Search

Mitapivat, a pyruvate kinase (PK) activator, shows great potential as a sickle cell disease (SCD)- modifying therapy. Safety and efficacy of mitapivat as a long-term maintenance therapy is currently being evaluated in two open-label studies. Here we apply a comprehensive multi-omics approach to investigate the impact of activating PK on red blood cells (RBCs) from 15 SCD patients. HbSS patients were enrolled in one of the open label, extended studies (NCT04610866). Leuko-depleted RBCs obtained from fresh whole blood at baseline (visit 1, V1), prior to drug initiation and longitudinal time points over the course of the study were processed for multiomics through a stepwise extraction of metabolites, lipids and proteins. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBCs. Mitapivat decreased 2,3-diphosphoglycerate (DPG) levels, increased adenosine triphosphate (ATP) levels, and improved hematologic and sickling parameters in patients with SCD. Agreement between omics measurements and clinical measurements confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of hemoglobin S (HbS) oxygen affinity (p50) and sickling kinetics (t50) during treatment. Mitapivat markedly reduced levels of proteins of mitochondrial origin within 2 weeks of initiation of drug treatment, with minimal changes in the reticulocyte counts. The first six months of treatment also witnessed transient elevation of lysophosphatidylcholines and oxylipins with depletion in free fatty acids, suggestive of an effect on membrane lipid remodeling. Multi-omics analysis of RBCs identified benefits for glycolysis, as well as activation of the Lands cycle.

Abstract Background There is a renewed interest in lower field magnetic resonance imaging (MRI) systems for cardiovascular magnetic resonance (CMR), due to their favorable physical properties, reduced costs, and increased accessibility to patients with implants. We sought to assess the diagnostic capabilities of high-performance low-field (0.55 T) CMR imaging for quantification of right and left ventricular volumes and systolic function in both healthy subjects and patients referred for clinical CMR. Methods Sixty-five subjects underwent paired exams at 1.5 T using a clinical CMR scanner and using an identical CMR system modified to operate at 0.55 T. Volumetric coverage of the right ventricle (RV) and left ventricles (LV) was obtained using either a breath-held cine balanced steady-state free-precession acquisition or a motion-corrected free-breathing re-binned cine acquisition. Bland-Altman analysis was used to compare LV and RV end-systolic volume (ESV), end-diastolic volume (EDV), ejection fraction (EF), and LV mass. Diagnostic confidence was scored on a Likert-type ordinal scale by blinded readers. Results There were no significant differences in LV and RV EDV between the two scanners (e.g., LVEDV: p = 0.77, bias = 0.40 mL, correlation coefficient = 0.99; RVEDV: p = 0.17, bias = − 1.6 mL, correlation coefficient = 0.98), and regional wall motion abnormality scoring was similar (kappa 0.99). Blood-myocardium contrast-to-noise ratio (CNR) at 0.55 T was 48 ± 7% of the 1.5 T CNR, and contrast was sufficient for endocardial segmentation in all cases. Diagnostic confidence of images was scored as “good” to “excellent” for the two field strengths in the majority of studies. Conclusion A high-performance 0.55 T system offers good bSSFP CMR image quality, and quantification of biventricular volumes and systolic function that is comparable to 1.5 T in patients. Trial registration Clinicaltrials.gov NCT03331380, NCT03581318.

