Human Sickle Cell Disease Research Articles

7568 Sickle Cell Variant Promotes Glycemic Dysregulation In A Mouse Model Of Human Sickle Cell Trait And Sickle Cell Disease

Abstract Disclosure: J.L. Zapater: None. B.T. Layden: None. Since cases of type 2 diabetes mellitus (T2DM) are rising due to environmental and societal factors, it is estimated that in the coming decades that T2DM will affect twenty percent of the United States population and that mortality from diabetic complications will continue to rise. It is therefore critical to accurately diagnose and treat T2DM early, however this proves difficult in certain populations. Sickle cell anemia is a common hemoglobinopathy in which changes in red blood cell turnover lead to inaccurate measurement of hemoglobin A1c and incongruencies between this and other glucose measures, making it difficult to establish a diagnosis of T2DM. The sickle cell variant has been linked to higher hemoglobin A1c, and with 1-in-10 African Americans having the sickle cell trait (SCT), an increasing prevalence of sickle cell disease (SCD), and a higher risk of diabetic complications in those with SCT or SCD, it is critical that we elucidate the precise mechanisms by which the sickle cell variant affects glycemic homeostasis. In this work, we utilized a mouse model of human sickle cell anemia to begin to determine the biochemical mechanisms governing glucose handling in the presence of the sickle cell variant by examining glycemic homeostasis under normal and obesogenic conditions. Townes sickle cell mice containing human hemoglobin with either two normal β-globin chains (littermate controls) or one normal and one sickle β-globin chain (SCT) were placed on either a normal chow diet or on a high fat diet (HFD) until 20 weeks of age to induce an obesogenic state. Although no overt glycemic phenotype was noted, 20-week-old HFD-fed SCT mice exhibited an increased glucose excursion following an oral glucose load compared to littermate controls. Though serum insulin levels were comparable, HFD-fed SCT mice also exhibited a reduced whole-body insulin sensitivity during insulin tolerance testing compared to controls, correlating with the noted increased glycemic excursion. With these findings, we have initiated work with Townes mice containing two normal α-chains and two sickle β-chains (SCD), which is found in human sickle cell anemia. Early results show that chow-fed SCD mice exhibit greater random and fasting glucose levels at 8 and 12 weeks of age, despite similar fasting insulin levels. These findings suggest that the presence of one sickle β-globin chain, as in SCT, promotes hyperglycemia in the obesogenic state through reduction of whole-body insulin sensitivity. Furthermore, the presence of two sickle β-globin chains, as in SCD, is sufficient to dysregulate glycemic control under normal conditions. In total, these findings demonstrate that the presence of the sickle cell variant promotes glucose dysregulation and hyperglycemia, meaning that it is essential to understand the glycemic regulation in patients with sickle cell trait and sickle cell anemia. Presentation: 6/1/2024

Inhibition of sodium-glucose cotransporter-2 improves anaemia in mice and humans with sickle cell disease, and reduces infarct size in a murine stroke model.

Sodium-glucose cotransporter-2 (SGLT-2) is expressed in the kidney and may contribute to anaemia and cardiovascular diseases. The effect of SGLT-2 inhibition on anaemia and vascular endpoints in sickle cell disease (SCD) is unknown. A murine model of SCD was studied to determine the effects of the SGLT-2 inhibitor, empagliflozin, on anaemia and stroke size. The University of Michigan's Precision Health Database was used to evaluate the effect of SGLT-2 inhibitors on anaemia in humans with SCD. SCD mice treated with daily empagliflozin for 8 weeks demonstrated increases in haemoglobin, haematocrit, erythrocyte counts, reticulocyte percentage and erythropoietin compared to vehicle-treated mice. Following photochemical-induced thrombosis of the middle cerebral artery, mice treated with empagliflozin demonstrated reduced stroke size compared to vehicle treated mice. In the electronic health records analysis, haemoglobin, haematocrit and erythrocyte counts increased in human SCD subjects treated with an SGLT-2 inhibitor. SGLT-2 inhibitor treatment of humans and mice with SCD is associated with improvement in anaemic parameters. Empagliflozin treatment is also associated with reduced stroke size in SCD mice suggesting SGLT-2 inhibitor treatment may be beneficial with regard to both anaemia and vascular complications in SCD patients.

Total Synthesis of a PSGL-1 Glycopeptide Analogue for Targeted Inhibition of P-Selectin.

The high affinity interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin is mediated by a multimotif glycosulfopeptide (GSP) recognition domain consisting of clustered tyrosine sulfates and a Core 2 O-glycan terminated with sialyl LewisX (C2-O-sLeX). These distinct GSP motifs are much more common than previously appreciated within a wide variety of functionally important domains involved in protein-protein interactions. However, despite the potential of GSPs to serve as tools for fundamental studies and prospects for drug discovery, their utility has been limited by the absence of chemical schemes for synthesis on scale. Herein, we report the total synthesis of GSnP-6, an analogue of the N-terminal domain of PSGL-1, and potent inhibitor of P-selectin. An efficient, scalable, hydrogenolysis-free synthesis of C2-O-sLeX-Thr-COOH was identified by both convergent and orthogonal one-pot assembly, which afforded this crucial building block, ready for direct use in solid phase peptide synthesis (SPPS). C2-O-sLeX-Thr-COOH was synthesized in 10 steps with an overall yield of 23% from the 4-O,5-N oxazolidinone thiosialoside donor. This synthesis represents an 80-fold improvement in reaction yield as compared to prior reports, achieving the first gram scale synthesis of SPPS ready C2-O-sLeX-Thr-COOH and enabling the scalable synthesis of GSnP-6 for preclinical evaluation. Significantly, we established that GSnP-6 displays dose-dependent inhibition of venous thrombosis in vivo and inhibits vaso-occlusive events in a human sickle cell disease equivalent microvasculature-on-a-chip system. The insights gained in formulating this design strategy can be broadly applied to the synthesis of a wide variety of biologically important oligosaccharides and O-glycan bearing glycopeptides.

Bone-marrow-homing lipid nanoparticles for genome editing in diseased and malignant haematopoietic stem cells.

