Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner.

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1-10 µM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca2+ influx. Yoda1 induced Ca2+ -dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo-/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.

Variation and impact of polygenic hematologic traits in monogenic sickle cell disease.

Several of the complications observed in sickle cell disease (SCD) are influenced by variation in hematologic traits (HT), such as fetal hemoglobin (HbF) level and neutrophil count. Previous large-scale genome-wide association studies carried out in largely healthy individuals have identified thousands of variants associated with HT, which have then been used to develop multi-ancestry polygenic trait scores (PTS). Here, we tested whether these PTS associate with HT in SCD patients and if they can improve statistical models associated with SCD-related complications. In 2,056 SCD patients, we found that the PTS predicted less HT variance than in non-SCD individuals of African ancestry. This was particularly striking at the Duffy/DARC locus, where we observed an epistatic interaction between the SCD genotype and the Duffy null variant (rs2814778) that led to a two-fold weaker effect on neutrophil count. PTS for these HT which are measured as part of routine practice were not associated with complications in SCD. In contrast, we found that a simple PTS for HbF that includes only six variants explained a large fraction of the phenotypic variation (20.5-27.1%), associated with acute chest syndrome and stroke risk, and improved the statistical modeling of the vaso-occlusive crisis rate. Using Mendelian randomization, we found that increasing HbF by 4.8% reduces stroke risk by 39% (P=0.0006). Taken together, our results highlight the importance of validating PTS in large diseased populations before proposing their implementation in the context of precision medicine initiatives.

Increased retention of functional mitochondria in mature sickle red blood cells is associated with increased sickling tendency, hemolysis and oxidative stress.

Abnormal retention of mitochondria in mature red blood cells (RBC) has been recently reported in sickle cell anemia (SCA) but their functionality and their role in the pathophysiology of SCA remain unknown. The presence of mitochondria within RBC was determined by flow cytometry in 61 SCA patients and ten healthy donors. Patients were classified according to the percentage of mature RBC with mitochondria contained in the whole RBC population: low (0-4%), moderate (>4% and <8%), or high level (>8%). RBC rheological, hematological, senescence and oxidative stress markers were compared between the three groups. RBC senescence and oxidative stress markers were also compared between mature RBC containing mitochondria and those without. The functionality of residual mitochondria in sickle RBC was measured by high-resolution respirometry assay and showed detectable mitochondrial oxygen consumption in sickle mature RBC but not in healthy RBC. Increased levels of mitochondrial reactive oxygen species were observed in mature sickle RBC when incubated with Antimycin A versus without. In addition, mature RBC retaining mitochondria exhibited greater levels of reactive oxygen species compared to RBC without mitochondria, as well as greater Ca2+, lower CD47 and greater phosphatidylserine exposure. Hematocrit and RBC deformability were lower, and the propensity of RBC to sickle under deoxygenation was higher, in the SCA group with a high percentage of mitochondria retention in mature RBC. This study showed the presence of functional mitochondria in mature sickle RBC, which could favor RBC sickling and accelerate RBC senescence, leading to increased cellular fragility and hemolysis.

Clinically relevant updates of the HbVar database of human hemoglobin variants and thalassemia mutations.

HbVar (http://globin.bx.psu.edu/hbvar) is a widely-used locus-specific database (LSDB) launched 20 years ago by a multi-center academic effort to provide timely information on the numerous genomic variants leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Here, we report several advances for the database. We made clinically relevant updates of HbVar, implemented as additional querying options in the HbVar query page, allowing the user to explore the clinical phenotype of compound heterozygous patients. We also made significant improvements to the HbVar front page, making comparative data querying, analysis and output more user-friendly. We continued to expand and enrich the regular data content, involving 1820 variants, 230 of which are new entries. We also increased the querying potential and expanded the usefulness of HbVar database in the clinical setting. These several additions, expansions and updates should improve the utility of HbVar both for the globin research community and in a clinical setting.

Sublingual Microcirculation Specificity of Sickle Cell Patients: Morphology of the Microvascular Bed, Blood Rheology, and Local Hemodynamics.