Cell membrane lipid composition, especially cholesterol, affects many functions of embedded enzymes, transporters and receptors in red blood cells (RBC). High membrane cholesterol content affects the RBCs’ main vital function, O2 and CO2 transport and delivery, with consequences on peripheral tissue physiology and pathology. A high degree of deformability of RBCs is required to accommodate the size of micro-vessels with diameters significantly lower than RBCs. The potential therapeutic role of high-density lipoproteins (HDL) in the removal of cholesterol and its activity regarding maintenance of an optimal concentration of RBC membrane cholesterol have not been well investigated. On the contrary, the focus for HDL research has mainly been on the clearance of cholesterol accumulated in atherosclerotic macrophages and plaques. Since all interventions aiming at decreasing cardiovascular diseases by increasing the plasma level of HDL cholesterol have failed so far in large outcome studies, we reviewed the potential role of HDL to remove excess membrane cholesterol from RBC, especially in sickle cell disease (SCD). Indeed, abundant literature supports a consistent decrease in cholesterol transported by all plasma lipoproteins in SCD, in addition to HDL, low- (LDL) and very low-density lipoproteins (VLDL). Unexpectedly, these decreases in plasma were associated with an increase in RBC membrane cholesterol. The concentration and activity of the main enzyme involved in the removal of cholesterol and generation of large HDL particles—lecithin cholesterol ester transferase (LCAT)—are also significantly decreased in SCD. These observations might partially explain the decrease in RBC deformability, diminished gas exchange and tendency of RBCs to aggregate in SCD. We showed that incubation of RBC from SCD patients with human HDL or the HDL-mimetic peptide Fx5A improves the impaired RBC deformability and decreases intracellular reactive oxygen species levels. We propose that the main physiological role of HDL is to regulate the cholesterol/phospholipid ratio (C/PL), which is fundamental to the transport of oxygen and its delivery to peripheral tissues.

Sickle cell disease (SCD) is an exemplar of bidirectional translational research, starting with a remarkable astute observation of the abnormally shaped red blood cells that motivated decades of bench research that have now translated into new drugs and genetic therapies. Introduction of hydroxyurea (HU) therapy, the only SCD-modifying treatment for >30 years and now standard care, was initiated through another clinical observation by a pediatrician. While the clinical efficacy of HU is primarily due to its fetal hemoglobin (HbF) induction, the exact mechanism of how it increases HbF remains not fully understood. Unraveling of the molecular mechanism of how HU increases HbF has provided insights on the development of new HbF-reactivating agents in the pipeline. HU has other salutary effects, reduction of cellular adhesion to the vascular endothelium and inflammation, and dissecting these mechanisms has informed bench—both cellular and animal—research for development of the 3 recently approved agents: endari, voxelotor, and crizanlizumab; truly, a bidirectional bench to bedside translation. Decades of research to understand the mechanisms of fetal to adult hemoglobin have also culminated in promising anti-sickling genetic therapies and the first-in-human studies of reactivating an endogenous (γ-globin) gene HBG utilizing innovative genomic approaches.

The formation of neutrophil extracellular traps (NETs) is thought to play a critical role in infections and propagating sterile inflammation. Histone citrullination is an essential and early step in NETs formation, detectable prior to the formation of the hallmark extracellular DNA-scaffolded strands. In addition to the classical microscopy method, new technologies are being developed for studies of NETs and their detection, both for research and clinical purposes. Classical microscopy studies of NETs are subjective, low throughput and semi-quantitative, and limited in their ability to capture the early steps. We have developed this novel Imaging Flow Cytometry (IFC) method that specifically identifies and quantifies citrullination of histone H4 as a NETs marker and its relationship with other alterations at nuclear and cellular level. These include nuclear decondensation and super-condensation, multi-lobulated nuclei versus 1-lobe nuclei and cell membrane damage. NETs markers can be quantified following variable periods of treatment with NETs inducers, prior to the formation of the specific extracellular DNA-scaffolded strands. Because these high throughput image-based cell analysis features can be performed with statistical rigor, this protocol is suited for both experimental and clinical applications as well as clinical evaluations of NETosis as a biomarker.