Therapeutic genome editing of haematopoietic stem cells (HSCs) would provide long-lasting treatments for multiple diseases. However, the in vivo delivery of genetic medicines to HSCs remains challenging, especially in diseased and malignant settings. Here we report on a series of bone-marrow-homing lipid nanoparticles that deliver mRNA to a broad group of at least 14 unique cell types in the bone marrow, including healthy and diseased HSCs, leukaemic stem cells, B cells, T cells, macrophages and leukaemia cells. CRISPR/Cas and base editing is achieved in a mouse model expressing human sickle cell disease phenotypes for potential foetal haemoglobin reactivation and conversion from sickle to non-sickle alleles. Bone-marrow-homing lipid nanoparticles were also able to achieve Cre-recombinase-mediated genetic deletion in bone-marrow-engrafted leukaemic stem cells and leukaemia cells. We show evidence that diverse cell types in the bone marrow niche can be edited using bone-marrow-homing lipid nanoparticles.

Evaluating sheep hemoglobins with MD simulations as an animal model for sickle cell disease

Sickle cell disease (SCD) affects millions worldwide, yet there are few therapeutic options. To develop effective treatments, preclinical models that recapitulate human physiology and SCD pathophysiology are needed. SCD arises from a single Glu-to-Val substitution at position 6 in the β subunit of hemoglobin (Hb), promoting Hb polymerization and subsequent disease. Sheep share important physiological and developmental characteristics with humans, including the same developmental pattern of fetal to adult Hb switching. Herein, we investigated whether introducing the SCD mutation into the sheep β-globin locus would recapitulate SCD’s complex pathophysiology by generating high quality SWISS-MODEL sheep Hb structures and performing MD simulations of normal/sickle human (huHbA/huHbS) and sheep (shHbB/shHbS) Hb, establishing how accurately shHbS mimics huHbS behavior. shHbS, like huHbS, remained stable with low RMSD, while huHbA and shHbB had higher and fluctuating RMSD. shHbB and shHbS also behaved identically to huHbA and huHbS with respect to β2-Glu6 and β1-Asp73 (β1-Asn72 in sheep) solvent interactions. These data demonstrate that introducing the single SCD-causing Glu-to-Val substitution into sheep β-globin causes alterations consistent with the Hb polymerization that drives RBC sickling, supporting the development of a SCD sheep model to pave the way for alternative cures for this debilitating, globally impactful disease.

Iron Restricted Erythropoiesis Under Hepcidin Mimetic Treatment (PN23114) Improved Disease Parameters in a Mouse Model for Sickle Cell Disease

Sickle Cell Disease (SCD) is an inherited hemolytic disorder that results from a single point mutation in the hemoglobin-beta (HBB) gene that encodes for the beta-globin subunit of hemoglobin, causing the production of abnormal hemoglobin S (HbS). HbS polymerizes under low oxygen conditions, causing sickling effects on red blood cells (RBCs). Sickling of RBCs initiates vaso-occlusion, downstream inflammatory responses, and hemolysis (Zhang D, et al., Blood. 2016;127:801-9). Previous studies have shown that polymerization under low oxygen conditions is extremely sensitive to HbS concentrations within the RBCs (Hofrichter J, et al, Proc Natl Acad Sci USA. 1974;71:4864-8; Sunshine HR, et al. Nature. 1978;275:238-40). Controlling iron delivery for erythropoiesis provides a mechanism to potentially down-modulate the HbS concentration, thereby reducing the potential for HbS polymerization and sickling of RBCs (Parrow NL, et al., Blood. 2021;137:1553-5). The potent hepcidin mimetic peptide PN23114 targets the iron exporter membrane protein ferroportin to trigger its degradation, thus preventing iron export from cells. We hypothesize that the resulting pharmacodynamic effect of limiting iron availability for erythropoiesis should reduce HbS concentrations within the RBCs that may prevent sickling and hemolysis. Townes mice (genetically altered to contain the human SCD HbSS homozygous mutation) are a well-established experimental model for human SCD (Li-Chen W, et al., Blood. 2006;108:1183-8). They exhibit severe hemolysis as indicated by elevated bilirubin and lactate dehydrogenase (LDH) levels, and a shortened RBC half-life compared to wild type (WT) mice. These mice also develop vaso-occlusive events and elevated inflammatory responses (for example, increases in peripheral blood white blood cell [WBC] and neutrophil counts). To test whether iron restriction mediated by a hepcidin mimetic may alter outcomes in this model, PN23114 was administered subcutaneously to Townes HbSS mice. Mice were treated over 4-weeks (3 times weekly) with vehicle or PN23114 at 1 or 2.5 mg/kg (vehicle treated WT mice served as controls). At the end of 4 weeks, total bilirubin was elevated in the HbSS-vehicle group compared to WT-vehicle (Figure 1A). Total bilirubin was dose-dependently reduced by PN23114 treatment compared to the HbSS-vehicle group (Figure 1A). LDH was also elevated in the HbSS-vehicle group and was dose-dependently reduced with PN23114 treatment (557±105 units/L in WT-vehicle, 809±145 units/L in HbSS-vehicle, 611±341 units/L in HbSS-PN23114-1mg/kg and 372±71 units/L in HbSS-PN23114-2.5mg/kg groups). Peripheral blood WBC counts were dose-dependently reduced by PN23114 treatment compared to HbSS-vehicle (Figure 1B), with concomitant decreases in peripheral blood neutrophil counts. Reduction in hemoglobin values was observed with PN23114 treatment in HbSS (5.9±0.9 g/dL in HbSS-1mg/kg group and 4.4±0.4 g/dL in HbSS-2.5 mg/kg group) compared to the HbSS-vehicle group (7.1±0.3 g/dL). These results suggest that pharmacologic iron restriction with PN23114, a hepcidin mimetic peptide, led to a reduction in hemolysis and inflammatory markers in a SCD model. Therefore, a hepcidin mimetic therapeutic could potentially be a novel approach to prevent hemolysis, inflammation and downstream clinical complications associated with human SCD.

