Fetal Blood Research Articles

Exploration of a Novel Noninvasive Prenatal Testing Approach for Monogenic Disorders Based on Fetal Nucleated Red Blood Cells.

Due to technical issues related to cell-specific capture methods, amplification, and sequencing, noninvasive prenatal testing (NIPT) based on fetal nucleated red blood cells (fNRBCs) has rarely been used for the detection of monogenic disorders. Maternal peripheral blood was collected from 11 families with hereditary hearing loss. After density gradient centrifugation and cellular immunostaining for multiple biomarkers, candidate individual fetal cells were harvested by micromanipulation and amplified by whole-genome amplification (WGA). Whole-exome sequencing/whole-genome sequencing (WGS) and Sanger sequencing were performed on the identified fNRBCs to determine the fetal genotype. The impact of single-cell and pooled WGA products on the sequencing quality and results was compared. A combined analysis strategy, encompassing whole-exome sequencing/WGS, haplotype analysis, and Sanger sequencing, was used to enhance the NIPT results. fNRBCs were harvested and identified in 81.8% (9/11) of families. The results of cell-based-NIPT (cb-NIPT) were consistent with those of invasive prenatal diagnosis in 8 families; the coincidence rate was 88.9% (8/9). The combined analysis strategy improved the success of cb-NIPT. The overall performance of pooled WGA products was better than that of individual cells. Due to a lack of alternative fetal cells or sufficient sequencing data, cb-NIPT failed in 3 families. We developed a novel fNRBC-based NIPT method for monogenic disorders. By combining multiple analysis strategies and multiple fetal cell WGA products, the problem of insufficient genome information in a single cell was remedied. Our method has promising prospects in the field of NIPT for the detection of monogenic disorders.

Tissue distribution, placental transfer and excretion of silver nanoparticles in pregnant rats after a single oral dose.

A quantitative assessment of silver nanoparticles (AgNPs) in fluids and some organs of pregnant rats as well as their fetal blood were carried out in this study. A single oral dose (1mg/kg) of AgNPs with a size range of 4-20 nm was administered to pregnant rats on the 19th of gestation. Five groups were euthanized after 10 min, 1, 6, 12, and 24 hr as well as the control group. Total Silver (Ag) contents were measured in bloods (maternal and fetal) and several organs using Inductive Coupled Plasma Optical Emission Spectroscopy (ICP-OES) followed by acid digestion. In maternal blood, AgNPs were found to increase time-dependently after 12 and 24 hr into 0.135 and 0.224 μg/ml, but it was slightly higher in fetal blood (0.32 and 0.31 μg/ml) after 10 min and 1 hr. In other samples: kidneys, liver, spleen, placenta, and uterus the data indicated that NPs were rapidly absorbed from the dosing site (gastrointestinal tract) as evidenced by the detection of Ag in the analyzed samples (fluids and tissues). On the other hand, the cumulative percentages of excretion level in urine was 8.25% which was higher than in feces (4.77%) after 24 hr. These findings indicate the ability of AgNPs to accumulate in pregnant rats and transfer to their fetus imposing adverse outcomes and male formation. Thus, further investigations must be followed for direct and/or indirect exposure to such NPs before decision for their practices.

The influence of insulin-related genetic variants on fetal growth, fetal blood flow, and placental weight in a prospective pregnancy cohort

The fetal insulin hypothesis proposes that low birthweight and type 2 diabetes (T2D) in adulthood may be two phenotypes of the same genotype. In this study we aimed to explore this theory further by testing the effects of GWAS-identified genetic variants related to insulin release and sensitivity on fetal growth and blood flow from week 20 of gestation to birth and on placental weight at birth. We calculated genetic risk scores (GRS) of first phase insulin release (FPIR), fasting insulin (FI), combined insulin resistance and dyslipidaemia (IR + DLD) and insulin sensitivity (IS) in a study population of 665 genotyped newborns. Two-dimensional ultrasound measurements with estimation of fetal weight and blood flow were carried out at week 20, 25, and 32 of gestation in all 665 pregnancies. Birthweight and placental weight were registered at birth. Associations between the GRSs and fetal growth, blood flow and placental weight were investigated using linear mixed models. The FPIR GRS was directly associated with fetal growth from week 20 to birth, and both the FI GRS, IR + DLD GRS, and IS GRS were associated with placental weight at birth. Our findings indicate that insulin-related genetic variants might primarily affect fetal growth via the placenta.