Neutrophil extracellular traps (NETs) formation has been implicated in an increasing number of infectious and non-infectious pathologies. NETosis is a tightly regulated process; the end-stage and read-out is the formation of DNA strands extruded from the nuclei, and traditionally assessed by fluorescence microscopy. Since NETosis has emerged as a possible biomarker of the inflammatory process, there is a need for less time-consuming, consistent, and quantitative approaches to improve its application in clinical assessment of pro-inflammatory conditions. Imaging Flow Cytometry (IFC) combines features of conventional flow cytometry with qualitative power of fluorescence microscopy and has an added advantage of the capability of assessing the early processes leading up to extrusion of the DNA-scaffolded strands. We explored the optimal imaging-based tools that can be used to measure citrullination of H4 in early NETosis. IFC identified and quantified histone 4 citrullination (H4cit3) induced with several known NETosis stimuli (Ionophore, PMA, LPS, Hemin, and IL-8) following treatment periods ranging from 2 to 60 min. Its relationship with other alterations at nuclear and cellular level, such as nuclear decondensation and super-condensation, multi-lobulated nuclei vs. 1-lobe nuclei and cell membrane damage, were also quantified. We show that the early progress of the H4cit3 response in NETosis depends on the stimulus. Our method identifies fast (Ionophore and Hemin), intermediate and slow (PMA) inducers and shows that H4cit3 appears to have a limited contribution to both early LPS- and IL-8-induced NETosis. While this method is rapid and of a higher throughput compared to fluorescence microscopy, detection and quantification is limited to H4cit3-mediated nuclear events and is likely to be stimulus- and signaling pathway dependent.

Patients with sickle cell disease (SCD) suffer from anemia and painful vaso-occlusive crisis (VOC) and sometimes need blood transfusions. Delayed hemolytic transfusion reaction (DHTR) is a rare life-threatening complication observed in SCD and mimics VOC. We describe a female SCD patient undergoing three surgical procedures during which DHTR developed following the first two. Prior to a planned tonsillectomy, she received transfusion and three days after surgery developed severe hemolysis as well as pain and respiratory symptoms. On suspicion of VOC, she received additional transfusions and became hemodynamically unstable, and her hemolytic anemia worsened. Gradually, she recovered and could be discharged after two weeks; DHTR was not suspected. Sixteen months later, an arthroplasty was performed due to avascular necrosis, and again she was transfused preoperatively. Similar to the initial surgery, she developed symptoms and signs of VOC after three days, but this time, DHTR was suspected and further transfusions were withheld. Although immunosuppressive medication did not alleviate the condition, she improved on combined treatment with darbepoietin, rituximab, and eculizumab. Six months later, a second arthroplasty was performed uneventfully after prophylaxis with rituximab and without transfusion. DHTR should be considered in the presence of severe, unexplained hemolysis following a recent transfusion, and additional transfusions in this setting should be given only on vital indication.

Abstract: Fetal hemoglobin (HbF) is a strong modifier of sickle cell disease (SCD) severity and is associated with 3 common genetic loci. Quantifying the genetic effects of the 3 loci would specifically address the benefits of HbF increases in patients. Here, we have applied statistical methods using the most representative variants: rs1427407 and rs6545816 in BCL11A, rs66650371 (3-bp deletion) and rs9376090 in HMIP-2A, rs9494142 and rs9494145 in HMIP-2B, and rs7482144 (Xmn1-HBG2 in the β-globin locus) to create g(HbF), a genetic quantitative variable for HbF in SCD. Only patients aged ≥5 years with complete genotype and HbF data were studied. Five hundred eighty-one patients with hemoglobin SS (HbSS) or HbSβ0 thalassemia formed the “discovery” cohort. Multiple linear regression modeling rationalized the 7 variants down to 4 markers (rs6545816, rs1427407, rs66650371, and rs7482144) each independently contributing HbF-boosting alleles, together accounting for 21.8% of HbF variability (r2) in the HbSS or HbSβ0 patients. The model was replicated with consistent r2 in 2 different cohorts: 27.5% in HbSC patients (N = 186) and 23% in 994 Tanzanian HbSS patients. g(HbF), our 4-variant model, provides a robust approach to account for the genetic component of HbF in SCD and is of potential utility in sickle genetic and clinical studies.