Patients with sickle cell disease (SCD) have poorly deformable red blood cells (RBC) that may impede blood flow into microcirculation. Very few studies have been able to directly visualize microcirculation in humans with SCD. Sublingual video microscopy was performed in eight healthy (HbAA genotype) and four sickle cell individuals (HbSS genotype). Their hematocrit, blood viscosity, red blood cell deformability, and aggregation were individually determined through blood sample collections. Their microcirculation morphology (vessel density and diameter) and microcirculation hemodynamics (local velocity, local viscosity, and local red blood cell deformability) were investigated. The De Backer score was higher (15.9 mm-1) in HbSS individuals compared to HbAA individuals (11.1 mm-1). RBC deformability, derived from their local hemodynamic condition, was lower in HbSS individuals compared to HbAA individuals for vessels < 20 µm. Despite the presence of more rigid RBCs in HbSS individuals, their lower hematocrit caused their viscosity to be lower in microcirculation compared to that of HbAA individuals. The shear stress for all the vessel diameters was not different between HbSS and HbAA individuals. The local velocity and shear rates tended to be higher in HbSS individuals than in HbAA individuals, notably so in the smallest vessels, which could limit RBC entrapment into microcirculation. Our study offered a novel approach to studying the pathophysiological mechanisms of SCD with new biological/physiological markers that could be useful for characterizing the disease activity.

The CirPAD, a circular 1.4†M hybrid pixel detector dedicated to X-ray diffraction measurements at Synchrotron SOLEIL.

One of the challenges of all synchrotron facilities is to offer the highest performance detectors for all their specific experiments, in particular for X-ray diffraction imaging and its high throughput data collection. In that context, the DiffAbs beamline, the Detectors and the Design and Engineering groups at Synchrotron SOLEIL, in collaboration with ImXPAD and Cegitek companies, have developed an original and unique detector with a circular shape. This detector is based on the hybrid pixel photon-counting technology and consists of the specific assembly of 20 hybrid pixel array detector (XPAD) modules. This article aims to demonstrate the main characteristics of the CirPAD (for Circular Pixel Array Detector) and its performance - i.e. excellent pixel quality, flat-field correction, high-count-rate performance, etc. Additionally, the powder X-ray diffraction pattern of an LaB6 reference sample is presented and refined. The obtained results demonstrate the high quality of the data recorded from the CirPAD, which allows the proposal of its use to all scientific communities interested in performing experiments at the DiffAbs beamline.

Increased blood viscosity and red blood cell aggregation in patients with COVID-19.

The aim of this study was to (1) analyze blood viscosity, red blood cell (RBC) deformability, and aggregation in hospitalized patients with Coronavirus disease 19 (COVID-19); (2) test the associations between impaired blood rheology and blood coagulation; and (3) test the associations between impaired blood rheology and several indicators of clinical severity. A total of 172 patients with COVID-19, hospitalized in COVID-unit of the Internal Medicine Department (Lyon, France) participated in this study between January and May 2021. Clinical parameters were collected for each patient. Routine hematological/biochemical parameters, blood viscosity, RBC deformability and aggregation, and RBC senescence markers were measured on the first day of hospitalization. A control group of 38 healthy individuals was constituted to compare the blood rheological and RBC profile. Rotational thromboelastography was performed in 76 patients to study clot formation dynamics. Our study demonstrated that patients with COVID-19 had increased blood viscosity despite lower hematocrit than healthy individuals, as well as increased RBC aggregation. In-vitro experiments demonstrated a strong contribution of plasma fibrinogen in this RBC hyper-aggregation. RBC aggregation correlated positively with clot firmness, negatively with clot formation time, and positively with the length of hospitalization. Patients with oxygen supplementation had higher RBC aggregation and blood viscosity than those without, and patients with pulmonary lesions had higher RBC aggregation and enhanced coagulation than those without. This study is the first to demonstrate blood hyper-viscosity and RBC hyper-aggregation in a large cohort of patients with COVID-19 and describe associations with enhanced coagulation and clinical outcomes.

