Effects of putative metformin targets on phenotypic age and leukocyte telomere length: a mendelian randomisation study using data from the UK Biobank.

BACKGROUND: Metformin, a first-line medication for type 2 diabetes, might also have a protective effect against ageing-related diseases, but so far little experimental evidence is available. We sought to assess the target-specific effect of metformin on biomarkers of ageing in the UK Biobank. METHODS: In this drug target mendelian randomisation study, we assessed the target-specific effect of four putative targets of metformin (AMPK, ETFDH, GPD1, and PEN2), involving ten genes. Genetic variants with evidence of causation of gene expression, glycated haemoglobin A1c (HbA1c), and colocalisation were used as instruments mimicking the target-specific effect of metformin via HbA1c lowering. The biomarkers of ageing considered were phenotypic age (PhenoAge) and leukocyte telomere length. To triangulate the evidence, we also assessed the effect of HbA1c on the outcomes using a polygenic mendelian randomisation design and assessed the effect of metformin use on these outcomes using a cross-sectional observational design. FINDINGS: GPD1-induced HbA1c lowering was associated with younger PhenoAge (ß -5·26, 95% CI -6·69 to -3·83) and longer leukocyte telomere length (ß 0·28, 0·03 to 0·53), and AMPKγ2 (PRKAG2)-induced HbA1c lowering was associated with younger PhenoAge (ß -4·88, -7·14 to -2·62) but not with longer leukocyte telomere length. Genetically predicted HbA1c lowering was associated with younger PhenoAge (ß -0·96 per SD lowering of HbA1c, 95% CI -1·19 to -0·74) but not associated with leukocyte telomere length. In the propensity score matched analysis, metformin use was associated with younger PhenoAge (ß -0·36, 95% CI -0·59 to -0·13) but not with leukocyte telomere length. INTERPRETATION: This study provides genetic validation evidence that metformin might promote healthy ageing via targets GPD1 and AMPKγ2 (PRKAG2), and the effect could be in part due to its glycaemic property. Our findings support further clinical research into metformin and longevity. FUNDING: Healthy Longevity Catalyst Award, National Academy of Medicine, and Seed Fund for Basic Research, The University of Hong Kong.

Sickle Cell Hemoglobin Genotypes Affect Malaria Parasite Growth and Correlate with Exosomal miR-451a and let-7i-5p Levels.

Malaria affects a significant portion of the global population, with 247 million cases in 2021, primarily in Africa. However, certain hemoglobinopathies, such as sickle cell trait (SCT), have been linked to lower mortality rates in malaria patients. Hemoglobin (Hb) mutations, including HbS and HbC, can cause sickle cell disease (SCD) when both alleles are inherited (HbSS and HbSC). In SCT, one allele is inherited and paired with a normal allele (HbAS, HbAC). The high prevalence of these alleles in Africa may be attributed to their protective effect against malaria. Biomarkers are crucial for SCD and malaria diagnosis and prognosis. Studies indicate that miRNAs, specifically miR-451a and let-7i-5p, are differentially expressed in HbSS and HbAS compared to controls. Our research examined the levels of exosomal miR-451a and let-7i-5p in red blood cells (RBCs) and infected red blood cells (iRBCs) from multiple sickle Hb genotypes and their impact on parasite growth. We assessed exosomal miR-451a and let-7i-5p levels in vitro in RBC and iRBC supernatants. Exosomal miRNAs exhibited distinct expression patterns in iRBCs from individuals with different sickle Hb genotypes. Additionally, we discovered a correlation between let-7i-5p levels and trophozoite count. Exosomal miR-451a and let-7i-5p could modulate SCD and malaria severity and serve as potential biomarkers for malaria vaccines and therapies.

Molecular characterization of a novel homozygous deletion in ß-globin cluster causing (δß)0-Thalassemia among Tunisian family.