Pharmacologic Reduction of Platelet Count Attenuates Whole Blood Hypofibrinolysis in a Mouse Model of Sickle Cell Disease

Increased risk of venous thrombosis is a hallmark of sickle cell disease (SCD). We previously reported that SCD dramatically alters the properties of venous clots, including resistance to tissue plasminogen activator (tPA)-mediated fibrinolysis. Using an internal fibrinolysis assay (tPA added to whole blood before clot formation), clot lysis time (CLT) was significantly (p<0.01) prolonged in clots formed ex vivo from whole blood of SCD patients (SS) (651±34 min) compared to race-matched controls (AA) (370±75). We then investigated how therapeutic RBC exchange affects CLT in SCD patients. Interestingly, despite hemoglobin A levels of approximately 50%, CLT for the pre-exchange SS clots (639±81 min) was similar to CLT recorded for blood clots from SS patients who did not undergo RBC exchange (see above). However, CLT for post-exchange SS clots was significantly shortened (240±34; p<0.05 vs pre-exchange) to the level observed for clots from AA controls. We noticed that the RBC exchange procedure not only changes the proportion of AA and SS RBCs, but also reduces both platelet counts and serum plasminogen activator inhibitor-1 (PAI-1) levels by approximately 50-60%. Importantly, exvivo restoration of the platelet number in post-exchange samples to the level observed in pre-exchange blood resulted in restoration of the hypofibrinolytic phenotype and a partial restoration of serum PAI-1 levels. Changes in platelet count after RBC exchange are quickly normalized. Therefore, in the current study, we investigated the effect of sustained reduction of platelet count on whole blood hypofibrinolysis in SCD mice. Using an external fibrinolysis assay (2.5 nM tPA added after clot formation) as described by Bonnard et al. (Sci Rep, 2017) we determined if hypofibrinolysis is also observed in Townes sickle cell mice (SS). Consistent with human SCD, the CLT was profoundly increased in ex vivo clots formed from whole blood of SS mice (n=13) compared to non-sickle (AA) control mice (n=12) (Figure 1A; first two bars). Next, we demonstrated that the cellular fraction of SS blood was responsible for resistance of the clots to tPA-mediated fibrinolysis, regardless of whether the plasma was from SS or AA mice (Figure 1A; last four bars). We then used a pharmacologic strategy to reduce platelet counts. Thrombopoietin (TPO) is a growth factor that promotes maturation of hematopoietic stem cells into megakaryocytes and thereby increases platelet production. We previously showed that silencing liver TPO expression using a liver-specific antisense oligonucleotide (TPO-ASO) resulted in a sustained 50% reduction in both plasma TPO levels and platelet counts without affecting hemostasis in both baboons and mice. In the present study, TPO-ASO (20 mg/kg subcutaneous) or vehicle control were administered to 3-month old AA and SS mice (n=3-6) on days 0, 3, 5 and 7, followed by weekly maintenance injections for the next 3 weeks. 4 weeks after initiation of treatment, mice were euthanized, and blood was collected to evaluate plasma levels of TPO and external whole blood fibrinolysis. Platelet, but no other cell count was significantly reduced in both AA (by 62%, p<0.05) and SS (by 47%, P<0.05) mice treated with TPO-ASO. Baseline plasma levels of TPO were reduced in SS mice (101±20 pg/ml) compared to AA controls (182±10; p<0.05). TPO-ASO treatment resulted in significant reduction of TPO plasma levels in both AA (21±16; p<0.01) and SS (17±9; p<0.05) mice. Interestingly, TPO-ASO-mediated reduction of platelet count did not affect CLT of ex vivo clots formed from whole blood of AA mice (Figure 1B). In contrast, TPO-ASO treatment dramatically reduced CLT in SS clots (p<0.05) (Figure 1B). In conclusion, we demonstrated that: (i) the resistance of ex vivo formed clots to tPA-mediated fibrinolysis observed in SCD patients can be reproduced in a mouse model of the disease; (i) the external fibrinolysis assay provides a robust and reliable model for studying whole blood hypofibrinolysis in SCD; (iii) hypofibrinolysis in SCD appears to be predominantly driven by the cellular fraction of blood; and (iv) platelets play an important role in the hypofibrinolytic phenotype in SCD, demonstrated by mitigation of the hypofibrinolytic phenotype in SS mice subjected to long-term reduction of platelet count. Further studies investigating the effect of platelet count reduction on venous thrombosis and vascular stasis in SS mice are currently ongoing.

Is Epigenetic Modulation to Induce Fetal Hemoglobin Translatable in Sickle Cell Mice?

Sickle cell disease (SCD) is a genetic disorder caused by a mutation in the β-globin subunit of hemoglobin, resulting in the formation of HbS. Under deoxygenation, HbS polymerizes leading to red blood cell sickling, increased hemolysis and multi-organ damage. During human fetal development, due to expression of the γ-globin gene, the predominant type of hemoglobin produced is fetal hemoglobin (HbF). However, after birth, this shifts to adult globin (HbA or HbS in SCD). This is driven by epigenetic repression of γ-globin due to an increased methylation of its gene promoters. DNA methyltransferase 1 (DNMT1) is an important enzyme for DNA methylation and is known to be pivotal in regulation of HbF expression. Reactivation of HbF provides a functional hemoglobin, and therefore has the potential to treat SCD symptoms ( Steinberg et al. JAMA 2003;289:1645-51). HbF induction is an accepted treatment strategy in SCD; even small increases result in decreased vaso-occlusive crises and mortality ( Kato et al. Nat Rev Dis Primers 2018;4:18010). Decitabine inhibits DNMT1 activity, thus promoting γ-globin expression and thereby increasing HbF levels. While inhibition of DNA methylation is known to cause HbF induction in humans and non-human primates, the effects observed in SCD mouse models are more ambiguous. This study investigated to which extent decitabine treatment could induce HbF expression in the Townes SCD model. In Townes mice, the murine globin genes have been substituted for their sickle human counterparts, HBA, HBB sickle and HBG1, and mimic many features of human SCD. Mice aged 4-5 weeks were dosed subcutaneously 3 times a week for 12 weeks with research-grade decitabine (0.6 mg/kg or 0.4 mg/kg) or vehicle. Blood samples were collected for analysis of HbF protein levels by HPLC (represented as % of total Hb) and F-cells by flow cytometry (represented as % of the total RBC population positive for HbF), complete blood counts, histology, and other markers of disease and organ damage. Results are presented as mean ± SD. Groups were compared using an ANCOVA model, adjusting for multiple comparisons (Dunnett) with p<0.05 considered statistically significant. For both treatment groups, HbF was significantly elevated compared to the control group, with the maximal response observed at 12 weeks (Figure 1). F-cells at the end of the study were increased in both 0.4 mg/kg (11.4±1.5%) and 0.6 mg/kg (11.8±1.4%) groups compared to vehicle (4.5±0.6%). The same was observed for protein levels: 0.4 mg/kg group (1.2±0.3%), 0.6 mg/kg (1.4±0.3%) compared to vehicle (0.4±0.1%). Interestingly, after 12 weeks RBC and reticulocyte counts were significantly decreased in treatment groups and mean corpuscular volume (MCV) was significantly increased. No changes on LDH, bilirubin and liver markers (ALP, ALT, AST) were observed at 6 or 12 weeks. The results showed that treatment groups had significantly higher HbF levels than the vehicle group. However, the level of induction was lower than observed for inhibition of DNA methylation in humans and primates. The decrease in RBCs and reticulocytes could be partially explained by a combination of HbF induction, reduction of sickle RBC, and inhibition of erythropoiesis. The lack of changes in disease markers suggests that the increase in HbF levels in Townes mice might not be sufficient to alleviate the disease. This observation is consistent with recently published data (Woodard et al. Dis Model Mech. 2022;15(6)) that indicates that Townes mice are less suitable for studying this mechanism of action since, even though they have all human globin genes and some proximal regulatory elements, they lack key cis-acting distant DNA elements that regulate the globin genes. However, in primates the globin structure and regulation mechanisms are similar to human (Hardison. Cold Spring Harb Perspect Med. 2012;2(12)). In conclusion, although the measured levels of HbF protein were no higher than 2%, we found that decitabine can induce HbF expression in the Townes mouse model of SCD. However, within the 12-weeks, there was no clear beneficial effect on the disease markers studied. The successful induction of HbF suggests that better outcomes might be achieved with further optimization of the dosing and duration of the study. This indicates that SCD mice may be useful for preclinical studies of HbF induction, but caution is needed when extrapolating results to humans.