Comparisons of oxygen gradient ektacytometry parameters between sickle cell patients with or without α-thalassaemia.

The present study tested the impact of α-thalassaemia on oxygen gradient ektacytometry in sickle cell anaemia (SCA). Three SCA groups were compared: (i) no α-thalassaemia (four α-genes, n = 62), (ii) silent α-thalassaemia (three α-genes, n = 35) and (iii) homozygous α-thalassaemia (two α-genes, n = 12). Red blood cell (RBC) deformability measured in normoxia was not different between the three groups. The lowest RBC deformability reached at low oxygen partial pressure (pO2 ) was greater and the pO2 at which RBC started to sickle was lower in the two α-genes group compared to the other groups. Our present study showed an effect of α-thalassaemia on oxygen gradient ektacytometry in SCA.

A Particular SORL1 Micro-haplotype May Prevent Severe Liver Disease in a French Cohort of Alpha 1-Antitrypsin-deficient Children.

ABSTRACT: The presence of modifier genes is now well recognized in severe liver disease outcome associated with alpha-1-antitrypsin deficiency (A1ATD) but their identification remains to be fully elucidated. To address this goal, we performed a candidate gene study with the SORL1 gene, already identified as risk gene in early-onset Alzheimer Disease families. A particular SORL1 micro-haplotype constituted with 3 SNPs (wild-type form TTG) was genotyped on 86 ZZ A1ATD children issued from 66 families. Interestingly, the mutated forms of this micro-haplotype (CAT most of the time) were associated with lower occurrence of severe liver disease and in cellulo studies showed that SORL1 influences Z-A1ATD cellular toxicity and biogenesis. These data suggest that the mutated CAT form of SORL1 micro-haplotype may partly prevent from severe liver disease in A1ATD children. Overall, these findings support a replication study on an independent cohort and additional in cellulo studies to confirm these promising results.

Impact of a submaximal mono-articular exercise on the skeletal muscle function of patients with sickle cell disease.

PURPOSE: Sickle cell disease (SCD) patients exhibit a limited exercise tolerance commonly attributed to anaemia, as well as hemorheological and cardio-respiratory abnormalities, but the functional status of skeletal muscle at exercise is unknown. Moreover, the effect of SCD genotype on exercise tolerance and skeletal muscle function has been poorly investigated. The aim of this study was to investigate skeletal muscle function and fatigue during a submaximal exercise in SCD patients. METHODS: Nineteen healthy individuals (AA), 28 patients with sickle cell anaemia (SS) and 18 with sickle cell-haemoglobin C disease (SC) performed repeated knee extensions exercise (FAT). Maximal isometric torque (Tmax) was measured before and after the FAT to quantify muscle fatigability. Electromyographic activity and oxygenation by near-infrared spectroscopy of the Vastus Lateralis were recorded. RESULTS: FAT caused a reduction in Tmax in SS (- 17.0 ± 12.1%, p < 0.001) and SC (- 21.5 ± 14.5%, p < 0.05) but not in AA (+ 0.58 ± 29.9%). Root-mean-squared value of EMG signal (RMS) decreased only in SS after FAT, while the median power frequency (MPF) was unchanged in all groups. Oxygenation kinetics were determined in SS and AA and were not different. CONCLUSION: These results show skeletal muscle dysfunction during exercise in SCD patients, and suggest different fatigue aetiology between SS and SC. The changes in EMG signal and oxygenation kinetics during exercise suggest that the greater skeletal muscle fatigue occurring in SCD patients would be rather due to intramuscular alterations modifications than decreased tissue oxygenation. Moreover, SS patients exhibit greater muscle fatigability than SC.

Goal-Oriented Monitoring of Cyclosporine Is Effective for Graft-versus-Host Disease Prevention after Hematopoietic Stem Cell Transplantation in Sickle Cell Disease and Thalassemia Major.

