HbAA Genotype Research Articles

Children with hemoglobin C or S trait have low serologic responses to a subset of malaria variant surface antigens

Children with hemoglobin AC or AS have decreased susceptibility to clinical malaria. Parasite variant surface antigen (VSA) presentation on the surface of infected erythrocytes is altered in erythrocytes with hemoglobin C (Hb AC) or sickle trait (Hb AS) mutations in vitro. The protective role of incomplete or altered VSA presentation against clinical malaria in individuals with Hb AC or AS is unclear. Using a high-throughput protein microarray, we sought to use serological responses to VSAs as a measure of host exposure to VSAs among Malian children with Hb AC, Hb AS, or wildtype hemoglobin (Hb AA). In uncomplicated malaria, when compared to Hb AA children, Hb AC children had significantly lower serological responses to extracellular Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) domains but did not differ in responses to intracellular PfEMP1 domains and other VSAs, including members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family. Healthy children with Hb AC and Hb AS genotypes recognized fewer extracellular PfEMP1s compared to children with Hb AA, especially CD36-binding PfEMP1s. These reduced serologic responses may reflect reduced VSA presentation or lower parasite exposure in children with Hb AC or AS and provide insights into mechanisms of protection.

Hemoglobin polymorphism and its association with morphometric and egg production traits in Ethiopian indigenous and Sasso chicken breeds.

The present study investigated the biochemical polymorphism of hemoglobin (Hb) and its relationship with performance traits of Ethiopian indigenous and Sasso chicken breeds. A total of 284 chickens reared in three agro-ecologies were examined for genetic diversity and associations with productive traits at Hb locus using agarose gel electrophoresis. The results showed that the HbA allele was dominant in both breeds, and a higher proportion of male chickens were HbAA genotypes, while females were predominantly HbBB types. In the highland agro-ecology, chickens with the HbAA genotype were the most dominant, whereas in mid- and low-land agro-ecologies, chickens with HbBB and HbAB genotypes were found to be more frequent. A moderate level of expected heterozygosity was obtained with 0.47 and 0.445 for indigenous and Sasso chickens, respectively, with an average effective number of alleles per locus of 1.89 and 1.80. Moreover, chickens with HbAA genotypes showed significantly (p ≤ 0.05) higher body weight and linear body measurements than those of HbAB and HbBB genotypes. However, for appendage body structures (comb and wattle dimensions), chickens with the HbAB and HbBB genotypes had higher mean values. Additionally, clutch size (14.2 ± 0.4), clutch length (21.8 ± 0.7), and eight-month egg production (84.1 ± 1.2) were significantly (p ≤ 0.05) higher for hens with HbBB genotypes, followed by those with HbAB-types. Therefore, the considerable hemoglobin variability and significant associations of Hb variants with the performance traits can be sought as guiding information for further genetic improvement interventions in the chicken breeds under investigation. Further microsatellite marker-based genotyping is recommended to validate the higher morphometric values for HbAA genotypes and the better egg production for HbBB and HbAB genotypes.

Immune responses to P falciparum antibodies in symptomatic malaria patients with variant hemoglobin genotypes in Ghana