Red Blood Cell Pyruvate Kinase Properties in Sickle Cell Disease - of Mice and Men

Background: Townes and Berkeley mouse models of sickle cell disease (SCD) are commonly used to study SCD pathophysiology and to develop novel therapies. Although the pathophysiology in these models is similar to the human disease, there are distinct differences. For example, Townes SS mice have higher ATP and lower 2,3-diphosphyglycerate (2,3-DPG) levels compared to Townes AA mice. This contrasts with SCD patients, who show lower ATP and higher 2,3-DPG levels, suggesting potential differences between human and murine red blood cell (RBC) metabolism. Pyruvate kinase (PK), a key ATP-generating enzyme in glycolysis, is a target for novel SCD therapies. While PK activators have been shown to increase hemoglobin in clinical trials, such treatments do not consistently lead to the same hematological change in mouse models, further indicating that PK properties may differ between SCD mouse models and human patients. A detailed comparative characterization of PK and glycolytic metabolites in murine SCD models and SCD patients is therefore important. Aim: To study PK properties in murine and human SCD RBCs. Methods: Wild-type (C57BL/6J), Townes (AA and SS) and Berkeley mice (non-sickling and sickling) were used. Human blood was obtained from healthy controls (HbAA) and untreated SCD patients (HbSS). Hematological parameters were measured using the Sysmex hematology analyzer. PK (Vmax, 5mM substrate) and hexokinase (HK) activity, and PK thermostability (53°C) were measured in purified RBCs. Levels of ATP, 2,3-DPG, as well as reduced glutathione (GSH) were measured by targeted LC-MS/MS and spectrophotometry, respectively. P50 (oxygen tension at which 50% of hemoglobin is saturated with oxygen) was measured with the Hemox analyzer (TCS). Statistical analysis was performed in Graphpad Prism 9.4.1. by Welch's t-test. Results: Samples from 17 wild-type mice, 21 Townes mice (10 AA, 11 SS), 15 Berkeley mice (7 non-sickling, 8 sickling) and 12 human subjects (6 HbAA, 6 HbSS) were included. Both Townes and Berkeley SCD mice showed increased PK activity (Table 1; p<0.0001). This may be attributed to their high reticulocyte counts (Townes SS vs. AA: 43% vs. 4%; Berkeley sickling vs. non-sickling: 30% vs. 6%), because when normalizing PK activity to that of HK, another RBC age-dependent enzyme, no genotype differences were found in these mouse models. This lack of PK/HK difference was in sharp contrast with our finding in human controls vs. SCD patients (Table 1). Notably, both Berkeley sickling and non-sickling mice had lower PK/HK ratio than their Townes counterparts (p<0.0001). Mouse PK thermostability was markedly reduced compared to that of human, and PK from the two sickling mice was less stable than their non-sickling controls (Fig. 1). Moreover, both sickling mice showed higher ATP/2,3-DPG ratio (Table 1; p<0.01). This was again in direct contrast with our observation using human blood, where SCD patients had lower ATP/2,3-DPG ratio than healthy controls (p<0.05). Even though there was no difference in ATP/2,3-DPG ratio between the two SCD models, Townes SS mice had significantly lower 2,3-DPG and higher ATP levels than Townes AA mice, whereas Berkeley sickling mice only had significantly higher ATP level compared to their controls. Furthermore, sickling mice had lower P50 value compared to non-sickling mice, reflecting a higher oxygen affinity. This also contrasted with human SCD patients, who had higher P50 value than healthy controls (Table 1). GSH levels were increased in both SCD patients and sickling mice, although the elevation was more pronounced in sickling mice (Table 1). Conclusions: This study reveals important distinctions between mouse models and humans: no differences in PK/HK ratio between sickling and non-sickling mice, and an overall lower PK thermostability in mice. Furthermore, this study shows significant changes in 2,3-DPG content (important for oxygen affinity), energy level (ATP), oxygen affinity (P50) and antioxidant level (GSH) when comparing sickling to non-sickling mice. Sickling mice also have higher ATP/2,3-DPG ratio, which contrasts with SCD patients. These differences should be considered when studying PK activators in SCD mouse models and extrapolating results from SCD mouse models to humans.

