -0.63, P=0.09) Monocytes increased in parallel with IV in SS/T117S (r = 0.73, P=0.16), but followed the opposite trajectory in SS/WT (r = -0.77, P=0.04). WalkPHaSST participants with T117S Cyb5R3 self-reported more ischemic stroke (7.4% vs 5.1%) relative to wild type. Conclusion: Cyb5R3 is an important modifier of the evolution and outcome of ischemic brain injury in SCD and its hematologic consequences. Our findings indicate a bidirectional relationship between stroke and anemia in SCD that may axially turn on Cyb5R3 activity.

scholarly journals Potential role of LSD1 inhibitors in the treatment of sickle cell disease: a review of preclinical animal model data

2018 ◽  
Vol 315 (4) ◽  
pp. R840-R847 ◽  
Author(s):  
Angela Rivers ◽  
Ramasamy Jagadeeswaran ◽  
Donald Lavelle
Keyword(s):  
Reactive Oxygen Species ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Reactive Oxygen ◽  
Organ Damage ◽  
The United States ◽  
Oxygen Species ◽  
Cell Disease ◽  
Hematopoietic Stem

Sickle cell disease (SCD) is caused by a mutation of the β-globin gene (Ingram VM. Nature 180: 326–328, 1957), which triggers the polymerization of deoxygenated sickle hemoglobin (HbS). Approximately 100,000 SCD patients in the United States and millions worldwide (Piel FB, et al. PLoS Med 10: e1001484, 2013) suffer from chronic hemolytic anemia, painful crises, multisystem organ damage, and reduced life expectancy (Rees DC, et al. Lancet 376: 2018–2031, 2010; Serjeant GR. Cold Spring Harb Perspect Med 3: a011783, 2013). Hematopoietic stem cell transplantation can be curative, but the majority of patients do not have a suitable donor (Talano JA, Cairo MS. Eur J Haematol 94: 391–399, 2015). Advanced gene-editing technologies also offer the possibility of a cure (Goodman MA, Malik P. Ther Adv Hematol 7: 302–315, 2016; Lettre G, Bauer DE. Lancet 387: 2554–2564, 2016), but the likelihood that these strategies can be mobilized to treat the large numbers of patients residing in developing countries is remote. A pharmacological treatment to increase fetal hemoglobin (HbF) as a therapy for SCD has been a long-sought goal, because increased levels of HbF (α2γ2) inhibit the polymerization of HbS (Poillin WN, et al. Proc Natl Acad Sci USA 90: 5039–5043, 1993; Sunshine HR, et al. J Mol Biol 133: 435–467, 1979) and are associated with reduced symptoms and increased lifespan of SCD patients (Platt OS, et al. N Engl J Med 330: 1639–1644, 1994; Platt OS, et al. N Engl J Med 325: 11–16, 1991). Only two drugs, hydroxyurea and l-glutamine, are approved by the US Food and Drug Administration for treatment of SCD. Hydroxyurea is ineffective at HbF induction in ~50% of patients (Charache S, et al. N Engl J Med 332: 1317–1322, 1995). While polymerization of HbS has been traditionally considered the driving force in the hemolysis of SCD, the excessive reactive oxygen species generated from red blood cells, with further amplification by intravascular hemolysis, also are a major contributor to SCD pathology. This review highlights a new class of drugs, lysine-specific demethylase (LSD1) inhibitors, that induce HbF and reduce reactive oxygen species.

scholarly journals Current and novel therapies for the prevention of vaso-occlusive crisis in sickle cell disease

2020 ◽  
Vol 11 ◽  
pp. 204062072095500
Author(s):  
Ifeyinwa Osunkwo ◽  
Deepa Manwani ◽  
Julie Kanter
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Severe Pain ◽  
Management Strategies ◽  
Organ Damage ◽  
Care Utilization ◽  
Cell Disease ◽  
High Resource ◽  
Emerging Treatments