Epigenetically-driven persistence of inflammatory and oxidative phenotypes in adolescents born to women with gestational diabetes

Abstract Background According to the theory of developmental origins of health and disease1, environmental changes during the in-utero period can permanently affect health and vulnerability to diseases later in life. As a matter of fact, the offspring of women with gestational diabetes (GD) exhibit a higher risk for developing obesity, type 2 diabetes (T2D), and atherosclerotic cardiovascular disease (ASCVD) than those individuals with a family history of diabetes2–4. Importantly, epigenetics has emerged as a crucial determinant for this transmission across generations5–8. Mixed Lineage Leukemia 1 (MLL1) histone methyltransferase activates inflammatory and oxidative pathways through trimethylation of histone 3 at lysine 4 amino (H3K4me3)9. Purpose To investigate whether such epigenetic mark regulates genes triggering inflammation and oxidative stress in GD women, and its potential transmission and persistence in their offspring. Methods We isolated peripheral blood mononuclear cells (PBMCs), fetal cord blood mononuclear cells (CBMCs), and umbilical cord vein endothelial cells (HUVECs) from GD and control pregnant women, as well as PBMCs and plasma samples from a retrospective cohort of adolescents (12-16 years old) born to control or GD women. Gene and protein expressions were investigated by RT-PCR, immunoblotting and/or confocal microscopy. H3K4me3 levels on NF-kB p65 and NADPH oxidase isoform 4 (NOX4) promoter regions were assessed by chromatin immunoprecipitation (ChIP) and RT-PCR. Mechanistic studies were performed in control- and GD-HUVECs using the MLL1 inhibitor MM-102 to assess downstream inflammatory mediators and superoxide anion (O2-) generation by electron spin resonance (ESR) spectroscopy. Results Our data showed upregulation of MLL1 and H3K4me3 levels in PBMCs, HUVECs, and CBMCs isolated from GD as compared to control women. ChIP analysis revealed H3K4me3 enrichment in the promoter region of the inflammatory and oxidative master regulatory genes NF-kB p65 and NOX4, respectively, in these cells. We also found an upregulation of NF-kB p65 target genes IL-6, MCP-1, ICAM-1, and VCAM-1, and increased monocyte adhesion and O2- production. The inhibition of MLL1 was able to reduce H3K4me3 levels blunting the inflammatory and oxidative phenotypes in GD-HUVECs. Interestingly enough, we demonstrated a persistent increase of H3K4me3 in the NF-kB p65 promoter region of PBMCs together with an elevation of inflammatory markers in plasma from adolescents born to GD women. Conclusions Our results suggest that GD may induce inflammation and oxidative stress through MLL1-dependent H3K4me3 mechanisms. Such epigenetic mark is transmitted to the offspring and, most importantly, can still be found in adolescence as a pro-inflammatory trigger. These findings pave the way for pharmacological reprogramming of adverse histone modifications to counteract early abnormal phenotypes in GD offspring which may accelerate ASCVD burden.

Inhibitory effects of SARS-CoV-2 spike protein and BNT162b2 vaccine on erythropoietin-induced globin gene expression in erythroid precursor cells from patients with β-thalassemia

During the recent coronavirus disease 2019 (COVID-19) pandemic several patients with β-thalassemia have been infected by severe acute respiratory syndrome coronavirus (SARS-CoV-2), and most patients were vaccinated against SARS-CoV-2. Recent studies demonstrate an impact of SARS-CoV-2 infection on the hematopoietic system. The main objective of this study was to verify the effects of exposure of erythroid precursor cells (ErPCs) from patients with β-thalassemia to SARS-CoV-2 spike protein (S-protein) and the BNT162b2 vaccine. Erythropoietin (EPO)-cultured ErPCs have been either untreated or treated with S-protein or BNT162b2 vaccine. The employed ErPCs were from a β-thalassemia cellular Biobank developed before the COVID-19 pandemic. The genotypes wereβ+-IVSI-110/β+-IVSI-110 (one patient), β039/β+-IVSI-110 (3 patients), and β039/β039 (2 patients). After treatment with S-protein or BNT162b2 for 5 days, lysates were analyzed by high performance liquid chromatography (HPLC), for hemoglobin production, and isolated RNA was assayed by RT-qPCR, for detection of globin gene expression. The main conclusions of the results obtained are that SARS-CoV-2 S-protein and BNT162b2 vaccine (a) inhibit fetal hemoglobin (HbF) production by β-thalassemic ErPCs and (b) inhibit γ-globin mRNA accumulation. In addition, we have performed in silico studies suggesting a high affinity of S-protein to HbF. Remarkably, the binding interaction energy of fetal hemoglobin to S-protein was comparable with that of angiotensin-converting enzyme 2 (ACE2). Our results are consistent with the hypothesis of a relevant impact of SARS-CoV-2 infection and COVID-19 vaccination on the hematopoietic system.