BackgroundHaemoglobin (Hb) variants such as sickle cell trait (SCT/HbAS) play a role in protecting against clinical malaria, but little is known about the development of immune responses against malaria parasite (Plasmodium falciparum surface protein 230 (Pfs230) and Plasmodium falciparum erythrocyte binding antigen 175 region-3 (PfEBA175-3R)) and vector (on the An. gambiae Salivary Gland Protein-6 peptide 1 (gSG6-P1)) antigens in individuals with variants Hb genotypes. This study assessed antibody (IgG) responses against malaria parasite, Pfs230 and PfEBA175-3R and vector, gSG6-P1 in febrile individuals with variant Hb genotypes.MethodsThe study was conducted on symptomatic malaria patients attending various healthcare facilities throughout Ghana. Microscopy and ELISA were used to determine the natural IgG antibody levels of gSG6-P1, PfEBA175-3R & Pfs230, and Capillarys 2 Flex Piercing was used for Hb variants determination.ResultsOf the 600 symptomatic malaria patients, 50.0% of the participants had malaria parasites by microscopy. The majority 79.0% (398/504) of the participants had Hb AA, followed by HbAS variant at 11.3% (57/504) and HbAC 6.7% (34/504). There were significantly (p < 0.0001) reduced levels of gSG6-P1 IgG in individuals with both HbAC and HbAS genotypes compared to the HbAA genotype. The levels of gSG6-P1 IgG were significantly (p < 0.0001) higher in HbAS compared to HbAC. Similarly, Pfs230 IgG and PfEBA-175-3R IgG distributions observed across the haemoglobin variants were significantly higher in HbAC relative to HbAS.ConclusionThe study has shown that haemoglobin variants significantly influence the pattern of anti-gSG6-P1, Pfs230, and PfEBA-175 IgG levels in malaria-endemic population. The HbAS genotype is suggested to confer protection against malaria infection. Reduced exposure to infection ultimately reduces the induction of antibodies targeted against P. falciparum antigens.

Evaluation of Red Cell Deformability in Sickle Cell Disease Patients, Sickle Cell Carriers, and Healthy Controls Using Lorrca-Derived Parameters

BACKGROUND Red blood cell (RBC) deformability plays a critical role in RBC elongation and viscosity, and when reduced, perfusion of peripheral tissues and oxygen delivery are impaired. Intracellular ATP maintains water and ion homeostasis in RBCs; reduced ATP leads to water and ion loss, dehydration and loss of RBC elasticity. RBC dehydration increases mean corpuscular hemoglobin concentration (MCHC) and also affects distribution of RBC width (RDW). Patients with sickle cell disease (SCD) have reduced RBC ATP, and markedly reduced RBC deformability when compared to healthy individuals. Here, we employed Laser-Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics) to evaluate deformability in RBCs from patients with SCD (HbSS) compared to RBCs from sickle cell carriers (HbAS) and ethnic-matched healthy controls (HbAA). We further analyzed how these LORRCA-derived parameters correlate with ATP, MCHC, and RDW. METHODS We enrolled adult subjects (age ≥ 18 years) of African-descent, and not recently transfused (within 8 weeks) under protocol NCT03685721 approved by the NHLBI Institutional Review Board. Fresh whole blood in EDTA was processed on the same day of collection. Cells were counted and diluted in polyvinylpyrrolidone (PVP) solution before undergoing LORRCA assays including deformability, osmoscan, and oxygenscan (HbSS subjects). The deformability assay uses the elongation Index (EI) to measure the cells' ability to undergo deformation with increasing shear stress at 0.95, 3, and 30 Pascals (Pa). The osmoscan assay measures the deformability under continuous osmotic changes (0-600mOsm/kg) and has several parameters: O min (RBC fragility); El max (maximum deformability and membrane flexibility); O max (osmolality where EI max is achieved) and O hyper (intracellular viscosity and maximum deformability in the hypertonic region). The oxygenscan assay measures the oxygen pressure at the time when RBCs start to become rigid (point of sickling). Whole blood levels of ATP were measured using LC-MS/MS with LLOQ at 50.0 μg/mL and converted to intracellular concentrations by dividing by the hematocrit (as a fraction). Descriptive statistics and correlation (Spearman) of ATP, RDW, and MCHC with each LORRCA parameter were performed using R (v4.2.3) and Prism(v9). RESULTS We studied 156 subjects which comprised of 63 HbSS (33 males), 61 HbAS (23 males) and 32 HbAA (8 males) genotypes. The average age in years (range) in HbSS was 32 (18-58); in HbAS, 43 (18-70), and in HbAA, 38 (19-72). As shear stress and osmotic pressure increased, the mean EI and osmolality were significantly (p&amp;lt;0.001, unpaired two-sample t-test) lower in the HbSS cohort compared to HbAS and HbAA (Figure 1). There was no significant difference in the mean EI and osmolality between HbAA and HbAS with increasing shear stresses and osmotic pressures. As shear stress increased, the correlation of deformability with reduced ATP got significantly stronger in the HbSS cohort (r= -0.351 at 0.95 Pa to r=-0.401 at 30 Pa) but the trend was reversed for the HbAS and HbAA cohort (Table 1). Under increasing osmotic pressure, a significant correlation was found between reduced ATP and maximal deformability (EI Max) in HbSS (p=0.008) compared to HbAS (p=0.896) and HbAA (p=0.114). MCHC was significantly correlated with RBC hydration state (O Hyper) in all 3 genotypic groups (HbSS, HbAS and HbAA). Similarly, RDW correlated with maximal deformability (EI Max) under increasing osmotic pressure in all 3 cohorts. In the HbSS subjects, there was a significant positive correlation of ATP and RDW with point of sickling (oxygenscan); MCHC was negatively correlated but was not significant. CONCLUSION RBC deformability was reduced in HbSS patients compared to HbAS and HbAA under increasing shear stress and osmotic pressure. ATP levels showed significant correlation with RBC deformability as measured by LORRCA, particularly in patients with SCD, suggesting increasing ATP levels as an important therapeutic strategy in patients with SCD. In keeping with published data, our analyses showed that RBC volume and size as well as intracellular hemoglobin concentration can affect deformability.