BACKGROUND: Deletions in the ß-globin cluster are uncommon and cause thalassemia (thal) with hereditary persistence of fetal hemoglobin. They constitute a heterogenous group of disorders characterized by absent or reduced synthesis of adult hemoglobin (Hb A) and increased synthesis of fetal hemoglobin (Hb F). Although the clinical severity of these disorders are asymptomatic owing to the increased Hb F levels, the molecular basis is very heterogenous due to the large deletions in the ß-globin cluster spanning both HBD and HBB genes. Here, we describe a Tunisian family carrying a novel deletion mutation causing (δß)°-thalassemia. METHODS: The amounts of hemoglobin fractions were measured by capillary electrophoresis of hemoglobin. Amplification and sequencing of different regions on the ß-gene cluster were performed by Sanger method. RESULTS: Family study and genetic analysis revealed a large deletion mutation in the ß-globin cluster of 14.5 kb (NG_000,007.3:g. 58,253 to g.72837del14584) at the homozygous state in the patient and at heterozygous state at the other members of the family. This deletion removes the HBD and HBB genes. CONCLUSIONS: In our knowledge, this new large deletion is described for the first time in the Tunisian population and in the world, designed Tunisian(δß)0 in Ithanet database (IthaID: 3971). Therefore, it is important to identify the deletion leading to δß-thalassemia carriers at the molecular level, to highlight the importance of recognizing the clinical features and implementing appropriate testing to clarify the diagnosis and manage the condition.

Decreased expression of HBA1 and HBB genes in acute myeloid leukemia patients and their inhibitory effects on growth of K562 cells.

BACKGROUND: Besides the traditional roles of HBA1 and HBB, recent findings suggest that hemoglobin genes may have roles in other contexts. OBJECTIVE: In the present study, we aim to investigate a possible tumor-suppressor role of HBA1 and HBB in acute myeloid leukemia. METHODS: Quantitative real-time PCR (RT-qPCR) was performed to detect the expression levels of HBA1 and HBB in acute myeloid leukemia patients and AML cell lines. The transfected cells were analyzed for Cell Counting Kit-8 (CCK-8), apoptosis, and cell cycle assay. RESULTS: HBA1 and HBB genes were significantly decreased in acute myeloid leukemia patients and AML cell lines. Furthermore, in vitro approaches showed that overexpression of HBA1 and HBB inhibited proliferation, induced cell apoptosis, and blocked cell cycle process at the G2/M phase in K562 cells. CONCLUSION: Our data indicated that HBA1 and HBB genes may be potential tumor-suppressor genes in acute myeloid leukemia.

LRF Promotes Indirectly Advantageous Chromatin Conformation via BGLT3-lncRNA Expression and Switch from Fetal to Adult Hemoglobin.

The hemoglobin switch from fetal (HbF) to adult (HbA) has been studied intensively as an essential model for gene expression regulation, but also as a beneficial therapeutic approach for ß-hemoglobinopathies, towards the objective of reactivating HbF. The transcription factor LRF (Leukemia/lymphoma-related), encoded from the ZBTB7A gene has been implicated in fetal hemoglobin silencing, though has a wide range of functions that have not been fully clarified. We thus established the LRF/ZBTB7A-overexpressing and ZBTB7A-knockdown K562 (human erythroleukemia cell line) clones to assess fetal vs. adult hemoglobin production pre- and post-induction. Transgenic K562 clones were further developed and studied under the influence of epigenetic chromatin regulators, such as DNA methyl transferase 3 (DNMT3) and Histone Deacetylase 1 (HDAC1), to evaluate LRF's potential disturbance upon the aberrant epigenetic background and provide valuable information of the preferable epigenetic frame, in which LRF unfolds its action on the ß-type globin's expression. The ChIP-seq analysis demonstrated that LRF binds to γ-globin genes (HBG2/1) and apparently associates BCL11A for their silencing, but also during erythropoiesis induction, LRF binds the BGLT3 gene, promoting BGLT3-lncRNA production through the γ-δ intergenic region of ß-type globin's locus, triggering the transcriptional events from γ- to ß-globin switch. Our findings are supported by an up-to-date looping model, which highlights chromatin alterations during erythropoiesis at late stages of gestation, to establish an "open" chromatin conformation across the γ-δ intergenic region and accomplish ß-globin expression and hemoglobin switch.

