The Gut Microbiome Regulates Psychological-Stress-Induced Inflammation.

Psychological stress has adverse effects on various human diseases, including those of the cardiovascular system. However, the mechanisms by which stress influences disease activity remain unclear. Here, using vaso-occlusive episodes (VOEs) of sickle cell disease as a vascular disease model, we show that stress promotes VOEs by eliciting a glucocorticoid hormonal response that augments gut permeability, leading to microbiota-dependent interleukin-17A (IL-17A) secretion from T helper 17 (Th17) cells of the lamina propria, followed by the expansion of the circulating pool of aged neutrophils that trigger VOEs. We identify segmented filamentous bacteria as the commensal essential for the stress-induced expansion of aged neutrophils that enhance VOEs in mice. Importantly, the inhibition of glucocorticoids synthesis, blockade of IL-17A, or depletion of the Th17 cell-inducing gut microbiota markedly reduces stress-induced VOEs. These results offer potential therapeutic targets to limit the impact of psychological stress on acute vascular occlusion.

ZNF410 Uniquely Activates the NuRD Component CHD4 to Silence Fetal Hemoglobin Expression.

Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplantation settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels.. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells, and its perturbation might offer new opportunities for treatment of hemoglobinopathies.

The genetic dissection of fetal haemoglobin persistence in sickle cell disease in Nigeria.

The clinical severity of sickle cell disease (SCD) is strongly influenced by the level of fetal haemoglobin (HbF) persistent in each patient. Three major HbF loci (BCL11A, HBS1L-MYB, and Xmn1-HBG2) have been reported, but a considerable hidden heritability remains. We conducted a genome-wide association study for HbF levels in 1006 Nigerian patients with SCD (HbSS/HbSß0), followed by a replication and meta-analysis exercise in four independent SCD cohorts (3,582 patients). To dissect association signals at the major loci, we performed stepwise conditional and haplotype association analyses and included public functional annotation datasets. Association signals were detected for BCL11A (lead SNP rs6706648, ß = -0.39, P = 4.96 × 10-34) and HBS1L-MYB (lead SNP rs61028892, ß = 0.73, P = 1.18 × 10-9), whereas the variant allele for Xmn1-HBG2 was found to be very rare. In addition, we detected three putative new trait-associated regions. Genetically, dissecting the two major loci BCL11A and HBS1L-MYB, we defined trait-increasing haplotypes (P < 0.0001) containing so far unidentified causal variants. At BCL11A, in addition to a haplotype harbouring the putative functional variant rs1427407-'T', we identified a second haplotype, tagged by the rs7565301-'A' allele, where a yet-to-be-discovered causal DNA variant may reside. Similarly, at HBS1L-MYB, one HbF-increasing haplotype contains the likely functional small indel rs66650371, and a second tagged by rs61028892-'C' is likely to harbour a presently unknown functional allele. Together, variants at BCL11A and HBS1L-MYB SNPs explained 24.1% of the trait variance. Our findings provide a path for further investigation of the causes of variable fetal haemoglobin persistence in sickle cell disease.

Sickle Cell Disease: From Genetics to Curative Approaches.

Sickle cell disease (SCD) is a monogenic blood disease caused by a point mutation in the gene coding for ß-globin. The abnormal hemoglobin [sickle hemoglobin (HbS)] polymerizes under low-oxygen conditions and causes red blood cells to sickle. The clinical presentation varies from very severe (with acute pain, chronic pain, and early mortality) to normal (few complications and a normal life span). The variability of SCD might be due (in part) to various genetic modulators. First, we review the main genetic factors, polymorphisms, and modifier genes that influence the expression of globin or otherwise modulate the severity of SCD. Considering SCD as a complex, multifactorial disorder is important for the development of appropriate pharmacological and genetic treatments. Second, we review the characteristics, advantages, and disadvantages of the latest advances in gene therapy for SCD, from lentiviral-vector-based approaches to gene-editing strategies.

High-throughput quantification of red blood cell deformability and oxygen saturation to probe mechanisms of sickle cell disease.