Abstract Background In addition to pain, sickle cell anaemia (HbSS) complications include neurocognitive difficulties in attention and processing speed associated with low daytime and night-time oxygen saturation compounded by obstructive sleep apnoea (OSA). In the general population OSA is treated with continuous positive airways pressure (CPAP). The aim of this single-blind, randomised, controlled phase II trial is to compare auto-adjusting CPAP (APAP) with standard care to standard care alone in individuals with HbSS to determine whether the intervention improves attention and processing speed, brain structure, pain and quality of life. Methods/Design Eligibility criteria include: ability to provide informed consent; age > 8 years; diagnosis of HbSS; and mean overnight saturation of 23 years); silent infarction on MRI; minimum overnight oxygen saturation > 90% or

Abstract Background Early detection and interventions have enabled patients with sickle cell disease (SCD) to live well into adulthood. Consequently, the chronicity of SCD allows for the insidious manifestation of multisystem complications, including renal damage. Cystic renal lesions are commonly incidentally discovered on ultrasound and computerised tomography (CT) imaging of the abdomen. Most are benign simple cysts, however, difficulties may be encountered if infection, rupture, haemorrhage or cancerous changes develop. We aimed to determine whether patients with SCD have a higher prevalence of simple renal cysts compared to non-SCD individuals. Methods Data for a group of 223 patients with SCD who had undergone an ultrasound and/or CT imaging of the abdomen were extracted for comparison with 180 control patients (haemoglobin genotype unknown), matched for age and ethnicity. Scans were evaluated for 198 SCD patients and 180 controls. Results Renal cysts were found in 58% of the SCD group and 20% of the controls (OR 5.4 (CI 2.6–11.0), RR 2.8 (CI 1.9–4.2)). Bilateral renal cysts were found in 28% of the SCD participants in comparison with 5% of the control group. In those who had one or more cysts identified, the average number of cysts was 3.76 for the SCD group and 1.94 for the controls. Men with SCD were more likely to develop cysts than women (66% vs 53%), as were men without SCD (22% vs 17%). Conclusions Simple renal cysts occur more frequently, are more abundant and develop at a younger age in patients with SCD than ethnically-matched controls. Further study of the mechanism underlying cyst formation may shed light on both sickle cell nephropathy and other cystic renal diseases.

Fetal hemoglobin (HbF) and peripheral hemoglobin oxygen saturation (SpO2) both predict clinical severity in sickle cell disease (SCD), while reticulocytosis is associated with vasculopathy, but there are few data on mechanisms. HbF, SpO2 and routine clinical and laboratory measures were available in a Tanzanian cohort of 1175 SCD individuals aged ≥ 5 years and the association with SpO2 (as response variable transformed to a Poisson distribution) was assessed by negative binomial model with age and sex as covariates. Increase in HbF was associated with increased SpO2 (rate ratio, RR = 1.19; 95% confidence intervals [CI] 1.04, 1.37 per natural log unit of HbF; p = 0.0004). In univariable analysis, SpO2 was inversely associated with age, reticulocyte count, and log (total bilirubin) and directly with pulse, SBP, hemoglobin, and log(HbF). In multivariable regression log(HbF) (RR 1.191; 95%CI 1.04, 1.37; p = 0.013), pulse (RR 1.01; 95%CI 1.00, 1.01; p = 0.026), SBP (RR 1.008; 95%CI 1.00, 1.02; p = 0.014), and hemoglobin (1.120; 95%CI 1.05, 1.19; p = 0.001) were positively and independently associated with SpO2 while reticulocyte count (RR 0.985; 95%CI 0.97, 0.99; p = 0.019) was independently inversely associated with SpO2. In SCD, improving SpO2, in part through cardiovascular compensation and associated with reduced reticulocytosis, may be a mechanism by which HbF reduces disease severity.