Genetic Variation and Sickle Cell Disease Severity

Sickle cell disease (SCD) is a monogenic disorder, yet clinical outcomes are influenced by additional genetic factors. Despite decades of research, the genetics of SCD remain poorly understood. To assess all reported genetic modifiers of SCD, evaluate the design of associated studies, and provide guidelines for future analyses according to modern genetic study recommendations. PubMed, Web of Science, and Scopus were searched through May 16, 2023, identifying 5290 publications. At least 2 reviewers identified 571 original, peer-reviewed English-language publications reporting genetic modifiers of human SCD phenotypes, wherein the outcome was not treatment response, and the comparison was not between SCD subtypes or including healthy controls. Data relevant to all genetic modifiers of SCD were extracted, evaluated, and presented following STREGA and PRISMA guidelines. Weighted z score meta-analyses and pathway analyses were conducted. Outcomes were aggregated into 25 categories, grouped as acute complications, chronic conditions, hematologic parameters or biomarkers, and general or mixed measures of SCD severity. The 571 included studies reported on 29 670 unique individuals (50% ≤ 18 years of age) from 43 countries. Of the 17 757 extracted results (4890 significant) in 1552 genes, 3675 results met the study criteria for meta-analysis: reported phenotype and genotype, association size and direction, variability measure, sample size, and statistical test. Only 173 results for 62 associations could be cross-study combined. The remaining associations could not be aggregated because they were only reported once or methods (eg, study design, reporting practice) and genotype or phenotype definitions were insufficiently harmonized. Gene variants regulating fetal hemoglobin and α-thalassemia (important markers for SCD severity) were frequently identified: 19 single-nucleotide variants in BCL11A, HBS1L-MYB, and HBG2 were significantly associated with fetal hemoglobin (absolute value of Z = 4.00 to 20.66; P = 8.63 × 10-95 to 6.19 × 10-5), and α-thalassemia deletions were significantly associated with increased hemoglobin level and reduced risk of albuminuria, abnormal transcranial Doppler velocity, and stroke (absolute value of Z = 3.43 to 5.16; P = 2.42 × 10-7 to 6.00 × 10-4). However, other associations remain unconfirmed. Pathway analyses of significant genes highlighted the importance of cellular adhesion, inflammation, oxidative and toxic stress, and blood vessel regulation in SCD (23 of the top 25 Gene Ontology pathways involve these processes) and suggested future research areas. The findings of this comprehensive systematic review and meta-analysis of all published genetic modifiers of SCD indicated that implementation of standardized phenotypes, statistical methods, and reporting practices should accelerate discovery and validation of genetic modifiers and development of clinically actionable genetic profiles.

Analysis of structural effects of sickle cell disease on brain vasculature of mice using three-dimensional quantitative phase imaging.

Although the molecular origins of sickle cell disease (SCD) have been extensively studied, the effects of SCD on the vasculature-which can influence blood clotting mechanisms, pain crises, and strokes-are not well understood. Improving this understanding can yield insight into the mechanisms and wide-ranging effects of this devastating disease. We aim to demonstrate the ability of a label-free 3D quantitative phase imaging technology, called quantitative oblique back-illumination microscopy (qOBM), to provide insight into the effects of SCD on brain vasculature. Using qOBM, we quantitatively analyze the vasculature of freshly excised, but otherwise unaltered, whole mouse brains. We use Townes sickle transgenic mice, which closely recapitulate the pathophysiology of human SCD, and sickle cell trait mice as controls. Two developmental time points are studied: 6-week-old mice and 20-week-old mice. Quantitative structural and biophysical parameters of the vessels (including the refractive index (RI), which is linearly proportional to dry mass) are extracted from the high-resolution images and analyzed. qOBM reveals structural differences in the brain blood vessel thickness (thinner for SCD in particular brain regions) and the RI of the vessel wall (higher and containing a larger variation throughout the brain for SCD). These changes were only significant in 20-week-old mice. Further, vessel breakages are observed in SCD mice at both time points. The vessel wall RI distribution near these breaks, up to away from the breaking point, shows an erratic behavior characterized by wide RI variations. Vessel diameter, tortuosity, texture within the vessel, and structural fractal patterns are found to not be statistically different. As with vessel breaks, we also observe blood vessel blockages only in mice brains with SCD. qOBM provides insight into the biophysical and structural composition of brain blood vessels in mice with SCD. Data suggest that the RI may be an indirect indicator of vessel rigidity, vessel strength, and/or tensions, which change with SCD. Future ex vivo and in vivo studies with qOBM could improve our understanding of SCD.

Moderate hypoxia induces metabolic divergence in circulating monocytes and tissue resident macrophages from Berkeley sickle cell anemia mice.

Human and murine sickle cell disease (SCD) associated pulmonary hypertension (PH) is defined by hemolysis, nitric oxide depletion, inflammation, and thrombosis. Further, hemoglobin (Hb), heme, and iron accumulation are consistently observed in pulmonary adventitial macrophages at autopsy and in hypoxia driven rodent models of SCD, which show distribution of ferric and ferrous Hb as well as HO-1 and ferritin heavy chain. The anatomic localization of these macrophages is consistent with areas of significant vascular remodeling. However, their contributions toward progressive disease may include unique, but also common mechanisms, that overlap with idiopathic and other forms of pulmonary hypertension. These processes likely extend to the vasculature of other organs that are consistently impaired in advanced SCD. To date, limited information is available on the metabolism of macrophages or monocytes isolated from lung, spleen, and peripheral blood in humans or murine models of SCD. Here we hypothesize that metabolism of macrophages and monocytes isolated from this triad of tissue differs between Berkley SCD mice exposed for ten weeks to moderate hypobaric hypoxia (simulated 8,000 ft, 15.4% O2) or normoxia (Denver altitude, 5000 ft) with normoxia exposed wild type mice evaluated as controls. This study represents an initial set of data that describes the metabolism in monocytes and macrophages isolated from moderately hypoxic SCD mice peripheral lung, spleen, and blood mononuclear cells.