Impact of Sickle Cell Trait Hemoglobin on the Intraerythrocytic Transcriptional Program of Plasmodium falciparum.

Sickle-trait hemoglobin (HbAS) confers nearly complete protection from severe, life-threatening falciparum malaria in African children. Despite this clear protection, the molecular mechanisms by which HbAS confers these protective phenotypes remain incompletely understood. As a forward genetic screen for aberrant parasite transcriptional responses associated with parasite neutralization in HbAS red blood cells (RBCs), we performed comparative transcriptomic analyses of Plasmodium falciparum in normal (HbAA) and HbAS erythrocytes during both in vitro cultivation of reference parasite strains and naturally occurring P. falciparum infections in Malian children with HbAA or HbAS. During in vitro cultivation, parasites matured normally in HbAS RBCs, and the temporal expression was largely unperturbed of the highly ordered transcriptional program that underlies the parasite's maturation throughout the intraerythrocytic development cycle (IDC). However, differential expression analysis identified hundreds of transcripts aberrantly expressed in HbAS, largely occurring late in the IDC. Surprisingly, transcripts encoding members of the Maurer's clefts were overexpressed in HbAS despite impaired parasite protein export in these RBCs, while parasites in HbAS RBCs underexpressed transcripts associated with the endoplasmic reticulum and those encoding serine repeat antigen proteases that promote parasite egress. Analyses of P. falciparum transcriptomes from 32 children with uncomplicated malaria identified stage-specific differential expression: among infections composed of ring-stage parasites, only cyclophilin 19B was underexpressed in children with HbAS, while trophozoite-stage infections identified a range of differentially expressed transcripts, including downregulation in HbAS of several transcripts associated with severe malaria in collateral studies. Collectively, our comparative transcriptomic screen in vitro and in vivo indicates that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Because HbAS consistently protects from severe P. falciparum, modulation of these responses may offer avenues by which to neutralize P. falciparum parasites. IMPORTANCE Sickle-trait hemoglobin (HbAS) confers nearly complete protection from severe, life-threatening malaria, yet the molecular mechanisms that underlie HbAS protection from severe malaria remain incompletely understood. Here, we used transcriptome sequencing (RNA-seq) to measure the impact of HbAS on the blood-stage transcriptome of Plasmodium falciparum in in vitro time series experiments and in vivo samples from natural infections. Our in vitro time series data reveal that, during its blood stage, P. falciparum's gene expression in HbAS is impacted primarily through alterations in the abundance of gene products as opposed to variations in the timing of gene expression. Collectively, our in vitro and in vivo data indicate that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Due to the persistent association of HbAS and protection from severe disease, these processes that are modified in HbAS may offer strategies to neutralize P. falciparum.

Transcription factor competition at the γ-globin promoters controls hemoglobin switching.

BCL11A, the major regulator of fetal hemoglobin (HbF, α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, α2ß2). To uncover how BCL11A initiates repression, we used CRISPR-Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)-globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb ß-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.

Hydroxyurea improves nitric oxide bioavailability in humanized sickle cell mice.