Neutrophilia is a feature of sickle cell disease (SCD) that has been consistently correlated with clinical severity and has been shown to remain highly activated even at steady state. In addition to induction of fetal hemoglobin (HbF), hydroxyurea (HU) leads to reduction in neutrophil count and their adhesion properties, which contributes to the clinical efficacy of HU in SCD. Although HU reduces the frequency and severity of acute vaso-occlusive crises (VOCs) and chest syndrome, HU therapy does not abolish these acute clinical events. In this study we investigated whether neutrophils in SCD patients whilst on HU therapy retained features of detrimental pro-inflammatory activity. Freshly isolated neutrophils from SCD patients on HU therapy at steady state and from ethnic-matched healthy controls were evaluated ex vivo for their degranulation response and production of neutrophil extracellular traps (NETs). Unstimulated SCD patient neutrophils already produced NETs within 30 minutes, compared to none for healthy neutrophils, and by 4 hours, these neutrophils produced significantly more NETs than the control neutrophils (P = 0.0079**). Higher numbers of neutrophils from SCD patients also showed higher degree of degranulation-related intracellular features compared to healthy neutrophils, including rough-textured cellular membranes (P = 0.03*), double-positivity for F-Actin and CD63 (P = 0.02*) and re-located CD63 within cytoplasm more efficiently than their healthy counterparts (P = 0.02*). The neutrophils from SCD donors released more myeloperoxidase (P = 0.02*) in the absence of any trigger. Our data showed that neutrophils from patients with SCD at steady state remained active during hydroxyurea treatment and are likely to be able to contribute to the SCD pro-inflammatory environment.

Silent cerebral infarction is the most common neurological abnormality in children with sickle cell anemia, affecting 30-40% of 14 year olds. There are no known biomarkers to identify children with silent cerebral infarcts, and the pathological basis is also unknown. We used an unbiased proteomic discovery approach to identify plasma proteins differing in concentration between children with and without silent cerebral infarcts. Clinical parameters and plasma samples were analysed from 51 children (mean age 11.8 years, range 6-18) with sickle cell anemia (HbSS). A total of 19 children had silent cerebral infarcts and 32 normal MRI; the children with silent infarcts had lower HbF levels (8.6 vs. 16.1%, P=0.049) and higher systolic blood pressures (115 vs. 108.6, P=0.027). Plasma proteomic analysis showed 13 proteins increased more than 1.3 fold in the SCI patients, including proteins involved in hypercoagulability (α2-antiplasmin, fibrinogen−γ chain, thrombospondin-4), inflammation (α2-macroglobulin, complement C1s and C3), and atherosclerosis (apolipoprotein B-100). Higher levels of gelsolin and retinol-binding protein 4 were also found in the population with silent infarcts, both of which have been linked to stroke. We investigated the genetic basis of these differences by studying 359 adults with sickle cell disease (199 with silent cerebral infarcts, 160 normal MRIs), who had previously undergone a genome-wide genotyping array. None of the genes coding for the differentially expressed proteins were significantly associated with silent infarction. Our study suggests that silent cerebral infarcts in sickle cell anemia may be associated with higher systolic blood pressure, lower HbF levels, hypercoagulability, inflammation and atherosclerotic lipoproteins.

Genetic variants at three quantitative trait loci (QTL) for fetal haemoglobin (HbF), BCL11A, HBS1L-MYB and the β-globin gene cluster, have attracted interest as potential targets of therapeutic strategies for HbF reactivation in sickle cell anaemia (SCA). We carried out the first systematic evaluation of critical single nucleotide polymorphisms at these disease modifier loci in Nigerian patients with SCA. Common variants for BCL11A and HBS1L-MYB were strongly associated with HbF levels. At both loci, secondary association signals were detected, illustrating the mapping resolution attainable in this population. For BCL11A, the two independent sites of association were represented by rs1427407 (primary site, p = 7.0 x 10(-10)) and rs6545816 (secondary site, conditioned on rs1427407: p = 0.02) and for HBS1L-MYB by rs9402686 (HMIP-2B, p = 1.23 x 10(-4)) and rs66650371 (HMIP-2A, p = 0.002). Haplotype analysis revealed similarities in the genetic architecture of BCL11A and HBS1L-MYB in Nigerian patients. Variants at both loci also alleviated anaemia. The variant allele for the γ globin gene promoter polymorphism XmnI-HBG2 was too infrequent in our patients to be evaluated in this relatively small study. Studying the large and diverse SCA patient populations in African countries such as Nigeria will be key for a clearer understanding of how these loci work and for the discovery of new disease modifier genes.