Differences in Cerebral Tissue Oxygenation in Preterm Neonates Receiving Adult or Cord Blood Red Blood Cell Transfusions

Repeated transfusions in preterm neonates with anemia of prematurity replace fetal hemoglobin (HbF) with adult Hb (HbA), which has a low oxygen affinity. The reduction of HbF is associated with a higher incidence of retinopathy of prematurity (ROP). To assess whether HbF and HbA are differently associated with cerebral tissue oxygenation in preterm neonates. This cohort study was a single-center, pilot study on cerebral oxygenation kinetics in preterm neonates with a gestational age between 24.0 weeks and 27.9 weeks who were admitted to the neonatal intensive care unit of Policlinico Universitario A. Gemelli IRCCS from December 27, 2021, to May 15, 2023. This study was ancillary to the ongoing, double-blind, multicenter Umbilical or Adult Donor Red Blood Cells in Extremely Low Gestational Age Neonates and Retinopathy of Prematurity (BORN) randomized clinical trial. The BORN trial outcome was ROP severity in neonates randomized to receive standard packed red blood cell (PRBC) transfusions obtained from RBCs of adult donors (A-RBCs) or from cord blood (CB-RBCs). According to standard procedures at the institute's neonatal intensive care unit, patients concurrently received continuous cerebral near-infrared spectroscopy (NIRS) monitoring. This cohort study was not prespecified in the trial protocol. Transfusion with A-RBCs or CB-RBCs. The main outcome was the kinetics of cerebral regional oxygen saturation (crSO2) and cerebral fraction of tissue oxygen extraction (cFTOE) associated with A-RBC or CB-RBC transfusions. Cerebral NIRS monitoring was performed by neonatologists and nurses, who were blinded to the PRBC type. The NIRS monitoring was conducted starting with the blood product order, during transfusion, and for the subsequent 24 hours after transfusion completion. The mean treatment effects of A-RBCs or CB-RBCs were quantified using a linear mixed model for repeated measures. Of 23 randomized neonates, 17 (11 male [64.7%]; median gestational age at birth, 25.6 weeks [IQR, 25.3-26.1 weeks]) with a median birth weight of 840 g (IQR, 580-900 g) were included in the study; NIRS was evaluated for 42 transfusion episodes, of which 22 were A-RBCs and 20 were CB-RBCs. Globally considering all posttransfusion time points, the overall crSO2 covariate-adjusted mean after CB-RBC transfusions was 5.27% lower (95% CI, 1.20%-9.34%; P = .01) than that after A-RBC transfusions, while the cFTOE after CB-RBC transfusions was 6.18% higher (95% CI, 1.66%-10.69%; P = .009) than that after A-RBCs. The findings of this cohort study suggest that A-RBC transfusions may be associated with more oxygen delivery to cerebral tissues of preterm neonates than transfusions from CB-RBCs. This finding may explain the previously observed association between low HbF and high ROP risk. It also suggests that use of CB to meet the RBC transfusion needs of neonates with a gestational age of less than 28 weeks may protect cerebral tissues from overexposure to oxygen.

240 Prolonged Maternofetal Inflammation in the Pregnant Ewe at Mid-Gestation Increases Subsequent Fetal Inflammatory Tone and Disrupts Skeletal Muscle Growth and Glucose Metabolism Near Term