Molecular Characterization of Haemoglobin E

Objectives: To detect mutation in cases having haemoglobin A2 level 7percent on high performance liquid chromatography. Method: The cross-sectional, descriptive study was conducted from July 2017 to December 2018 at the Department of Haematology and Human Genetics and Molecular Biology, University of Health Sciences, Lahore, Pakistan, and comprised patients of either gender with haemoglobin A2 7percent. The samples were collected from different cities of Punjab in collaboration with the Punjab Thalassemia Prevention Programme, Lahore. The samples were subjected to complete blood count and high performance liquid chromatography using automated haematology analysers and a beta thalassemia short programme, respectively. To analyse haemoglobin E mutations at the molecular level, polymerase chain reaction-restriction fragment length polymorphism (PCR_RFLP) was performed using a type IIS restriction endonuclease known as Mnl1 (derived from Moraxella nonliquefaciens) to cleave DNA at specific sites and the results were further confirmed on randomly selected samples using Sanger sequencing. Data was analysed using SPSS 25. Results: Of the 39 patients, 15(38.5percent) were males and 24(61.5percent) were females. The overall median age was 14 (23) years. There were 29 (74.4percent) patients a thalassemia family history, and 22(56.4percent) had a positive family history of transfusion related to thalassemia, while no patient had a family history of iron therapy. The media haemoglobin A, haemoglobin A2 and haemoglobin F levels were 72.2 (65.2-79.1) percent, 26.6 (19.1-34.0) percent and 0.9 (-0.8-2.6) percent, respectively. After molecular investigation, HbAE mutation was found in 23(59percent) patients, while wild type HbAA genotype was found in 16(41percent). The heterozygous HbE mutation was present in 23(59percent) patients. Conclusions: Frequently missed/undiagnosed cases of haemoglobin E that co-elute with haemoglobin A2 in the same high performance liquid chromatography window were detected among those with haemoglobin A2 7percent. Key Words: Haemoglobin E, Beta-thalassemia, High performance liquid chromatography, Restriction fragment length polymorphism.