Sickle cell disease is an increasing global health burden. This inherited disease is characterized by a remarkable phenotypic heterogeneity, which can only partly be explained by genetic factors. Environmental factors are likely to play an important role but studies of their impact on disease severity are limited and their results are often inconsistent. This study investigated associations between a range of environmental factors and hospital admissions of young patients with sickle cell disease in London and in Paris between 2008 and 2012. Specific analyses were conducted for subgroups of patients with different genotypes and for the main reasons for admissions. Generalized additive models and distributed lag non-linear models were used to assess the magnitude of the associations and to calculate relative risks. Some environmental factors significantly influence the numbers of hospital admissions of children with sickle cell disease, although the associations identified are complicated. Our study suggests that meteorological factors are more likely to be associated with hospital admissions for sickle cell disease than air pollutants. It confirms previous reports of risks associated with wind speed (risk ratio: 1.06/standard deviation; 95% confidence interval: 1.00–1.12) and also with rainfall (1.06/standard deviation; 95% confidence interval: 1.01–1.12). Maximum atmospheric pressure was found to be a protective factor (0.93/standard deviation; 95% confidence interval: 0.88–0.99). Weak or no associations were found with temperature. Divergent associations were identified for different genotypes or reasons for admissions, which could partly explain the lack of consistency in earlier studies. Advice to patients with sickle cell disease usually includes avoiding a range of environmental conditions that are believed to trigger acute complications, including extreme temperatures and high altitudes. Scientific evidence to support such advice is limited and sometimes confusing. This study shows that environmental factors do explain some of the variations in rates of admission to hospital with acute symptoms in sickle cell disease, but the associations are complex, and likely to be specific to different environments and the individual’s exposure to them. Furthermore, this study highlights the need for prospective studies with large numbers of patients and standardized protocols across Europe.

HbSC disease is the second commonest form of sickle cell disease, with poorly understood pathophysiology and few treatments. We studied the role of K-Cl cotransport activity in determining clinical and laboratory features, and investigated its potential role as a biomarker. Samples were collected from 110 patients with HbSC disease and 41 with sickle cell anemia (HbSS). K-Cl cotransport activity was measured in the oxygenated (K-Cl cotransport100) and deoxygenated (K-Cl cotransport0) states, using radioactive tracer studies. K-Cl cotransport activity was high in HbSC and decreased significantly on deoxygenation. K-Cl cotransport activity correlated significantly and positively with the formation of sickle cells. On multiple regression analysis, K-Cl cotransport increased significantly and independently with increasing reticulocyte count and age. K-Cl cotransport activity was increased in patients who attended hospital with acute pain in 2011 compared to those who did not (K-Cl cotransport100: mean 3.87 versus 3.20, P=0.009, independent samples T-test; K-Cl cotransport0: mean 0.96 versus 0.68, P=0.037). On logistic regression only K-Cl cotransport was associated with hospital attendance. Increased K-Cl cotransport activity was associated with the presence of retinopathy, but this effect was confounded by age. This study links variability in a fundamental aspect of cellular pathology with a clinical outcome, suggesting that K-Cl cotransport is central to the pathology of HbSC disease. Increased K-Cl cotransport activity is associated with increasing age, which may be of pathophysiological significance. Effective inhibition of K-Cl cotransport activity is likely to be of therapeutic benefit.