The complex, systemic pathology of sickle cell disease is driven by multiple mechanisms including red blood cells (RBCs) stiffened by polymerized fibers of deoxygenated sickle hemoglobin. A critical step toward understanding the pathologic role of polymer-containing RBCs is quantifying the biophysical changes in these cells in physiologically relevant oxygen environments. We have developed a microfluidic platform capable of simultaneously measuring single RBC deformability and oxygen saturation under controlled oxygen and shear stress. We found that RBCs with detectable amounts of polymer have decreased oxygen affinity and decreased deformability. Surprisingly, the deformability of the polymer-containing cells is oxygen-independent, while the fraction of these cells increases as oxygen decreases. We also find that some fraction of these cells is present at most physiologic oxygen tensions, suggesting a role for these cells in the systemic pathologies. Additionally, the ability to measure these pathological cells should provide clearer targets for evaluating therapies.

Effective therapies for sickle cell disease: are we there yet?

Sickle cell disease (SCD) is a common genetic blood disorder associated with acute and chronic pain, progressive multiorgan damage, and early mortality. Recent advances in technologies to manipulate the human genome, a century of research and the development of techniques enabling the isolation, efficient genetic modification, and reimplantation of autologous patient hematopoietic stem cells (HSCs), mean that curing most patients with SCD could soon be a reality in wealthy countries. In parallel, ongoing research is pursuing more facile treatments, such as in-vivo-delivered genetic therapies and new drugs that can eventually be administered in low- and middle-income countries where most SCD patients reside.

Diverse Approaches to Gene Therapy of Sickle Cell Disease.

Sickle cell disease (SCD) results from a single base pair change in the sixth codon of the ß-globin chain of hemoglobin, which promotes aggregation of deoxyhemoglobin, increasing rigidity of red blood cells and causing vaso-occlusive and hemolytic complications. Allogeneic transplant of hematopoietic stem cells (HSCs) can eliminate SCD manifestations but is limited by absence of well-matched donors and immune complications. Gene therapy with transplantation of autologous HSCs that are gene-modified may provide similar benefits without the immune complications. Much progress has been made, and patients are realizing significant clinical improvements in multiple trials using different approaches with lentiviral vector-mediated gene addition to inhibit hemoglobin aggregation. Gene editing approaches are under development to provide additional therapeutic opportunities. Gene therapy for SCD has advanced from an attractive concept to clinical reality.

Accurate long-read sequencing allows assembly of the duplicated RHD and RHCE genes harboring variants relevant to blood transfusion.

Next-generation sequencing (NGS) technologies have transformed medical genetics. However, short-read lengths pose a limitation on identification of structural variants, sequencing repetitive regions, phasing of distant nucleotide changes, and distinguishing highly homologous genomic regions. Long-read sequencing technologies may offer improvements in the characterization of genes that are currently difficult to assess. We used a combination of targeted DNA capture, long-read sequencing, and a customized bioinformatics pipeline to fully assemble the RH region, which harbors variation relevant to red cell donor-recipient mismatch, particularly among patients with sickle cell disease. RHD and RHCE are a pair of duplicated genes located within an ∼175 kb region on human chromosome 1 that have high sequence similarity and frequent structural variations. To achieve the assembly, we utilized palindrome repeats in PacBio SMRT reads to obtain consensus sequences of 2.1 to 2.9 kb average length with over 99% accuracy. We used these long consensus sequences to identify 771 assembly markers and to phase the RHD-RHCE region with high confidence. The dataset enabled direct linkage between coding and intronic variants, phasing of distant SNPs to determine RHD-RHCE haplotypes, and identification of known and novel structural variations along with the breakpoints. A limiting factor in phasing is the frequency of heterozygous assembly markers and therefore was most successful in samples from African Black individuals with increased heterogeneity at the RH locus. Overall, this approach allows RH genotyping and de novo assembly in an unbiased and comprehensive manner that is necessary to expand application of NGS technology to high-resolution RH typing.

Autologous gene therapy for hemoglobinopathies: From bench to patient's bedside.