Treatment with recombinant ADAMTS13, alleviates hypoxia/reoxygenation-induced pathologies in a mouse model of human sickle cell disease

BackgroundSickle cell disease (SCD) is an inherited red blood cell disorder with a causative substitution in the beta-globin gene that encodes beta-globin in hemoglobin. Furthermore, the ensuing vasculopathy in the microvasculature involves heightened endothelial cell adhesion, inflammation, and coagulopathy, all of which contribute to vaso-occlusive crisis (VOC) and the sequelae of SCD. In particular, dysregulation of the von Willebrand factor (VWF) and a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13) axis has been implicated in human SCD pathology. ObjectivesTo investigate the beneficial potential of treatment with recombinant ADAMTS13 (rADAMTS13) to alleviate VOC. MethodsPharmacologic treatment with rADAMTS13 in vitro or in vivo was performed in a humanized mouse model of SCD that was exposed to hypoxia/reoxygenation stress as a model of VOC. Then, pharmacokinetic, pharmacodynamic, and behavioral analyses were performed. ResultsAdministration of rADAMTS13 to SCD mice dose-dependently increased plasma ADAMTS13 activity, reduced VWF activity/antigen ratios, and reduced baseline hemolysis (free hemoglobin and total bilirubin) within 24 hours. rADAMTS13 was administered in SCD mice, followed by hypoxia/reoxygenation stress, and reduced VWF activity/antigen ratios in parallel to significantly (p < .01) improved recovery during the reoxygenation phase. Consistent with the results in SCD mice, we demonstrate in a human in vitro system that treatment with rADAMTS13 counteracts the inhibitory activity of hemoglobin on the VWF/ADAMTS13-axis. ConclusionCollectively, our data provide evidence that relative ADAMTS13 insufficiency in SCD mice is corrected by pharmacologic treatment with rADAMTS13 and provides an effective disease-modifying approach in a human SCD mouse model.

Endothelial Extracellular Vesicles Regulate Platelet Purinergic Signaling in Sickle Cell Disease

Introduction. Acute chest syndrome (ACS) is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. ACS is often preceded by thrombocytopenia and involves massive thrombosis across pulmonary artery branches. Although, released during hemolysis, adenosine diphosphate (ADP) is known to activate platelets by stimulating their P2Y1 and P2Y12 purinergic receptors, antagonists of P2Y12 have not shown any benefit in ACS therapy, justifying the need for better understanding of purinergic signaling in SCD. CD39 maintains ADP homeostasis by degrading excessive ADP. Though CD39 inhibits ADP-dependent platelet activation, its role in the pathophysiology of SCD is still unidentified. Methods. To evaluate SCD platelet response to ADP in vivo we used a state-of-the-art intravital lung microscopy and a novel in vivo model of ADP-triggered thrombocytopenia in transgenic humanized SCD mice. Additionally, both mouse and human SCD platelet ADP-dependent aggregation was examined using in vitro turbidimetric aggregation assay. Hemin, a major host-derived damage associated molecular pattern associated with SCD, was incubated with Human Lung Micro-Vascular Endothelial Cells (HMVEC-L). Extracellular vesicles (EVs) were obtained from mouse and human plasma samples, and cell culture medium samples using size exclusion chromatography. Obtained EVs were subjected to nanoparticle tracking analysis. EVs CD39 levels and activity were determined using ELISA, western blot and malachite green phosphate assays, respectively. Results. Intravital lung microscopy and in vivo thrombocytopenia studies revealed that intravascular administration of ADP triggered acute pulmonary thrombosis in control but not in SCD mice. In vitro aggregation study confirmed our in vivo findings and demonstrated impaired SCD mouse and human platelet response to ADP, which was, further, significantly augmented by a specific CD39 inhibitor. Hemin triggered shedding of CD39-bearing EVs by HMVEC-L. Indeed, we discovered that isolated from mouse and human plasma endothelial cell-derived SCD EVs expressed higher CD39 levels and activity in comparison to control EVs. Conclusions. Our findings suggest that CD39-bearing EVs prevent ADP-mediated platelet aggregation and pulmonary thrombosis in SCD. Current study explains why P2Y12 blockers are not effective in SCD therapy.

Novel Sheep Model of Sickle Cell Disease Reproduces Human Clinical and Laboratory Parameters

Sickle cell disease (SCD), the most common form of an inherited blood disorder, affects nearly 100,000 patients in the US and millions worldwide. SCD is caused by a point mutation in the β-globin gene that results in the replacement of the glutamic acid at position 6 of the protein with valine, causing polymerization of hemoglobin and red blood cell (RBC) sickling. The sheep β-globin gene shares approximately 87.5% identity with the human gene, and sheep hematopoietic development parallels that of humans, including a developmental γ- to β-globin switch, making sheep an ideal model in which to study SCD. After performing protein modeling to ascertain that insertion of the mutation present in human SCD patients into the sheep β-globin gene induced conformational changes consistent with human sickling, we used CRISPR/Cas9 editing technologies and subsequent somatic cell nuclear transfer (SCNT) to introduce the single nucleotide mutation present in human SCD into the β-globin locus of male sheep fibroblasts (both in a single allele and biallelically). Fibroblasts harboring the desired biallelic mutation were clonally expanded, and SCNT and subsequent embryo transfers were performed, that resulted in 3 SCD lambs. Here, we report on the hematological evaluation of these 3 animals. Blood from three sickle cell edited lambs (SCEL) and age-matched controls (AMC) were evaluated by the same diagnostic methods used in humans: hematocrit, blood smears, reticulocyte counts, alkaline and capillary hemoglobin (Hb) electrophoresis, Hb solubility tests, and ektacytometry. Peripheral blood (PB) smears in SCEL exhibit sickled erythrocytes, acanthocytes, target cells, normoblasts, and polychromasia, while AMC displayed normal PB smears. Reticulocyte counts via flow cytometry were slightly elevated in SCEL when compared to AMC. All 3 SCEL displayed positive SickleDex tests at birth and at all time points analyzed thereafter - these tests were consistently negative in AMC. Alkaline and capillary Hb electrophoresis demonstrated the presence of an abnormal Hb with the same migration pattern as the human HbS, and the presence of HbF at birth. HbF waned progressively to disappear by 3 months, being replaced by HbS in SCEL and normal adult Hb in AMC. In addition, ektacytometry demonstrated a leftward shift of the elongation index-osmolality curves. These data indicate that introduction of the human SCD mutation in the sheep β-globin gene results in a similar phenotype at the blood level, thereby potentially producing a similar disease phenotype and progression in the sheep model. Our findings thus support the translational relevance of the SCD sheep model and highlight the potential for its future use in the development of novel therapies for SCD.