Patients with sickle cell disease have significant morbidity and mortality. Pulmonary hypertension is suggested to be an important contributor but its nature and severity in these patients and how best to non-invasively assess it are controversial. We hypothesised that a high-output state rather than primary pulmonary vascular pathology may be the major abnormality in sickle cell disease. This study aimed to evaluate the characteristics and severity of pulmonary hypertension in patients with sickle cell disease using detailed echocardiography.We undertook a prospective study in 122 consecutive stable outpatients with sickle cell disease and 30 age, gender and ethnicity-matched healthy controls. Echocardiographic evaluation included 3D ventricular volumes, sphericity, tissue Doppler, and non-invasive estimation of pulmonary vascular resistance. 36% of patients had a tricuspid regurgitant velocity ≥2.5 m.s(-1) but only 2% had elevated pulmonary vascular resistance and the prevalence of right ventricular dysfunction was very low. Patients with raised tricuspid regurgitant velocity had significantly elevated biventricular volumes and globular left ventricular remodelling, related primarily to anaemia. In a subgroup of patients who underwent cardiac catheterization, invasive pulmonary haemodynamics confirmed the echocardiographic findings.Elevated cardiac output and left ventricular volume overload secondary to chronic anaemia may be the dominant factor responsible for abnormal cardiopulmonary haemodynamics in patients with sickle cell disease. 3D echocardiography with non-invasive estimation of pulmonary vascular resistance represents a valuable approach for initial evaluation of cardiopulmonary haemodynamics in sickle cell disease.

Fetal hemoglobin (HbF) is an important modulator of sickle cell disease (SCD). HbF has previously been shown to be affected by variants at three loci on chromosomes 2, 6 and 11, but it is likely that additional loci remain to be discovered.We conducted a genome-wide association study (GWAS) in 1,213 SCA (HbSS/HbSβ0) patients in Tanzania. Genotyping was done with Illumina Omni2.5 array and imputation using 1000 Genomes Phase I release data. Association with HbF was analysed using a linear mixed model to control for complex population structure within our study. We successfully replicated known associations for HbF near BCL11A and the HBS1L-MYB intergenic polymorphisms (HMIP), including multiple independent effects near BCL11A, consistent with previous reports. We observed eight additional associations with P

Studies have questioned whether renal dysfunction in sickle cell disease is linked to hemolysis-associated vasculopathy. We have investigated renal function and markers of hemolysis in a cohort of 424 adult African-British patients with sickle cell disease. While significant associations were found in HbSS and HbSβ0 (sickle cell anemia) patients with and without controlling for covariates between hemolytic markers and albuminuria, the associations were not significant in patients with HbSC. Estimated glomerular filtration rate, a marker of renal function, correlated significantly with reticulocyte count and bilirubin. Alpha thalassemia, present in 34% of the sickle cell anaemia patients, had a protective effect against albuminuria in this group. Altogether, the incidence of hyperfiltration was 71% and microalbuminuria 37%, making nephropathy a common complication of sickle cell anemia.

White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count-6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count-17q21; basophil count- 3p21 near RPN1 and C3orf27; lymphocyte count-6p21, 19p13 at EPS15L1; monocyte count-2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.

Sickle cell anemia is caused by a single type of mutation, a homozygous A→T substitution in the ß globin gene. Clinical severity is diverse, partially due to additional, disease-modifying genetic factors. We are studying one such modifier locus, HMIP (HBS1L-MYB intergenic polymorphism, chromosome 6q23.3). Working with a genetically admixed patient population, we have encountered the necessity to generate haplotype signatures of genetic markers to label genomic fragments with distinct genealogical origin at this locus. With the goal to generate haplotype signatures from patients experimentally, we have investigated the suitability of an existing nanofluidic assay platform to perform phase alignment with single-nucleotide polymorphism alleles.Patient DNA samples were loaded onto Fluidigm Digital Arrays and individual DNA molecules were assayed with allele-specific probes for SNP markers. Here we present data showing the utility of the nanofluidic approach, yielding haplotype data identical to those obtained with a family-based method. We then determined haplotype composition in a group of patients with sickle cell disease, including in those where a mathematical inference approach gave ambiguous or misleading results. Experimental phasing of genotypes across 3.8 kb for rs9399137, rs9402685, and rs11759553 created unequivocal haplotype signatures for each of the patients. In 68 patients, we found 8 copies of a haplotype signature ('C-C-T'), which is known to be prevalent in Europeans but to be absent in West African populations. We have confirmed the identity of our phased allele pairs by single-molecule sequencing and have demonstrated, in principle, that three-allele phasing (using three colors) is a potential extension to this method.Phased haplotypes yield more information than the individual marker genotypes. Procedures such as the one described here would therefore benefit genetic mapping and functional studies as well as diagnostic procedures where the identity or parental origin of short genetic fragments is of importance.