In recent years, a growing number of clinical trials have been initiated to evaluate gene therapy approaches for the treatment of patients with transfusion-dependent ß-thalassemia and sickle cell disease (SCD). Therapeutic modalities being assessed in these trials utilize different molecular techniques, including lentiviral vectors to add functional copies of the gene encoding the hemoglobin ß subunit in defective cells and CRISPR-Cas9, transcription activator-like effector protein nuclease, and zinc finger nuclease gene editing strategies to either directly address the underlying genetic cause of disease or induce fetal hemoglobin production by gene disruption. Here, we review the mechanisms of action of these various gene addition and gene editing approaches and describe the status of clinical trials designed to evaluate the potentially for these approaches to provide one-time functional cures to patients with transfusion-dependent ß-thalassemia and SCD.

Endothelial VWF is critical for the pathogenesis of vaso-occlusive episode in a mouse model of sickle cell disease.

Vaso-occlusive episode (VOE) is a common and critical complication of sickle cell disease (SCD). Its pathogenesis is incompletely understood. von Willebrand factor (VWF), a multimeric plasma hemostatic protein synthesized and secreted by endothelial cells and platelets, is increased during a VOE. However, whether and how VWF contributes to the pathogenesis of VOE is not fully understood. In this study, we found increased VWF levels during tumor necrosis factor (TNF)-induced VOE in a humanized mouse model of SCD. Deletion of endothelial VWF decreased hemolysis, vascular occlusion, and organ damage caused by TNF-induced VOE in SCD mice. Moreover, administering ADAMTS13, the VWF-cleaving plasma protease, reduced plasma VWF levels, decreased inflammation and vaso-occlusion, and alleviated organ damage during VOE. These data suggest that promoting VWF cleavage via ADAMTS13 may be an effective treatment for reducing hemolysis, inflammation, and vaso-occlusion during VOE.

Phenotypic screening of the ReFRAME drug repurposing library to discover new drugs for treating sickle cell disease.

Stem cell transplantation and genetic therapies offer potential cures for patients with sickle cell disease (SCD), but these options require advanced medical facilities and are expensive. Consequently, these treatments will not be available for many years to the majority of patients suffering from this disease. What is urgently needed now is an inexpensive oral drug in addition to hydroxyurea, the only drug approved by the FDA that inhibits sickle-hemoglobin polymerization. Here, we report the results of the first phase of our phenotypic screen of the 12,657 compounds of the Scripps ReFRAME drug repurposing library using a recently developed high-throughput assay to measure sickling times following deoxygenation to 0% oxygen of red cells from sickle trait individuals. The ReFRAME library is a very important collection because the compounds are either FDA-approved drugs or have been tested in clinical trials. From dose-response measurements, 106 of the 12,657 compounds exhibit statistically significant antisickling at concentrations ranging from 31 nM to 10 µM. Compounds that inhibit sickling of trait cells are also effective with SCD cells. As many as 21 of the 106 antisickling compounds emerge as potential drugs. This estimate is based on a comparison of inhibitory concentrations with free concentrations of oral drugs in human serum. Moreover, the expected therapeutic potential for each level of inhibition can be predicted from measurements of sickling times for cells from individuals with sickle syndromes of varying severity. Our results should motivate others to develop one or more of these 106 compounds into drugs for treating SCD.

Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis induced hepatobiliary injury in sickle cell disease.

Sickle cell disease (SCD) associated chronic hemolysis promotes oxidative stress, inflammation and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD related hepatobiliary injury has not been fully elucidated yet. Here, we show that chronic liver injury in SCD patients is associated with elevated levels of hepatic membrane bound CD163. Hemolysis and increase in hepatic heme, hemoglobin and iron levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that HO-1 positively regulates membrane bound CD163 expression independent of NRF2 signaling in SCD liver. We further demonstrate that of the interaction between CD163 and HO-1 is not dependent on CD163-hemoglobin binding. These findings indicate that CD163 is a potential biomarker of SCD associated hepatobiliary injury. Understanding the role of HO-1 in membrane bound CD163 regulation may help identify novel therapeutic targets for hemolysis induced chronic liver injury.