Individuals with sickle cell disease (SCD) are living further into adulthood in high-resource countries. However, despite increased quantity of life, recurrent, acute painful episodes cause significant morbidity for affected individuals. These SCD-related painful episodes, also referred to as vaso-occlusive crises (VOCs), have multifactorial causes, and they often occur as a result of multicellular aggregation and vascular adherence of red blood cells, neutrophils, and platelets, leading to recurrent and unpredictable occlusion of the microcirculation. In addition to severe pain, long-term complications of vaso-occlusion may include damage to muscle and/or bone, in addition to vital organs such as the liver, spleen, kidneys, and brain. Severe pain associated with VOCs also has a substantial detrimental impact on quality of life for individuals with SCD, and is associated with increased health care utilization, financial hardship, and impairments in education and vocation attainment. Previous treatments have targeted primarily SCD symptom management, or were broad nontargeted therapies, and include oral or parenteral hydration, analgesics (including opioids), nonsteroidal anti-inflammatory agents, and various other types of nonpharmacologic pain management strategies to treat the pain associated with VOC. With increased understanding of the pathophysiology of VOCs, there are several new potential therapies that specifically target the pathologic process of vaso-occlusion. These new therapies may reduce cell adhesion and inflammation, leading to decreased incidence of VOCs and prevention of end-organ damage. In this review, we consider the benefits and limitations of current treatments to reduce the occurrence of VOCs in individuals with SCD and the potential impact of emerging treatments on future disease management.

Reduced Oxidative Stress and Enhanced Nitric Oxide (NO) Bioavailability in Transgenic-Knockout Sickle Mice Expressing Fetal Hemoglobin (HbF) Is Mediated by Decreased Sickling and Hemolysis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 842-842
Author(s):  
Trisha Dasgupta ◽  
Mary E. Fabry ◽  
Dhananjay K. Kaul
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Genetic Manipulation ◽  
Fetal Hemoglobin ◽  
Organ Damage ◽  
Multiple Organ ◽  
Protective Effects ◽  
Cell Disease ◽  
No Bioavailability

Abstract The primary event in the vaso-occlusive pathophysiology of sickle cell disease (SCD) is polymerization of hemoglobin S under deoxygenated conditions. In SCD, sub-clinical transient vaso-occlusive events caused by red cell sickling are likely to be more frequent resulting in “reperfusion injury” that generates reactive oxygen species and results in chronic oxidative stress that will contribute to multiple organ damage. In fact, previous studies have suggested that sickling is etiologic to repefusion injury and oxidative stress (Kaul and Hebbel, JCI, 2000), although the effect of antisickling therapy on oxidative stress has not been evaluated. Increasing the levels of antisickling fetal hemoglobin (HbF) by hydroxyurea therapy markedly reduces polymer formation. HbF exerts an ameliorating effect in sickle cell disease patients both on red cells and in the prevention of multiple organ damage. Here, we hypothesize that induction of HbF by genetic manipulation (in the absence of pharmacological manipulation) will reduce organ oxidative stress by reducing sickling and hemolysis, and thereby increase NO bioavailability. To test our hypothesis, we measured activity of selected antioxidants and lipid peroxidation (LPO) in BERK mice expressing exclusively human α- and βS-globins and varying levels of HbF, i.e., BERK (<1% HbF), BERKγM (20% HbF) and BERKγH (40% HbF). Percent sickled cells in venous samples (drawn in 2.5% glutaraldehyde solution in 0.1M cacodylate buffer) showed a distinct decrease with increased %HbF (P<0.05, multiple comparisons). Consistent with maximal sickling, BERK mice showed 5.4–6.9-fold increase in LPO in various tissues (muscle, kidney and liver) compared with C57BL controls (P<0.001). In contrast, BERKγM and BERKγH mice showed a marked decrease (73% and 80%, respectively) in LPO compared with BERK mice (P<0.001). Also, activity/levels of antioxidants (superoxide dismutase [SOD], catalase, glutathione peroxidase [GPx] and reduced glutathione [GSH]) showed significant decreases in BERK mice (P<0.001–0.00001). On the other hand, BERKγM and BERKγH mice showed significant increases in antioxidant activity (P<0.05–0.0001). Induction of HbF was associated with increased levels of NO metabolites (NOx) and reduced hemolysis; the latter is in agreement with our previous observations in BERKγM mice (Kaul et al. JCI, 2004). These results strongly suggest that reduced sickling and hemolysis in the presence of HbF cause increased NO bioavailability. NO is well known to exert antioxidative effects. Thus, we show for the first time that the induction of antisickling HbF leads to an increase in NO bioavailability and a decrease in oxidative stress, and that these protective effects are mediated primarily by reduced intravascular sickling.

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