Abstract Chronic maternofetal inflammation in late gestation (i.e., peak fetal growth) causes fetal intrauterine growth restriction (IUGR) in sheep, which was associated with deficits in growth, body composition, and metabolic function before and after birth. However, the impact of sustained maternofetal inflammation during mid-gestation (i.e., peak placental growth and development) has not been explored. Therefore, our objective was to determine the effects of sustained maternofetal inflammation during this critical window on subsequent fetal inflammatory tone, metabolism, and myoblast (i.e., muscle stem cell) function. Pregnant ewes were injected every 3rd day from the 50th to 65th d of gestational age (dGA; term = 150 dGA) with saline (control; n = 12) or bacterial lipopolysaccharide endotoxin to induce maternofetal inflammation and IUGR (MI-IUGR; n = 12). Fetuses were surgically catheterized on dGA 118 to collect blood samples, and a hyperinsulinemic-euglycemic clamp (HEC) was performed on dGA 123. Fetuses were necropsied on dGA 125 to collect semitendinosus muscle samples. Fetal plasma TNFα concentrations tended to be greater (P = 0.07) for MI-IUGR fetuses (440 ± 45 pg/m l) than for controls (348 ± 24 pg/mL). Fetal blood O2 partial pressures were 12% less (P < 0.05) and maternofetal O2 gradients tended to be 11% greater (P = 0.08) for MI-IUGR fetuses than for controls. Hindlimb-specific glucose uptake rates did not differ between fetal groups, but hindlimb-specific glucose oxidation rates were reduced (P < 0.05) by 15% under basal conditions and by 27% under hyperinsulinemic conditions for MI-IUGR fetuses compared with controls. Semitendinosus glycogen content and β2 adrenoceptor content did not differ between fetal groups, but semitendinosus content of the TNFα receptor, TNFR1, was 41% greater (P < 0.05) for MI-IUGR fetuses than for controls. Immunohistochemistry revealed 18% fewer (P < 0.05) proliferating myoblasts (pax7+/Ki67+) and 38% fewer (P < 0.05) differentiated myoblasts (myogenin+) in semitendinosus from MI-IUGR fetuses compared with controls, which coincided with 13% smaller (P < 0.05) muscle fibers. From these findings, we conclude that sustained maternofetal inflammation during peak placental development at mid-gestation resulted in enhanced fetal inflammatory tone near term, which in turn impaired myoblast function, muscle fiber hypertrophy, and muscle glucose metabolism.

Targeted Degradation of the Wiz Transcription Factor for Gamma Globin De-Repression

Sickle cell disease (SCD) is a life-threatening genetic condition arising from a point mutation in the β-globin gene that causes hemoglobin polymerization and vaso-occlusive sickling of erythrocytes. Hereditary persistence or pharmacologic induction of gamma-globin (HBG1 or HBG2) expression leads to increases in fetal hemoglobin (HbF) that biophysically oppose hemoglobin polymerization. Indeed, the standard-of-care, hydroxyurea (HU), increases expression of HbF in the erythroblast, and in patients the magnitude of response (9-24% HbF) correlates with a reduction in pain crises and acute complications. However, the use of HU is limited by variable response, myelosuppression, GI side effects and carcinogenicity risk. New targeted therapeutic approaches to induce HbF are therefore being intently pursued, leveraging the increasingly detailed functional characterization of the globin locus. The recent discovery of BCL11A as a repressor of HBG has prompted the widespread innovation of genetically engineered hematopoietic stem cells that suppress BCL11A expression or function. While a major advance in SCD therapeutics, the complexity and clinical implementation of stem cell transplantation may limit access and impact, especially in developing countries where SCD burden is greatest. We therefore undertook to discover a small-molecule HbF inducer, focusing on transcription factors that repress gamma-globin expression. Although transcription factors as a target class have historically been considered "undruggable," recent mechanistic characterization of phthalimide medicines as serendipitous, cereblon (CRBN)-dependent degraders of IKZF1 and IKZF3 by our organization and others, suggested the plausible degradation of new zinc-finger containing transcription factors by this emerging class of molecular glues. Therefore, we elaborated a large library of CRBN-biased ligands for study in target-directed and phenotypic drug discovery campaigns. Here we report for the first time the phenotypic discovery and structure-based optimization of two molecular glue degraders of the transcription factor, WIZ, which lead to pronounced de-repression of HbF. Using a chemical biology approach, the first molecule, dWIZ-1, was identified by phenotypic screening of a CRBN-biased chemical library for compounds that induce HbF without deleterious effect on erythroid cell proliferation and differentiation. Subsequent target deconvolution by global proteomics revealed WIZ as the target. WIZ knockout using two independent sgRNA elevated the proportion of γ-globin mRNA, HbF protein levels and HbF + cells (Figure 1) in vitro in primary human erythroblasts, validating WIZ as a previously unrecognized repressor of HbF. Optimization of dWIZ-1 led to a second tool compound, dWIZ-2, which was suitable to assess WIZ degradation and HbF induction in vivo due to its superior pharmacokinetic properties. dWIZ-2 treatment increased the proportion of γ-globin mRNA, HbF + cells and HbF protein levels in vitro in sickle cell disease patient-derived erythroblasts (Figure 2). Because many IMiDs lack activity in rodents, we used a humanized mouse model to measure the impact of dWIZ-2 on WIZ degradation and HbF expression in human erythroblasts in vivo. NBSGW mice humanized with CD34 + hematopoietic stem and progenitor cells from healthy human donors were orally dosed once daily with dWIZ-2 for 21 days. dWIZ-2 treatment resulted in robust dose-dependent WIZ degradation and an increase in total HbF and the proportion of HbF + human erythroblasts in the bone marrow. To further study the kinetics of HbF induction in the peripheral blood, and assess the effects of dWIZ-2 on steady-state hematopoiesis, we orally dosed naïve healthy cynomolgus monkeys with vehicle or 30 mg/kg dWIZ-2, once daily for 28 days. We detected elevated levels of γ-globin mRNA in the blood on day 28, reaching up to 37% of β-like globins. Additionally, on day 28, we observed up to 95% HbF + reticulocytes.dWIZ-2 was well-tolerated based on the absence of notable clinical observations during the study and consistent body weight for all animals. There were no dWIZ-2-related changes in hematology, coagulation or clinical chemistry measurements, specifically including reticulocytes, hematocrit and neutrophil counts. These findings support the development of an oral WIZ degrader as a globally accessible therapeutic approach for SCD.