Antiviral Response and HIV-1 Inhibition in Sickle Cell Disease

BACKGROUND: A number of host restriction factors controls HIV-1 infection. While many of these factors have been investigated in vitro, their role in controlling HIV-1 infection in vivo remains to be elucidated. Here, we investigate a naturally occurring HIV-1 restriction in Sickle cell disease (SCD), a hereditary disorder characterized by sickle cell hemoglobin polymerization and red blood cells hemolysis. We previously showed that SCD PBMCs express higher levels of ferroportin (FPN), and that reduced intracellular iron leads to the upregulation of antiviral restriction factors. Previously, HIV-1 infection was shown to inversely correlate with the expression levels of transferrin receptor (TFR), a marker of reduced intracellular iron. Iron chelation also potently inhibits HIV-1 replication as shown by us and others. Thus, iron metabolism is intrinsically connected to the HIV-1 infection and disease progression. Our study in vitro suggests that SCD environment leads to the antiviral state of T cells and macrophages, and suppression of HIV-1 infection. Here we analyzed HIV infection of SCD mice using mouse adapted EcoHIV and compared antiviral factors in SCD mouse spleenocytes and human SCD PBMCs. METHODS: The study was approved by Howard University IRB and IACUC committees. Townes SCD mice carrying human normal α (hα) and sickle hemoglobin beta (βS) genes and control Townes mice carrying human normal hα and βA genes were used in the study. EcoHIV virus obtained from David Volsky (Albert Einstein University) was constructed in pNL4-3 vector with the substitution of gp120 with the envelope-coding gp80 region from ectopic MLV. Whole blood samples were obtained from 9 SCD patients and 9 healthy individuals. RNA Seq analysis was conducted on Illumina® NextSeq 500 and the data were mapped and analyzed using Dragen software. Normalized gene counts were used for gene set enrichment analysis (GSEA) and Gene ontology (GO) analysis. RNA expression was measured by quantitative RT-PCR (Roche). Protein expression in mouse splenocytes was determined by ELISA. RESULTS: Infection of control mice with EcoHIV virus, which contained MLV envelope and was able to infect mouse cells, led to robust viral replication in spleen at 7 days post infection, as detected by the expression of HIV-1 Nef mRNA (Fig. 1) and Gag mRNA. We also detected HIV-1 p24 in splenic tissue. Comparing to the control, SCD mice showed significantly reduced EcoHIV replication both at the levels of RNA expression (Figs 1) and p24 production. GSEA analysis against the 72 genes that included all known HIV-1 restriction factors revealed strong enrichment of these genes in the human activated SCD PBMCs compared to the activated control PBMCs (Fig.2). These genes included APOBEC3A, HMOX1, CH25H, IFIT3, CDKN1A, APOBEC3B, IFITM3, SAMHD1 and TSPO. GSEA analysis also showed upregulation of several pathogen-associated molecular patterns sensing genes including NOD1, NLRC4, TRL7 and TLR9 in human PBMCS. In SCD mice splenocytes, we detected overexpression of FPN, and transferrin receptor (Tfr) genes indicating upregulation of iron metabolism similar to previously observed in human SCD PBMCs. In mouse splenocytes we detected upregulation of CDKN1A, HMOX1, IFITM3, CH25H and TSPO mRNAs. Protein expression analysis showed upregulation of FPN, TFR, HO-1, APOBEC, CH25H, IFIT3 and TSPO in SCD mouse splenocytes. CONCLUSION: Together, these data reveal, for the first time, that HIV-1 infection is suppressed in vivo in SCD mouse infected with the mouse-adapted EcoHIV. SCD mouse splenocytes expressed higher levels of FPN and TFR, and exhibited higher expression of antiviral factor, which were also observed in human SCD PBMCs. The observed upregulation of similar sets of antiviral factors in SCD PBCMs and SCD mice splenocytes suggests that these factors upregulation might be driven by the SCD-related processes such as hemolysis. Taken together, our findings point to a novel mechanism of upregulation of antiviral factors mediated by SCD that included induction of antiviral, heme- and iron- regulatory pathways. ACKNOWLEDGEMENTS: We thank Dr. David Volsky for the gift of EcoHIV and the George Washington University SPH Genomics Core for library preparation and sequencing. This work was supported by NIH grants 1R01HL125005, 5U54MD007597, 1P30AI117970-06 and 1SC1HL150685. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sickle Cell Disease Patient Plasma Sensitizes iPSC-Derived Sensory Neurons from Sickle Cell Disease Patients