Graft-versus-host disease (GVHD) is an important challenge and a major cause of morbidity and mortality in children after hematopoietic stem cell transplant (HSCT). Herein we report our institution's experience of goal-oriented Bayesian monitoring for cyclosporine (CsA) used alone as GVHD prophylaxis during the post-transplant period in pediatric patients with thalassemia major (TM) or sickle cell anemia (SCA) undergoing HLA-matched HSCT. We also studied evolution of chimerism. Twenty-six consecutive patients (SCA, 14; TM, 12) underwent matched sibling donor (MSD) HSCT from 2004 to 2014. All patients received a myeloablative conditioning regimen. GVHD prophylaxis consisted of 20 mg/kg antithymocyte globulin in the conditioning regimens and then CsA alone in the post-transplant period. Target CsA trough blood concentration (TBC) was 150 ± 20 ng/mL. At last follow-up, all patients were alive and free of disease, even in cases of mixed chimerism. Engraftment occurred in all patients. No patient developed grades II to IV acute GVHD, 4 patients developed acute grade I skin GVHD, and only 1 presented with chronic pulmonary GVHD. A better control of GVHD and immunosuppression by a strict monitoring of CsA TBC as described herein is promising and could play a crucial role. Further investigations are required, but this study opens new perspectives to improve survival and safety of HSCT from alternative donors in TM and SCA to levels compatible with that obtained with MSDs.

Appropriate thresholds for accurate screening for ß-thalassemias in the newborn period: results from a French center for newborn screening.

Objectives: Newborn screening (NBS) for ß-thalassemia is based on measuring the expression of the hemoglobin A (HbA) fraction. An absence or very low level of HbA at birth may indicate ß-thalassemia. The difficulty is that the HbA fraction at birth is correlated with gestational age (GA) and highly variable between individuals. We used HbA expressed in multiples of the normal (MoM) to evaluate relevant thresholds for NBS of ß-thalassemia. Methods: The chosen threshold (HbA≤0.25 MoM) was prospectively applied for 32 months in our regional NBS program for sickle cell disease, for all tests performed, to identify patients at risk of ß-thalassemia. Reliability of this threshold was evaluated at the end of the study. Results: In all, 343,036 newborns were tested, and 84 suspected cases of ß-thalassemia were detected by applying the threshold of HbA≤0.25 MoM. Among the n=64 cases with confirmatory tests, 14 were confirmed using molecular analysis as ß-thalassemia diseases, 37 were confirmed as ß-thalassemia trait and 13 were false-positive. Determination of the optimum threshold for ß-thalassemia screening showed that HbA≤0.16 MoM had a sensitivity of 100% and a specificity of 95.3%, whatever the GA. Conclusions: NBS for ß-thalassemia diseases is effective, regardless of the birth term, using the single robust threshold of HbA≤0.16 MoM. A higher threshold would also allow screening for carriers, which could be interesting when ß-thalassemia constitutes a public health problem.

Impact of a 10 km running trial on eryptosis, red blood cell rheology, and electrophysiology in endurance trained athletes: a pilot study.

PURPOSE: Blood rheology is a key determinant of blood flow and tissue perfusion. There are still large discrepancies regarding the effects of an acute running exercise on blood rheological properties and red blood cell (RBC) physiology. We investigated the effect of a 10 km running trial on markers of blood rheology and RBC physiology in endurance trained athletes. METHODS: Blood was sampled before and after the exercise to measure lactate and glucose, hematological and hemorheological parameters (blood viscosity, RBC deformability, and aggregation), eryptosis markers (phosphatidylserine and CD47 exposure, RBC reactive oxygen species), RBC-derived microparticles (RBC-MPs), and RBC electrophysiological activity. Weight was measured before and after exercise. Peripheral oxygen saturation and heart rate were monitored before and during the trial. RESULTS: Blood lactate and glucose levels increased after exercise and subjects significantly lost weight. All athletes experienced a significant fall in oxygen saturation. Mean corpuscular volume (MCV) was increased from 95.1 ± 3.2 to 96.0 ± 3.3 and mean corpuscular hemoglobin concentration (MCHC) decreased after exercise suggesting a slight RBC rehydration. Exercise increased RBC deformability from 0.344 ± 0.04 to 0.378 ± 0.07, decreased RBC aggregates strength and blood viscosity, while hematocrit (Hct) remained unaffected. While RBC electrophysiological recording suggested a modulation in RBC calcium content and/or chloride conductance, eryptosis markers and RBC-MPs were not modified by the exercise. CONCLUSION: A 10 km acute running exercise had no effect on RBC senescence and membrane blebbing. In contrast, this exercise increased RBC deformability, probably through rehydration process which resulted in a decrease in blood viscosity.