Nontargeted Plasma Proteomic Analysis of Renal Disease and Pulmonary Hypertension in Patients with Sickle Cell Disease.

Sickle cell disease (SCD) is characterized by red blood cell sickling, vaso-occlusion, hemolytic anemia, damage to multiple organ systems, and, as a result, shortened life expectancy. Sickle cell disease nephropathy (SCDN) and pulmonary hypertension (pHTN) are common and frequently co-occurring complications of SCD; both are associated with markedly accelerated mortality. To identify candidate circulating biomarkers of SCDN and pHTN, we used mass spectrometry to quantify the relative abundance of >1000 proteins in plasma samples from 189 adults with SCD from the Outcome Modifying Genes in SCD (OMG-SCD) cohort (ProteomeXchange identifier PXD048716). Forty-four proteins were differentially abundant in SCDN, most significantly cystatin-C and collagen α-1(XVIII) chain (COIA1), and 55 proteins were dysregulated in patients with SCDN and pHTN, most significantly insulin-like growth factor-binding protein 6 (IBP6). Network analysis identified a module of 133 coregulated proteins significantly associated with SCDN, that was enriched for extracellular matrix proteins, insulin-like growth factor binding proteins, cell adhesion proteins, EGF-like calcium binding proteins, and several cadherin family members. Collectively, these data provide a comprehensive understanding of plasma protein changes in SCDN and pHTN which validate numerous studies of chronic kidney disease and suggest shared profiles of protein disruption in kidney dysfunction and pHTN among SCD patients.

The dawn of a cure for sickle cell disease through CRISPR-based treatment: A critical test of equity in public health genomics.

Equity in access to genomic technologies, resources, and products remains a great challenge. This was evident especially during the coronavirus disease 2019 (COVID-19) pandemic when the majority of lower middle-income countries were unable to achieve at least 10% population vaccination coverage during initial COVID-19 vaccine rollouts, despite the rapid development of those vaccines. Sickle cell disease (SCD) is an inherited monogenic red blood cell disorder that affects hemoglobin, the protein that carries oxygen through the body. Globally, the African continent carries the highest burden of SCD with at least 240,000 children born each year with the disease. SCD has evolved from a treatable to a curable disease. Recently, the UK medical regulator approved its cure through clustered regularly interspaced short palindromic repeat (CRISPR)-based treatment, whereas the US Food and Drug Administration has equally approved two SCD gene therapies. This presents a remarkable opportunity to demonstrate equity in public health genomics. This CRISPR-based treatment is expensive and therefore, a need for an ambitious action to ensure that they are affordable and accessible where they are needed most and stand to save millions of lives.

Lessons learnt in the screening and diagnosis of haemoglobinopathies.

For this paper, cases reported formally and anecdotally to the authors in their screening and diagnostic roles have been selected to demonstrate areas where errors have occurred, and caution should be exercised. The cases demonstrate that it is vital that the performance and limitations of the techniques used, along with the phenotypic presentation of cases where haemoglobin variants and/or thalassaemias are coinherited are understood by those performing result interpretation. Those who deliver the service as well as those who receive reports and give results and counselling should be aware of the complexity of the topic.

Seeing haemoglobin SC: Challenging the misperceptions.

Historically understudied and regarded as a mild type of sickle cell disease, HbSC can be associated with significant, progressive complications. Prospective studies are urgently needed to address treatment gaps for HbSC disease. Commentary on: Nelson et al. The clinical spectrum of HbSC sickle cell disease-not a benign condition. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19523.

Determinants of the haemoglobin level in patients with sickle cell disease living in sub-Saharan Africa: Major impact of the country of residence and independent effects of leucocyte and platelet counts and haemolysis.