Despite advancements in disease management, sickle cell nephropathy, a major contributor to mortality and morbidity in patients, has limited therapeutic options. Previous studies indicate hydroxyurea, a commonly prescribed therapy for sickle cell disease (SCD), can reduce renal injury in SCD but the mechanisms are uncertain. Because SCD is associated with reduced nitric oxide (NO) bioavailability, we hypothesized that hydroxyurea treatment would improve NO bioavailability in the humanized sickle cell mouse. Humanized male 12-wk-old sickle (HbSS) and genetic control (HbAA) mice were treated with hydroxyurea or regular tap water for 2 wk before renal and systemic NO bioavailability as well as renal injury were assessed. Untreated HbSS mice exhibited increased proteinuria, elevated plasma endothelin-1 (ET-1), and reduced urine concentrating ability compared with HbAA mice. Hydroxyurea reduced proteinuria and plasma ET-1 levels in HbSS mice. Untreated HbSS mice had reduced plasma nitrite and elevated plasma arginase concentrations compared with HbAA mice. Hydroxyurea treatment augmented plasma nitrite and attenuated plasma arginase in HbSS mice. Renal vessels isolated from HbSS mice also had elevated nitric oxide synthase 3 (NOS3) and arginase 2 expression compared with untreated HbAA mice. Hydroxyurea treatment did not alter renal vascular NOS3, however, renal vascular arginase 2 expression was significantly reduced. These data support the hypothesis that hydroxyurea treatment augments renal and systemic NO bioavailability by reducing arginase activity as a potential mechanism for the improvement on renal injury seen in SCD mice.

The Shift of HbF to HbA under Influence of SKA2 Gene; A Possible Link between Cortisol and Hematopoietic Maturation in Term and Preterm Newborns.

BACKGROUND: We hypothesized that the SKA2 gene can convert hemoglobin F to A leading to the maturity of the hematopoietic system by glucocorticoid hormone; so, the present study aimed to investigate the health outcome of newborns by using the effect of SKA2 gene on hematopoietic maturation. METHODS: At first, 142 samples were divided into term and preterm. After sampling from the umbilical cord blood, the expression of SKA2 genes and HbA and F were evaluated by quantitative RT-PCR. The blood gases were measured by Campact 3 device. Finally, the cortisol level was measured by ELISA method and HbA and F levels were investigated by capillary electrophoresis. RESULTS: The blood gases and Apgar scores were more favorable in term newborns (P <0.001). Levels of protein/expression of HbF in newborns with Apgar score greater than 7 was lower than that of the newborns with Apgar score below 7 (P <0.001). Cortisol and HbA levels were considerably higher in term newborns compared to the preterm ones (P <0.001). In the preterm and term groups, SKA2 gene expression had a positive and significant relationship with cortisol and HbA levels as well as a negative relationship with the HbF level. In the preterm group, a positive and significant relationship was observed between the expression of SKA2 and HbF genes. CONCLUSION: The results revealed that the SKA2 gene affected hematopoietic maturation in preterm and term newborns and the health outcome of newborns improved by increasing HbA level.

Association between haematological parameters and sickle cell genotypes in children with Plasmodium falciparum malaria resident in Kisumu County in Western Kenya.

BACKGROUND: Sickle cell disease (SCD) is a monogenic disorder due to point mutation in the ß-globin gene resulting in substitution of Valine for Glutamic acid. The SCD is prevalent in P. falciparum endemic regions such as western Kenya. Carriage of different sickle cell genotypes may influence haematological parameter during malaria. Children resident in malaria holoendemic regions suffer more from malaria-related complications and this is moderated by the presence of the SCD. In the current study, we determined the association between sickle cell genotypes and haematological parameters in children with P. falciparum malaria resident in Kisumu County in Western Kenya. METHODOLOGY: Children (n = 217, aged 1-192 months) with acute febrile condition were recruited at Jaramogi Oginga Odinga Teaching and Referral Hospital. Chi-square (χ2) analysis was used to determine differences between proportions. Differences in haematological parameters were compared across groups using Kruskal Wallis test and between groups using Mann Whitney U test. Multivariate logistic regression analysis controlling for infection status was used to determine the association between sickle cell genotypes and haematological parameters. RESULTS: Using HbAA as the reference group, multivariate logistic regression analysis revealed that carriage of HbSS was associated with reduced haemoglobin [OR = 0.310, 95% CI = 0.101-0.956, P = 0.041], reduced haematocrit [OR = 0.318, 95% CI = 0.128-0.793, P = 0.014], reduced RBC count [OR = 0.124, 95% CI = 0.045-0.337, P = 0.001], reduced MCHC [OR = 0.325, 95% CI = 0.118-0.892, P = 0.029], increased leucocytosis [OR = 9.283, 95% CI = 3.167-27.210, P = 0.001] and reduced monocytosis [OR = 0.319, 95% CI = 0.123-0.830, P = 0.019]. However, carriage of HbAS was only associated with increased micro-platelets [OR = 3.629, 95% CI = 1.291-8.276, P = 0.012]. CONCLUSION: Results show that carriage of HbSS in children influence the levels of haemoglobin, haematocrit, RBC, MCHC, WBC and Monocytes. Therefore prior knowledge of HbSS should be considered to improve clinical management of haematological alterations during malaria in children.