Impact of Trail Running Races on Blood Viscosity and Its Determinants: Effects of Distance.

Blood rheology is a key determinant of tissue perfusion at rest and during exercise. The present study investigated the effects of race distance on hematological, blood rheological, and red blood cell (RBC) senescence parameters. Eleven runners participated in the Martigny-Combes à Chamonix 40 km race (MCC, elevation gain: 2300 m) and 12 others in the Ultra-Trail du Mont Blanc (UTMB, 171 km, elevation gain: 10,000 m). Blood samples were collected before and after the races. After the UTMB, the percentage of RBC phosphatidylserine (PS) exposure was not affected while RBC CD235a levels decreased and RBC-derived microparticles increased. In contrast, after the MCC, RBC PS exposure increased, while RBC CD235a and RBC-derived microparticles levels were not affected. The free hemoglobin and hemolysis rate did not change during the races. RBC aggregation and blood viscosity at moderate shear rates increased after the MCC. RBC deformability, blood viscosity at a high shear rate, and hematocrit decreased after the UTMB but not after the MCC. Our results indicate that blood rheology behavior is different between a 40 km and a 171 km mountain race. The low blood viscosity after the ultra-marathon might facilitate blood flow to the muscles and optimize aerobic performance.

Liver disease related to alpha1-antitrypsin deficiency in French children: The DEFI-ALPHA cohort.

BACKGROUND & AIMS: To identify prognostic factors for liver disease in children with alpha-1 antitrypsin deficiency, irrespective of phenotype, using the DEFI-ALPHA cohort. METHODS: Retrospective, then prospective from 2010, multicentre study including children known to have alpha-1 antitrypsin blood concentration below 0.8 g/L, born in France since 1989. Clinical and biological data were collected. Liver disease was classified as "severe" (portal hypertension, liver failure, liver transplantation or death); "moderate" (persistent abnormal liver biology without portal hypertension); and "mild/none" (normal or almost normal liver biology and native liver). Prognostic factors for severe liver disease were evaluated using a Cox semiparametric model. RESULTS: In January 2017, 153 patients from 19 centres had been included; genotypes were PIZZ in 81.9%, PISZ in 8.1%, other in 10.0%. Mean ± SD follow-up was 4.7 ± 2.1 years. Half of patients had moderate liver disease. Twenty-eight children (18.3%) had severe liver disease (mean age 2.5 years, range: 0-11.6): diagnosis of alpha-1 antitrypsin deficiency was made before two months of age in 65.4%, genotypes were PIZZ in 25 (89.3%), PISZ in 2, PIMlike Z in 1, 15 children underwent liver transplantation, 1 child died at 3 years of age. Neonatal cholestasis was significantly associated with severe liver disease (P = 0.007). CONCLUSION: Alpha-1 antitrypsin-deficient patients presenting with neonatal cholestasis were likely to develop severe liver disease. Some patients with non-homozygous ZZ genotype can develop severe liver disease, such as PISZ and M variants, when associated with predisposing factors. Further genetic studies will help to identify other factors involved in the development of liver complications.

Combined and differential effects of alpha-thalassemia and HbF-quantitative trait loci in Senegalese hydroxyurea-free children with sickle cell anemia.