The effects of inflammation and acidosis on placental blood vessels reactivity

IntroductionInflammation and acidosis are two stress stimuli that correspond to pathophysiological processes occurring in placental-mediated vascular disorders. We aimed to investigate the effects of these stimuli on placental chorionic blood vessels reactivity using the ex-vivo placental perfusion model. MethodsTerm placentas were obtained immediately after cesarean deliveries, and selected cotyledons were cannulated and dually perfused ex-vivo. Placentas were perfused with three different protocols: culture medium (M199-controls, n = 5), culture medium with lipopolysaccharide (inflammatory stimuli) (LPS,1 μg/ml, n = 7), and acidotic culture medium (M − 199, pH: 6.9–7, n = 6). Each perfusion experiment was maintained for 180 min. Fetal perfusion pressure was continuously measured. Measurements in response to angiotensin II (AT II) at the end of the perfusion were compared between the treatment groups, including amplitude of the contraction response, relaxation factor, time to maximal constriction and the area under the pressure curve (AUC). ResultsIn response to ATII there was a significant difference in the amplitude of the contraction and the AUC between the treatment groups, (p = 0.049, p = 0.015, respectively). As compared with control perfused cotyledon, the inflammatory stimuli significantly increased the vasoconstriction response to ATII in fetal placental blood vessels, as expressed by increased AUC - median (IQR): 555 (235–1184) vs. 133 (118–207), respectively, p = 0.017. The time to maximal constriction and the relaxation factor did not differ between the groups. DiscussionInflammatory stimuli but not acidosis impact fetal-placental vasculature in response to ATII, suggesting that inflammation can compromise vascular function.

Acute splenic sequestration in HbSS: observations from the Jamaican birth cohort

ObjectiveTo document the prevalence, clinical features, haematology and outcome of acute splenic sequestration (ASS) in homozygous sickle cell disease (HbSS).Study designA cohort study from birth.SettingThe Medical Research Council Laboratories at...

Activating transcription factor 4 in erythroid development and [Formula: see text]-thalassemia: a powerful regulator with therapeutic potential.

Activating transcription factor 4 (ATF4) is a fundamental basic region/leucine zipper transcription factor, responds to various stress signals, and plays crucial roles in normal metabolic and stress response processes. Although its functions in human health and disease are not completely understood, compelling evidence underscores ATF4 is indispensable for multiple stages and lineages of erythroid development, including the regulation of fetal liver hematopoietic stem cells, induction of terminal erythroid differentiation, and maintenance of erythroid homeostasis. [Formula: see text]-Thalassemia is a blood disorder arising from mutations in the [Formula: see text]-globin gene. Reactivating the expression of the [Formula: see text]-globin gene in adult patients has emerged as a promising therapeutic strategy for ameliorating clinical symptoms associated with [Formula: see text]-thalassemia. Recent research has suggested that ATF4 contributes to decreased fetal hemoglobin (HbF) level through its binding to potent negative regulators of HbF, such as BCL11A and MYB. Notably, evidence also suggests a contrasting outcome where increased ATF4 protein levels are associated with enhanced HbF at the transcriptional level. Consequently, the identification of mechanisms that modulate ATF4-mediated [Formula: see text]-globin transcription and its effects on erythroid development may unveil novel targets for [Formula: see text]-thalassemia treatment.