Effect of Mutations on mRNA and Globin Stability: The Cases of Hb Bernalda/Groene Hart and Hb Southern Italy.

We identified two unstable variants in the third exon of α-globin genes: Hb Bernalda/Groene Hart (HBA1:c.358C>T), and Hb Caserta (HBA2:c.79G>A) in cis to Hb Sun Prairie (HBA2:c.391G>C), also named Hb Southern Italy. These mutations occurred in the H helix of the α-globin that is involved in heme contacting, specific recognition of α-hemoglobin-stabilizing protein (AHSP), and α1ß1 interactions. The carriers showed α-thalassemia phenotype, but one also jaundice and cholelithiasis. Molecular identification of clusters of families in Southern Italy encouraged molecular characterization of mRNA, globin chain analyses, molecular modeling studies, and comparison with globin variants to understand the mechanisms causing the α-thalassemia phenotype. A normal amount of Hb Bernalda/Groene Hart mRNA were found, and molecular modeling highlighted additional H bonds with AHSP. For Hb Southern Italy, showing an unexpected α/ß biosynthetic ratio typical of the ß-thalassemia type, two different molecular mechanisms were shown: Reduction of the variant mRNA, likely due to the No-Go Decay for the presence of unused triplet ACG at cod 26, and protein instability due to the impairment of AHSP interaction. The UDP glucuronosyltransferase 1A (UGT1A1) genotyping was conclusive in the case of jaundice and cholelithiasis. Multiple approaches are needed to properly identify the mechanisms leading to unstable variants and the effect of a mutation.

The Effect of Five Single Nucleotide Polymorphisms on Hb F Variation of ß-Thalassemia Traits and Hematologically Normal Individuals in Southeast Turkey.

ß-Thalassemia (ß-thal) is caused by deficiency of ß-globin chain synthesis and leads to the accumulation of unstable globin chain production. This results in a higher Hb F level in order to neutralize the excess α chains. In addition, γ-globin gene expression, due to genetic factors after birth, leads to increased Hb F levels in adulthood [hereditary persistence of fetal hemoglobin (Hb) (HPFH)]. In this study, the relationship between ß-thal trait and individuals with suspected HPFH and a control group was investigated in Adiyaman, Turkey. Single nucleotide polymorphism (SNP) analyses were performed in five different polymorphic regions using real-time polymerase chain reaction (qPCR) methods [rs4671393 (G>A), rs766432 (A>C), rs9402686 (G>A), rs28384513 (T>G), rs1609812 (A>G)]. No significant difference was found between the control and ß-thal group in the codominant inheritance model in the rs1609812 (A>G) polymorphism region only, while all the other polymorphic regions were found to be statistically significant. It was found that different genotype models increased Hb F levels between 1.6- and 3.06-fold in four studied polymorphic regions [rs4671393 (G>A), rs766432 (A>C), rs9402686 (G>A), rs28384513 (T>G)]. All of the polymorphic regions increased the Hb F levels from 1.86- to 24.76-fold, except rs9402686 (G>A) and rs28384513 (T>G) over dominant and rs1609812 (A>G) codominant inheritance models. The AC and AA genotypes increased Hb F levels in the B-cell CLL/lymphoma 11 A haplotype studies. It was determined that both haplotypes 2 and 4 increased Hb F levels. As a result, SNPs strongly affect the Hb F levels in both healthy individuals and ß-thal trait.