BACKGROUND: Our objective was to investigate the combined and differential effects of alpha-thalassemia -3.7 kb deletion and HbF-promoting quantitative trait loci (HbF-QTL) in Senegalese hydroxyurea (HU)-free children and young adults with sickle cell anemia (SCA). PROCEDURE: Steady-state biological parameters and vaso-occlusive crises (VOC) requiring emergency admission were recorded over a 2-year period in 301 children with SCA. The age of the first hospitalized VOC was also recorded. These data were correlated with the alpha-globin and HbF-QTL genotypes. For the latter, three different genetic loci were studied (XmnI, rs7482144; BCL11A, rs1427407; and the HBS1L-MYB region, rs28384513) and a composite score was calculated, ranging from zero (none of these three polymorphisms) to six (all three polymorphisms at the homozygous state). RESULTS: A positive clinical impact of the HbF-QTL score on VOC rate, HbF, leucocytes, and C-reactive protein levels was observed only for patients without alpha-thalassemia deletion. Conversely, combination of homozygous -3.7 kb deletion with three to six HbF-QTL was associated with a higher VOC rate. The age of the first hospitalized VOC was delayed for patients with one or two alpha-thalassemia deletions and at least two HbF-QTL. CONCLUSION: Alpha-thalassemia -3.7 kb deletion and HbF-QTL are modulating factors of SCA clinical severity that interact with each other. They should be studied and interpreted together and not separately, at least in HU-free children.

An Additional Case of Hb Saint Nazaire [ß103(G5)Phe→Ile; HBB: c.310T>A] Leading to Moderate Erythrocytosis in a French Family.

We report two members of a French family who are carriers of a rare hemoglobin (Hb) variant leading to erythrocytosis: Hb Saint Nazaire [ß103(G5)Phe→Ile; HBB: c.310T>A]. The proband is a 38-year-old woman referred to our institution for a moderate but persistent polycythemia without any clinical consequence. As her mother had a similar blood count, a diagnosis of a Hb variant with high oxygen affinity was proposed. The variant was difficult to detect by capillary electrophoresis (CE) and not distinguishable by high performance liquid chromatography (HPLC) and isoelectric focusing. Finally, a heterozygous mutation on the HBB gene corresponding to Hb Saint Nazaire was identified. This case report illustrates that this rare cause of erythrocytosis can be easily under or misdiagnosed unless several Hb separation techniques are used.

Genetic Background of ß-Thalassemia in Northeast Algeria with Assessment of the Thalassemia Severity Score and Description of a new ß0-Thalassemia Frameshift Mutation (HBB: c.374dup; p.Pro126Thrfs*15).

ß-Thalassemia (ß-thal) is a genetic disorder representing a major health problem in Algeria. Our first objective was to determine the allelic frequencies and molecular spectrum of ß-thal mutations in patients with major hemoglobinopathies [ß-thal major (ß-TM) and sickle cell disease] in three provinces of northeast Algeria. Our second objective was to assess if the clinical management of ß-TM patients depended on their region of origin. Our last objective was to assess a population originating from Maghreb, the reliability of the thalassemia severity score (TSS) for patients with homozygous ß-thal. Sanger HBB gene sequencing was performed on 59 patients with sickle cell disease and 60 with ß-TM. For the latter patients, the genetic modifiers of the TSS were genotyped: α-thalassemia (α-thal) deletions and four Hb F-inducing polymorphisms (XmnI, rs1427407 and rs10189857 for BCL11A and rs9399137 for HMIP). Eleven different ß-thal mutations were found but two of them (HBB: c.118C>T and HBB: c.93-21G>A) accounted for about 70.0% of the ß-thal alleles. A relatively high proportion of Hb S (HBB: c.20A>T)/ß-thal genotypes (27.0%) was found in our sickle cell disease cohort where a new frameshift ß0-thal mutation (HBB: c.374dup; p.Pro126Thrfs*15) was identified. No difference was found in the three provinces. Of the 60 ß-TM patients, those with a high or very high TSS were significantly younger at the age of first transfusion, thus assessing the reliability of this scoring system in a Maghrebin cohort. Trends for a lower age of splenectomy and high ferritin levels were also detected for the higher TSS categories.