The Effect of Maternal Lower Limb Compression on Amniotic Fluid Index, Uteroplacental Perfusion, and Fetal Blood Flow in Isolated Oligohydramnios

Introduction: The aim of this study was to examine the efficacy of pneumatic compression of the maternal lower extremities in increasing the amniotic fluid index (AFI) in pregnancies complicated by isolated oligohydramnios. Methods: Women with isolated oligohydramnios (AFI <5 cm) at 32–41 weeks of pregnancy were connected to a sequential compression device for 60 min. Prior and after the application, AFI and the pulsatility index (PI) of a number of arteries were measured. Results: The median (interquartile range) maternal age of the 21 women included was 29 years (26.50–32.00), the median parity was 1 (1–2), and the median gestational age at intervention was 37.60 weeks (37.00–39.40). The median AFI increased after the application from 4.00 (3.62–4.50) to 6.08 cm (4.90–7.03) (p < 0.001). The median PI of the fetal renal artery decreased from 2.30 (2.01–2.88) to 2.26 (1.68–2.71) (p = 0.01). The hourly fetal urine production did not increase. Changes were not significant in the PI of the umbilical artery, the middle cerebral artery, and the bilateral uterine arteries. Conclusion: Short-term activation of pneumatic compression on maternal lower extremities could increase the AFI in women with isolated oligohydramnios.

Maternal Risk Factors and Their Effect on Outcome and Procedure-Related Complications in Cordocentesis: A Multicenter Retrospective Study.

Cordocentesis is used in clinical situations in which lower-risk diagnostic procedures do not deliver the desired results. The aim of this study was to evaluate the risk for procedure-related complications and fetal loss in correlation to maternal risk factors. This is a multicenter retrospective study investigating the complications, risk factors and perinatal outcome of diagnostic cordocentesis between 1998 and 2019 in three different centers. A total of 1806 cordocenteses were performed and procedure-related complications (IUFD within 48 h, contractions, bradycardia, unsuccessful puncture, chorioamniotic separation) were noted in 1.6% of cases. Fetuses with chromosomal aberrations, intrauterine growth restriction and hydropic fetuses had a significantly higher rate of fetal loss compared to other indications. Fetal blood sampling (FBS) performed before 17+0 weeks of gestation was associated with a higher risk of procedure-related complications. Maternal BMI ≥ 40 increased the risk for fetal loss, whereas maternal age, number of previous miscarriages, number of previous abortions, history of vaginal bleeding or nicotine abuse did not affect the risk for complications or overall fetal loss rate. In the hands of experienced operators, FBS is a safe way to further fetal diagnostics, and the risk of complications is low.

Agreement between youth and caregiver report of pain and functioning in pediatric sickle cell disease: PedsQL sickle cell disease module.

Pain is a primary symptom of sickle cell disease (SCD) and is often severe and chronic. To treat SCD-related pain, proper assessment of SCD pain among youth, including the degree of concordance or agreement between youth and caregiver reports of pain, is essential but has not yet been adequately evaluated. In this study, 525 youth with SCD and their parents were evaluated as part of the Sickle Cell Clinical Research and Intervention Program (SCCRIP) to examine pain rating concordance and predictors of concordance. Youth and parents completed the Pediatric Quality of Life Inventory Sickle Cell Disease module (PedsQL-SCD) to measure pain, pain interference, and pain-related constructs. Disease, clinical, and demographic variables were obtained from the SCCRIP database. Intraclass correlations demonstrated moderate-to-poor consistency between youth and caregiver reports of pain and pain interference (ICCs range from 0.17 to 0.54). Analysis of covariance and regression models found that patient age, frequency of hospitalizations and emergency department (ED) visits, economic hardship, and fetal hemoglobin levels were significantly associated with varying pain-rating agreement levels among parent proxy and child self-report pain. Concordance of pain assessments among youth with SCD and their caregivers using the PedsQL-SCD Module was moderate at best, corroborating prior research. Youth factors predicting discordance among pain-related factors included increased ED visits, older age, and female sex. Collectively, these results bolster the use of integrated pain assessments to reduce parent-child discrepancies, thereby improving the adequacy of SCD-related pain assessment and treatment.