α-Thalassemia Intermedia Results from Interactions of Unstable Hb Prato [α31(B12)Arg→Ser (HBA1 or HBA2 c.96G>T or C)] with the α-Thalassemia-1 [- -SEA (Southeast Asian)] Deletion in Thailand.

The clinical consequences of many abnormal hemoglobins (Hbs) interacting with α- or ß-thalassemia (α- or ß-thal) or other hemoglobinopathies have not been described. We evaluated a 75-year-old Thai woman and her 45-year-old daughter. Hematological data was obtained on an automated cell counter. Hemoglobin (Hb) analysis was carried out using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) assays. Mutations and globin haplotypes were identified by appropriated DNA techniques. The proband presented with moderate anemia and inclusion bodies in most of the red blood cells (RBCs), while altered RBC parameters were absent in her daughter. Hemoglobin analysis showed an abnormal Hb peak only in the proband. DNA analysis identified a G>T substitution at codon 31 of the α1-globin gene, corresponding to Hb Prato [α31(B12)Arg→Ser (HBA1 or HBA2 c.96G>T or C)] in both subjects. The α-thal-1 [- -SEA (Southeast Asian)] deletion was also identified in the proband, but not in her daughter. These mutations could be identified using newly developed allele-specific polymerase chain reaction (ASPCR) assays. The α haplotypic analysis demonstrated the Thai Hb Prato allele was associated with haplotype [+ - S + - + -] [the S represents the inter ζ hypervariable region (HVR)]. The combination of the unstable Hb Prato with α-thal-1 result in α-thal intermedia (α-TI) phenotypes. A simple DNA method is essential for detection, and a haplotypic α-globin gene cluster are presented.

ß-Thalassemia pathogenic variants in a cohort of children from the East African coast.

BACKGROUND: ß-Thalassemia is rare in sub-Saharan Africa. Previous studies have suggested that it is limited to specific parts of West Africa. Based on hemoglobin A2 (HbA2 ) concentrations measured by HPLC, we recently speculated that ß-thalassemia might also be present on the East African coast of Kenya. Here, we follow this up using molecular methods. METHODS: We used raised hemoglobin A2 (HbA2 ) values (> 4.0% of total Hb) to target all HbAA members of a cohort study in Kilifi, Kenya, for HBB sequencing for ß-thalassemia (n = 99) together with a sample of HbAA subjects with lower HbA2 levels. Because HbA2 values are artifactually raised in subjects carrying sickle hemoglobin (HbS) we sequenced all participants with an HPLC pattern showing HbS without HbA (n = 116) and a sample with a pattern showing both HbA and HbS. RESULTS: Overall, we identified 83 carriers of four separate ß-thalassemia pathogenic variants: three ß0 -thalassemia [CD22 (GAA→TAA), initiation codon (ATG→ACG), and IVS1-3' end del 25bp] and one ß+ -thalassemia pathogenic variants (IVS-I-110 (G→A)). We estimated the minimum allele frequency of all variants combined within the study population at 0.3%. CONCLUSIONS: ß-Thalassemia is present in Kilifi, Kenya, an observation that has implications for the diagnosis and clinical care of children from the East Africa region.

A New α Chain Variant, Hb Heilongjiang (HBA2: c.49A>C), Found During Hb A1c Measurement.