The degree of anaemia in sickle cell disease (SCD) is a well-known contributor to morbidity and mortality. We aimed to explore the factors affecting haemoglobin (Hb) level in African SCD patients, considering haemolysis biomarkers (LDH and bilirubin level, and reticulocyte count), leucocyte and platelet counts and socio-demographic characteristics (gender, age group, country of residence and BMI). The research was part of the CADRE multinational cohort and involved 3699 SCD patients living in Mali, Senegal, Ivory Coast, Democratic Republic of Congo, Gabon and Cameroon: 2936 SS/Sß0, 587 SC and 176 Sß + patients with median Hb level of 8, 11.3 and 11.2 g/dL respectively (p < 0.001). In multivariate analysis conducted in 1394 SS/Sß0 patients, living in Cameroon, female gender, lower BMI, higher haemolysis markers (especially LDH) and higher leucocyte and platelet counts were independently associated with lower Hb level (all p < 0.05). In 497 SC and 156 Sß + patients, female gender (p < 0.001), lower BMI (p < 0.05) and higher platelet counts (p < 0.001) were independently associated with lower Hb level. Anaemia in African SCD patients is not only associated with haemolysis but also with the country of residence, lower BMI and leucocyte or platelet counts which might reflect inflammation related to infectious burden in the region.

Low vitamin B12 blood levels in sickle cell disease: Data from a large cohort study in Tanzania.

Sickle cell disease (SCD) is associated with high rates of undernutrition and stunting. Undernutrition in combination with chronic haemolysis may lead to deficiencies in micronutrients necessary for erythropoiesis. Here we examined selected levels of ferritin, vitamins B2 , B6 , B9 and B12 , and vitamin C that were measured in blood samples from 820 SCD patients from Tanzania with no history of hospital admission, infections or painful episodes in the previous 30 days. We studied children (0-8 years), early adolescents (9-14 years), late adolescents (15-17 years) and adults (≥18 years). Severely low levels of vitamin B12 were observed across the four age groups. Despite the lowered vitamin B12 concentrations, total homocysteine concentrations were normal across both genders in all age groups. We found no significant gender-related differences between the other measured micronutrients. In this large SCD population, spanning the whole life cycle, a low level of vitamin B12 was consistently found across both genders and all age groups. Given the pivotal role of vitamin B12 in cellular metabolism, particularly in erythropoiesis, more studies are required to unravel how to better detect clinically relevant vitamin B12 deficiency among SCD patients, and thus to identify more precisely those who need supplementation of vitamin B12 .

Screening for haemoglobin disorders: One size may not fit all.

Accurate laboratory screening for sickle cell disease and other haemoglobin disorders is expanding worldwide. Two new reports describe different methods and strategies for screening in Mali and Denmark, respectively, and their encouraging results suggest that countries should tailor their screening programmes according to local needs, resources and opportunities. Commentary on: Guindo et al. Potential for a large-scale newborn screening strategy for sickle cell disease in Mali: a comparative diagnostic performance study of two rapid diagnostic tests (SickleScan® and HemotypeSC®) on cord blood. Br J Haematol 2024;204:337-345 and Gravholt et al. The Danish national haemoglobinopathy screening programme: report from 16 years of screening in a low-prevalence, non-endemic region. Br J Haematol 2024;204:329-336.

UK Haemoglobin Disorders Peer Review: A Quality Standards-based review programme for sickle cell disease and thalassaemia.

We evaluated the impact of peer reviews in driving improvement in healthcare quality for people with haemoglobinopathy in the United Kingdom. We analysed compliance to four Quality Standards (QS)-based peer reviews from 2010 to 2020 to evaluate its impact in driving healthcare quality. Seventeen paediatric and 29 adult haemoglobinopathy centres were reviewed in 2010/11 and 2012/13 respectively; 33 paediatric and 33 adult centres were reviewed in 2014/16, and 32 paediatric and 32 adult centres were reviewed in 2018/2020. Compliance with QS and participant feedback were analysed to assess the impact of peer review programmes to drive improvement in quality of care. We noted that haemoglobinopathy centres significantly improved their compliance to QS between the first two review programmes, but not in the final review programme. In comparison to other disease-group reviews, the haemoglobinopathy departments were less able to address critical peer review recommendations in their own institutions. The peer review programme was unable to drive sustained improvement in healthcare quality, underscoring the need for sustained development and support for haemoglobinopathy services in the National Health Service. Further work is needed to understand why disparities exist among peer review-driven improvement initiatives within different disease groups.