In Silico Molecular Docking and Dynamics Simulation Analysis of Potential Histone Lysine Methyl Transferase Inhibitors for Managing β-Thalassemia

A decreased hemoglobin synthesis is contemplated as a pathological indication of β-thalassemia. Recent studies show that EPZ035544 from Epizyme could induce fetal hemoglobin (HbF) levels due to its proven capability to inhibit euchromatin histone lysine methyl transferase (EHMT2). Therefore, the development of EHMT2 inhibitors is considered promising in managing β-thalassemia. Our strategy to find novel compounds that are EHMT2 inhibitors relies on the virtual screening of ligands that have a structural similarity to N2-[4-methoxy-3-(2,3,4,7-tetrahydro-1H-azepin-5-yl) phenyl]-N4,6-dimethyl-pyrimidine-2,4-diamine (F80) using the PubChem database. In silico docking studies using Autodock Vina were employed to screen a library of 985 compounds and evaluate their binding ability with EHMT2. The selection of hit compounds was based on the docking score and mode of interaction with the protein. The top two ranked compounds were selected for further investigations, including pharmacokinetic properties analysis and molecular dynamics simulations (MDS). Based on the obtained docking score and interaction analysis, N-(4-methoxy-3-methylphenyl)-4,6-diphenylpyrimidin-2-amine (TP1) and 2-N-[4-methoxy-3-(5-methoxy-3H-indol-2-yl)phenyl]-4-N,6-dimethylpyrimidine-2,4-diamine (TP2) were found to be promising candidates, and TP1 exhibited better stability in the MDS study compared to TP2. In summary, our approach helps identify potential EHMT2 inhibitors, and further validation using in vitro and in vivo experiments could certainly enable this molecule to be used as a therapeutic drug in managing β-thalassemia disease.

CRISPR/Cas-based gene editing in therapeutic strategies for beta-thalassemia.

Beta-thalassemia (β-thalassemia) is an autosomal recessive disorder caused by point mutations, insertions, and deletions in the HBB gene cluster, resulting in the underproduction of β-globin chains. The most severe type may demonstrate complications including massive hepatosplenomegaly, bone deformities, and severe growth retardation in children. Treatments for β-thalassemia include blood transfusion, splenectomy, and allogeneic hematopoietic stem cell transplantation (HSCT). However, long-term blood transfusions require regular iron removal therapy. For allogeneic HSCT, human lymphocyte antigen (HLA)-matched donors are rarely available, and acute graft-versus-host disease (GVHD) may occur after the transplantation. Thus, these conventional treatments are facing significant challenges. In recent years, with the advent and advancement of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) gene editing technology, precise genome editing has achieved encouraging successes in basic and clinical studies for treating various genetic disorders, including β-thalassemia. Target gene-edited autogeneic HSCT helps patients avoid graft rejection and GVHD, making it a promising curative therapy for transfusion-dependent β-thalassemia (TDT).In this review, we introduce the development and mechanisms of CRISPR/Cas9. Recent advances on feasible strategies of CRISPR/Cas9 targeting three globin genes (HBB, HBG, and HBA) and targeting cell selections for β-thalassemia therapy are highlighted. Current CRISPR-based clinical trials in the treatment of β-thalassemia are summarized, which are focused on γ-globin reactivation and fetal hemoglobin reproduction in hematopoietic stem cells. Lastly, the applications of other promising CRISPR-based technologies, such as base editing and prime editing, in treating β-thalassemia and the limitations of the CRISPR/Cas system in therapeutic applications are discussed.