Individuals living with sickle cell disease (SCD) experience severe recurrent acute and chronic pain. The underlying neurobiology of the complex pain of SCD is not well understood. Limited and often ineffective treatment options and multifactorial healthcare disparities create an immediate need to develop non-opioid based SCD pain therapies. However, in order to develop novel therapies, we must better understand the neurobiological mechanisms underlying SCD pain. Although humanized mouse models of SCD do exist, growing evidence indicates there are striking differences in gene expression and functional responsiveness between human and mouse dorsal root ganglia (DRG) sensory neurons. Human DRG tissues are extremely limited in availability, postmortem tissues only provide static, endpoint molecular signatures, and obtaining DRGs from patients with SCD is untenable. These are all barriers to gaining mechanistic insight into pathogenic pain processes. Human induced pluripotent stem cell (iPSC)-derived sensory neurons (iSNs) have recently been used to model the altered function and expression of molecules in native DRG neurons in chronic pain disorders. These iPSC-derived SNs express canonical markers of human nociceptors and exhibit functional responses to noxious stimuli similar to those properties of native mammalian nociceptors. The objective of this study was to utilize iSNs differentiated from SCD patient iPSCs as well as age- and race-matched healthy controls (HCs) to characterize baseline receptor expression and function of SCD iSNs compared to HC iSNs. Our goal is to assess the intrinsic properties of SCD nociceptors that may contribute to the persistent sensitization of nociceptors in SCD patients with pain. To this end, both HC and SCD iSNs expressed transcripts for canonical markers of mature sensory neurons (NF200, CGRP), immune receptors (CCR1/2/3/5, CD4, CRLF2, CSF2Ra/b), and functional ion channels (HCN1, Nav1.7, P2X3/4, CACNA2D1, CHRNA9) (Table 1). Further, they expressed transcripts for ion channels relevant to SCD pain (Piezo1, Piezo2, TRPA1, TRPC5, TRPM3, TRPM8, TRPV1, TRPV4). We confirmed the expression of Peripherin, CGRP, TRPV1 at the protein level using immunocytochemistry. There were no significant differences in expression for any of these targets in iSNs derived from SCD patients compared to those from HCs. However, we did identify trends for higher endothelin 1 receptor type A (ETRa) and CC motif chemokine ligand 2 (CCL2) transcript production by SCD iSNs compared to HC iSNs (Table 1). Additionally, we observed robust responses with no significant differences in baseline function between HC and SCD iSNs when stimulated with known agonists of human SNs (KCl, Glutamate, ab-meATP, capsaicin) in traditional or high-throughput calcium imaging studies. To begin defining extrinsic mechanisms that may sensitize human SCD SNs, we tested the effects of SCD patient plasma (n=4 from SCD baseline [SS BL] and acute pain [SS IP]- paired samples, n=4 Black controls [HC]) on iSN hypersensitivity by exposing iSNs in vitro to human plasma diluted 1:10 in maintenance media for 30 minutes before analyzing calcium flux. Plasma samples from SCD patients experiencing acute pain (SS IP) were found to significantly increase SCD iSN response to capsaicin (p<0.05) compared to HC iSNs treated with SS IP plasma, while race-matched HC plasma or SCD patient plasma at baseline pain states (SS BL) did not induce this effect (Figure 1). Excitingly, these findings reproduce results identified in native DRG neurons from SCD mice and suggest that iSNs derived from patient iPSCs can be used in parallel with native SCD neurons from mouse models to investigate mechanisms underlying the chronic sensory neuron sensitization in SCD. They also identify potential intrinsic mechanisms of SCD pain which may extend beyond a blood-based disease pathology. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Extended Spectrum β-Lactamase Producing Lactose Fermenting Bacteria Colonizing Children with Human Immunodeficiency Virus, Sickle Cell Disease and Diabetes Mellitus in Mwanza City, Tanzania: A Cross-Sectional Study.

Rectal carriage of extended spectrum β-lactamase-lactose fermenters (ESBL-LF) is the major risk factor for the development of subsequent endogenous infections. This study determined the patterns and factors associated with the rectal carriage of ESBL-LF among children with Human Immunodeficiency Virus (HIV), Diabetes Mellitus (DM), and Sickle Cell Disease (SCD) attending clinics at different health care facilities in the city of Mwanza, Tanzania. A cross-sectional study was conducted among children living with HIV (n = 236), DM (n = 42) and SCD (n = 126) between July and September 2021. Socio-demographic and clinical data were collected using a structured questionnaire. Rectal swabs/stool samples were collected and processed to detect the rectal carriage of ESBL-LF following laboratory standard operating procedures (SOPs). Descriptive statistical analysis was conducted using STATA 13.0. The overall prevalence of ESBL-LF carriage was 94/404 (23.3%). Significantly higher resistance was observed to ampicillin, trimethoprim-sulfamethoxazole, and tetracycline among Enterobacteriaceae isolated from HIV infected children than in non-HIV infected children (p < 0.05). The commonest ESBL allele 45/62 (72.6%) detected was blaCTX-M. Generally, a parent’s low education level was found to be associated with ESBL-LF colonization among children living with HIV; (OR 4.60 [95%CI] [1.04–20], p = 0.044). A higher proportion of ESBL-LF from DM 10/10 (100%) carried ESBL genes than ESBL-LF from HIV 37/56 (66.1%) and SCD 15/28 (53.6%), p = 0.02. There is a need to collect more data regarding trimethoprim-sulfamethoxazole (SXT) prophylaxis and antibiotic resistance to guide the decision of providing SXT prophylaxis in HIV-infected children especially at this time, when testing and treatment is carried out.

Salubrinal induces fetal hemoglobin expression via the stress-signaling pathway in human sickle erythroid progenitors and sickle cell disease mice.

Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, chronic hemolysis, and progressive organ damage. This disease affects ~100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2α and ATF4 trans-activation in the γ-globin gene promoter. Sickle erythroid progenitors treated with 24μM SAL increased F-cells levels 1.4-fold (p = 0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p = 0.0441), along with dose-dependent increases of p-eIF2α and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p = 0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.

Trends and causes of maternal death at the Lagos University teaching hospital, Lagos, Nigeria (2007\u20132019)

BackgroundNigeria has one of the worst global maternal mortality ratios (MMR). Institutional audit is invaluable in providing useful information for formulating preventive interventions.ObjectiveTo evaluate the trends, causes and socio-demographic characteristics of maternal mortality at a tertiary institution in South-western Nigeria over a period of 13 years (2007–2019).MethodologyCross sectional and temporal trend analysis of maternal deaths were conducted using chart reviews from 2007 to 2019. Socio-biological characteristics, booking status and ranking of clinical causes of maternal deaths were derived from patients’ chart review (2007–2019). Bivariate analysis and Annual percent change (APC) of the observed trends was respectively conducted using Stata version 17 and Joinpoint version 4.5.0.1 software respectively.ResultsIn the period 2007–2019, the mean age at maternal death was 30.8 ± 5.9 years and 88.8% of mortalities occurred among the “unbooked” women. The leading causes of direct maternal mortality were Hypertension (27.0%), Sepsis (20.6%) and haemorrhage (18.7%), while anaemia in pregnancy (3.2%), Human Immunodeficiency Virus (3.2%) and Sickle Cell Disease (2.4%) were the leading indirect causes of maternal mortality Joinpoint estimates showed a statistically significant increase in MMR of about 3.4% per annum from 2211 per 100,000 live births in 2007 to 3555.6 per 100,000 live births in 2019 (APC: +3.4%, P-value < 0.001).ConclusionContrary to some other reports, there was an increase in the institutional MMR between 2007 and 2019 even though the leading causes of death remained similar. Targeted interventions based on accurate data are urgently required in order to achieve the Sustainable Development Goal (SDG) 3.1.