Background Sickle cell anemia is one of the commonest causes of stroke in children. It is usually, but not always, associated with intracranial vasculopathy. We have assessed the value of ultrasound screening for extracranial internal carotid artery disease.Design and Methods Using Doppler ultrasound scanning, we assessed peak systolic blood velocity, tortuosity and stenosis in the extracranial internal carotid arteries of 236 children with sickle cell anemia. Seventeen of the children had previously had a stroke. All measurements were performed as part of routine clinical care.Results The median extracranial internal carotid artery velocity was 148cm/s (5th centile 84, 95th centile 236). Higher velocities were significantly correlated with younger age, higher white blood cell counts and higher rates of hemolysis. Fourteen (5.9%) had tortuous extracranial internal carotid arteries and 13 (5.4%) had stenosis or occlusion. None of the children with tortuous vessels but 8 of those with stenosis had previously had a stroke; the presence of stenosis was strongly associated with overt clinical stroke (OR 35.9, 95% C.I. 9.77–132, P

Background Following a clinical evaluation of deferasirox (Exjade®) it was concluded that, in addition to baseline body iron burden, ongoing transfusional iron intake should be considered when selecting doses. The 1-year EPIC study, the largest ever investigation conducted for an iron chelator, is the first to evaluate whether fixed starting doses of deferasirox, based on transfusional iron intake, with dose titration guided by serum ferritin trends and safety markers, provides clinically acceptable chelation in patients (aged ≥2 years) with transfusional hemosiderosis from various types of anemia.Design and Methods The recommended initial dose was 20 mg/kg/day for patients receiving 2–4 packed red blood cell units/month and 10 or 30 mg/kg/day was recommended for patients receiving less or more frequent transfusions, respectively. Dose adjustments were based on 3-month serum ferritin trends and continuous assessment of safety markers. The primary efficacy end-point was change in serum ferritin after 52 weeks compared with baseline.Results The 1744 patients enrolled had the following conditions; thalassemia (n=1115), myelodysplastic syndromes (n=341), aplastic anemia (n=116), sickle cell disease (n=80), rare anemias (n=43) and other transfused anemias (n=49). Overall, there was a significant reduction in serum ferritin from baseline (−264 ng/mL; P5%) adverse events were gastrointestinal disturbances (28%) and skin rash (10%).Conclusions Analysis of this large, prospectively collected data set confirms the response to chelation therapy across various anemias, supporting initial deferasirox doses based on transfusional iron intake, with subsequent dose titration guided by trends in serum ferritin and safety markers (clinicaltrials.gov identifier: NCT00171821).

Fetal haemoglobin (HbF) is a major ameliorating factor in sickle cell disease. We investigated if a quantitative trait locus on chromosome 6q23 was significantly associated with HbF and F cell levels in individuals of African descent. Single nucleotide polymorphisms (SNPs) in a 24-kb intergenic region, 33-kb upstream of the HBS1L gene and 80-kb upstream of the MYB gene, were typed in 177 healthy Afro-Caribbean subjects (AC) of approximately 7% European admixture, 631 healthy Afro-Germans (AG, a group of African and German descendents located in rural Jamaica with about 20% European admixture), 87 West African and Afro-Caribbean individuals with sickle cell anaemia (HbSS), as well as 75 Northern Europeans, which served as a contrasting population. Association with a tag SNP for the locus was detected in all four groups (AC, P = 0.005, AG, P = 0.002, HbSS patients, P = 0.019, Europeans, P = 1.5 x 10(-7)). The association signal varied across the interval in the African-descended groups, while it is more uniform in Europeans. The 6q QTL for HbF traits is present in populations of African origin and is also acting in sickle cell anaemia patients. We have started to distinguish effects originating from European and African ancestral populations in our admixed study populations.