A large cohort of deletional high hemoglobin F determinants in Thailand: A molecular revisited and identification of a novel mutation

Background and aimsHigh hemoglobin F determinants can be classified into hereditary persistence of fetal hemoglobin (HPFH) and δβ-thalassemia with different phenotype. We report the molecular basis and hematological features in a large cohort of deletional high Hb F determinants in Thailand. Materials and methodsSubjects (n = 28,177) encountered during 2015–2022 were reviewed, and those with phenotypically suspected of having high Hb F determinants were selected. Combined PCR, multiplex ligation-dependent probe amplification, next-generation sequencing, and DNA sequencing were used to identify the mutations. ResultsAmong 28,177 subjects investigated, 300 (1.06 %) were found to carry deletional high Hb F determinants in a total of 302 alleles, including heterozygote, compound heterozygote with β-hemoglobinopathies, and homozygote. DNA analysis identified eight different DNA deletions, including δβ0-thalassemia (12.6 kb deletion) (73.8 %), HPFH-6 (14.9 %), Indian deletion-inversion Aγδβ0-thalassemia (3.6 %), Thai deletion-inversion-insertion Aγδβ0-thalassemia (3.0 %), SEA-HPFH (3.0 %), Chinese Aγδβ0-thalassemia (1.0 %), Thai δβ0-thalassemia (11.3 kb deletion) (0.3 %), and a novel δβ0-thalassemia (137.1 kb deletion) (0.3 %). In addition, three novel genetic interactions, including Chinese Aγδβ0-thalassemia/Hb E, δβ0-thalassemia/Indian deletion-inversion Aγδβ0-thalassemia, and homozygous δβ0-thalassemia were found. Hematological features and Hb analysis results of 20 different genotypes were recorded. Multiplex gap-PCR assays for detection of these genetic determinants were described. ConclusionsDeletional high Hb F determinants are common and heterogeneous in Thailand. Data on the prevalence, molecular spectrum, phenotypic expression, and complex interactions of these genetic determinants should prove useful in the study and a prevention and control program of hemoglobinopathies in the region.

Prenatal exposure to acetaminophen at different doses, courses and time causes testicular dysplasia in offspring mice and its mechanism

Epidemiological investigation suggested that the use of acetaminophen during pregnancy may cause offspring testicular dysplasia, but no systematic study has been conducted. In this study, Kunming mice were given acetaminophen at different doses (100/200/400 mg/kg.d), courses (single/multiple), time (second/third trimester) during pregnancy. Fetal blood and testes were collected on gestaional day 18 for detection. The results indicated abnormal testicular development in the PAcE (prenatal acetaminophen exposure) groups. The maximum diameter/cross-sectional area decreased, the interstitial space widened, and decreased proliferation/increased apoptosis were observed, especially in the high-dose, multi-course and second-trimester groups. Meanwhile, the serum testosterone level decreased in PAcE groups, and the steroid synthesis function in Leydig cells, Sertoli and spermatogenic cell function were inhibited, it was more significant in high-dose, multi-course and second-trimester groups. Furthermore, Wnt signal pathway was activated but Notch signal pathway was inhibited in the PAcE groups. Finally, in vitro experiment, acetaminophen could inhibit spermatogonial cell proliferation, enhance apoptosis, and change Wnt/Notch signal pathway. In conclusion, this study confirmed that PAcE can change fetal testicular development in a dose, course and time-dependent manner, and found that multicellular function impaired. This study provides theoretical and experimental basis for systematically elucidating the developmental toxicity of acetaminophen in testis.

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.

Accuracy of a portable hemoglobinometer (HemoCue) to measure fetal hemoglobin values during in utero transfusion

Objectives The current recommended treatment for severe fetal anemia is in utero transfusion (IUT). During this procedure, the evaluation of the necessary volume of transfused blood is based on regular measurement of fetal hemoglobin (FHb) concentration. The gold standard measurement is performed in the biology laboratory. A rapid medical test such as HemoCue® is an effective way to predict FHb concentration. It would reduce the time to obtain results and therefore the procedure duration. To evaluate the accuracy of HemoCue® to measure FHb during IUT, we compared Hb levels obtained by HemoCue® and by our biology laboratory. Methods This retrospective study involved all pregnant women who had undergone an IUT in the university hospital of Clermont-Ferrand, France, during the period from 1 January 2010 to 6 June 2021. The FHb level was evaluated by two methods, a rapid medical test, HemoCue®, and a standard method in the biology laboratory. Results We obtained 244 pairs of results from HemoCue® and our laboratory, of 90 IUT procedures. The correlation between the two sets of results was excellent, with Lin’s concordance correlation coefficient of 0.979. However, we established that the measurements were not significantly modified by IUT number, puncture time, cause of fetal anemia, estimated fetal weight, gestational age, and delay between two IUT or middle cerebral artery peak systolic velocity values. Conclusion Our results allowed to extend the relevance of FHb measurements by HemoCue® during IUT.