We here report a new hemoglobin (Hb) variant found in a Chinese woman. The presence of the Hb variant can be easily recognized by HbA1c procedures based on ion exchange high performance liquid chromatography (HPLC) or capillary electrophoresis (CE) techniques. DNA sequencing revealed a new point mutation (HBA2: c.49A>C) at codon 16, resulting in an amino acid substitution from lysine to glutamine. Moreover, the Hb variant affected Hb A1c determination by VARIANT II Turbo 2.0 and D100. We named the new Hb variant Hb Heilongjiang for the birthplace of the proband.

Understanding heterogeneity of fetal hemoglobin induction through comparative analysis of F and A erythroblasts.

Reversing the developmental switch from fetal hemoglobin (HbF, α2γ2) to adult hemoglobin (HbA, α2ß2) is an important therapeutic approach in sickle cell disease (SCD) and ß-thalassemia. In healthy individuals, SCD patients, and patients treated with pharmacologic HbF inducers, HbF is present only in a subset of red blood cells known as F cells. Despite more than 50 years of observations, the cause for this heterocellular HbF expression pattern, even among genetically identical cells, remains unknown. Adult F cells might represent a reversion of a given cell to a fetal-like epigenetic and transcriptional state. Alternatively, isolated transcriptional or posttranscriptional events at the γ-globin genes might underlie heterocellularity. Here, we set out to understand the heterogeneity of HbF activation by developing techniques to purify and profile differentiation stage-matched late erythroblast F cells and non-F cells (A cells) from the human HUDEP2 erythroid cell line and primary human erythroid cultures. Transcriptional and proteomic profiling of these cells demonstrated very few differences between F and A cells at the RNA level either under baseline conditions or after treatment with HbF inducers hydroxyurea or pomalidomide. Surprisingly, we did not find differences in expression of any known HbF regulators, including BCL11A or LRF, that would account for HbF activation. Our analysis shows that F erythroblasts are not significantly different from non-HbF-expressing cells and that the primary differences likely occur at the transcriptional level at the ß-globin locus.

Expanded carrier screening for preconception reproductive risk assessment: Prevalence of carrier status in a Mexican population.

OBJECTIVE: Genetic carrier screening has the potential to identify couples at risk of having a child affected with an autosomal recessive or X-linked disorder. However, the current prevalence of carrier status for these conditions in developing countries is not well defined. This study assesses the prevalence of carrier status of selected genetic conditions utilizing an expanded, pan-ethnic genetic carrier screening panel (ECS) in a large population of Mexican patients. METHODS: Retrospective chart review of all patients tested with a single ECS panel at an international infertility center from 2012 to 2018 were included, and the prevalence of positive carrier status in a Mexican population was evaluated. RESULTS: Eight hundred five individuals were analyzed with ECS testing for 283 genetic conditions. Three hundred fifty-two carriers (43.7%) were identified with 503 pathogenic variants in 145 different genes. Seventeen of the 391 participating couples (4.34%) were identified as being at-risk couples. The most prevalent alleles found were associated with alpha thalassemia, cystic fibrosis, GJB2 nonsyndromic hearing loss, biotinidase deficiency, and familial Mediterranean fever. CONCLUSION: Based on the prevalence and severity of Mendelian disorders, we recommend that couples who wish to conceive regardless of their ethnicity background explore carrier screening and genetic counseling prior to reproductive medical treatment.

Impact of ET-1 and sex in glomerular hyperfiltration in humanized sickle cell mice.

Hyperfiltration, highly prevalent early in sickle cell disease (SCD), is in part driven by an increase in ultrafiltration coefficient (Kf). The increase in Kf may be due to enlarged filtration surface area and/or increased glomerular permeability (Palb). Previous studies have demonstrated that endothelin-1 (ET-1) contributes to Palb changes in models of diabetes and SCD. Thus, we performed longitudinal studies of renal function to determine the relationship between ET-1 and glomerular size and Palb that may contribute to hyperfiltration in humanized sickle cell (HbSS) and control (HbAA) mice at 8-32 weeks of age. HbSS mice were characterized by significant increases in plasma and glomerular ET-1 expression in both sexes although this increase was significantly greater in males. HbSS glomeruli of both males and females presented with a progressive and significant increase in glomerular size, volume, and Kf During the onset of hyperfiltration, plasma and glomerular ET-1 expression were associated with a greater increase in glomerular size and Kf in HbSS mice, regardless of sex. The pattern of Palb augmentation during the hyperfiltration was also associated with an increase in glomerular ET-1 expression, in both male and female HbSS mice. However, the increase in Palb was significantly greater in males and delayed in time in females. Additionally, selective endothelin A receptor (ETA) antagonist prevented hyperfiltration in HbSS, regardless of sex. These results suggest that marked sex disparity in glomerular hyperfiltration may be driven, in part, by ET-1-dependent ultra-structural changes in filtration barrier components contributing to glomerular hyperfiltration in HbSS mice.

Urinary dysfunction in transgenic sickle cell mice: model of idiopathic overactive bladder syndrome.

Voiding abnormalities are common among the sickle cell disease (SCD) population, among which overactive bladder (OAB) syndrome is observed at rates as high as 39%. Although detrusor overactivity is the most common cause of OAB, its molecular pathophysiology is not well elucidated. The nitric oxide (NO) signaling pathway has been implicated in the regulation of lower genitourinary tract function. In the present study, we evaluated the role of the NO signaling pathway in voiding function of transgenic SCD mice compared with combined endothelial and neuronal NO synthase gene-deficient mice, both serving as models of NO deficiency. Mice underwent void spot assay and cystometry, and bladder and urethral specimens were studied using in vitro tissue myography. Both mouse models exhibited increased void volumes; increased nonvoiding and voiding contraction frequencies; decreased bladder compliance; increased detrusor smooth muscle contraction responses to electrical field stimulation, KCl, and carbachol; and increased urethral smooth muscle relaxation responses to sodium nitroprusside compared with WT mice. In conclusion, our comprehensive behavioral and functional study of the SCD mouse lower genitourinary tract, in correlation with that of the NO-deficient mouse, reveals NO effector actions in voiding function and suggests that NO signaling derangements are associated with an OAB phenotype. These findings may allow further study of molecular targets for the characterization and evaluation of OAB.

Haematological values in steady-state sickle cell anaemia patients and matched heamoglobin AA Controls in a Rural Area of Eastern Gabon.

BACKGROUND: In Gabon, universal neonatal screening of sickle cell disease is not carried out in rural areas, often leading to late detection of the disease. However, complete blood counts are available in rural areas. MATERIALS AND METHODS: We evaluated the haematological parameters of 45 homozygous steady-state sickle cell anaemia (SCA) patients and compared them with 45 sex- and age-matched Haemoglobin AA controls in Koula-Moutou, a rural area in Eastern Gabon. RESULTS: Homozygous SCA patients had low erythrocyte values (red blood cells: 2.50 × 1012/L, haemoglobin: 7.20 g/dL and haematocrit: 20.70%) and high leucocyte values (white blood cells: 14.40 × 109/L, lymphocytes: 5.24 × 109/L and monocytes: 1.60 × 109/L). Most of the SCA patients had severe anaemia (67%), normochromia (76%), lymphocytosis (73%) and monocytosis (84%). A haemoglobin level of < 8.5 g/dL together with a leucocyte level above 9.5 × 109 cells/L was used as screening test to detect homozygous SCA patients, with sensitivity of 84.4% and specificity of 97.8%. CONCLUSION: The values for erythrocyte and leucocyte cell lines of SCA patients in steady state are clearly different from those of the matched HbA/A controls. This makes it possible to set up a tool to detect SCA based on the haemogram in a rural area that does not possess haemoglobin electrophoresis. This tool could be used by healthcare workers in the absence of universal newborn screening for SCA.