Exploring Mitochondrial DNA Heteroplasmy in a Mouse Model of Sickle Cell Disease

BACKGROUND: Mitochondria are important in the pathology of sickle cell disease (SCD). They are abnormally retained in sickle red blood cells (RBCs) and the likely source of cell-free mitochondrial DNA (mtDNA), an erythrocytic-DAMP that triggered formation of neutrophil extracellular traps (Tumburu et al, 2022). Acquired mutations in the mitochondrial genome, referred to mtDNA heteroplasmy, are emerging markers of aging and inflammation. Using whole genome sequence data from peripheral blood DNA, we previously established that patients with SCD not only have increased mtDNA heteroplasmy, but the burden also varied with the sickle genotype [Ahmad et. al, 2021]. The burden of mtDNA heteroplasmy is known to vary across different organs and tissues. Here, we utilized the Townes mouse model of SCD to explore differential mtDNA heteroplasmy burden across the different tissues and sickle genotypes in SCD. METHODS: Genomic DNA was extracted from 9 different organs (heart, brain, muscle, kidney, blood, bone marrow, liver, lungs, spleen) of littermate mice (3 male and 3 female) of three genotypes (HbAA, HbAS, HbSS) in three replica sets (total number of mice = 18, 9 different tissue samples per mouse, total sample number = 162). Mitochondria DNA was enriched from genomic DNA by long range PCR in 2 amplicons of 9kb and 7kb sizes (Misa Hirose et al, 2018). The enriched mtDNA PCR amplicons were mixed together and subjected to library preparation for deep sequencing using Illumina platform. Sequence reads aligned to mtDNA were mapped and called for variants using LoFreq variant calling tool. RESULTS: Of the 162 samples, 1 sample (HbAA, lung) was excluded from analysis due to low coverage. Using LoFreq variant caller tool (VAF cut-off 4% &amp; Coverage Filter 3500X), and after removing the repeating variants, 11 unique variants spanning 16kb mouse mitochondrial genome, were found in 106 of the 161 (65.8%) samples. The variants were differentially distributed across the tissues and genotypes with the variant at mito-position 9820 (MT: 9820) being most frequent, present in 19, 16 and 18 samples in AA, AS and SS mice, respectively (Fig. 1). Two variants at MT: 11866 and MT: 6881 were shared between AA and AS mice. Four variants were unique to SS mice: MT: 6753 in COX1 gene causing Phe476-Leu substitution, MT: 6794 in intergenic region upstream to COX2 gene, MT: 1220 in intergenic region upstream to ND1 gene, and MT425 in regulatory/D-Loop region (Table -1). The SS-unique variants were present in all tissues except for Cox1 Phe476-Leu (MT: 6753), a single variant that was found only in the spleen. Overall, HbSS had the highest concentration of variants distributed among 41/54 (75.9%) followed by AS 38 /54 (70.3 %) and AA 27/53 (50.9%). Female mice had higher mtDNA heteroplasmy burden compared to male mice in all genotypes. Across all genotypes and tissues, spleen had the highest number of variants (16.03%) and liver, the lowest (9.4%). CONCLUSION: In keeping with data in patients with SCD, HbSS mice had the highest mtDNA heteroplasmic burden followed by HbAS and HbAA genotypes. Four mito-variants were unique to HbSS. We also found differentially higher mtDNA heteroplasmic burden in splenic tissues and speculate that this could arise from metabolic stress conditions in the spleen.

Influence of the sickle cell trait on Plasmodium falciparum infectivity from naturally infected gametocyte carriers

BackgroundSickle cell trait (SCT) refers to the carriage of one abnormal copy of the β-globin gene, the HbS allele. SCT offers protection against malaria, controlling parasite density and preventing progression to symptomatic malaria. However, it remains unclear whether SCT also affects transmission stages and mosquito infection parameters. Deciphering the impact of the SCT on human to mosquito malaria transmission is key to understanding mechanisms that maintain the trait in malaria endemic areas.MethodsThe study was conducted from June to July 2017 among asymptomatic children living in the locality of Mfou, Cameroon. Blood samples were collected from asymptomatic children to perform malaria diagnosis by microscopy, Plasmodium species by PCR and hemoglobin typing by RFLP. Infectiousness of gametocytes to mosquitoes was assessed by membrane feeding assays using blood from gametocyte carriers of HbAA and HbAS genotypes. A zero-inflated model was fitted to predict distribution of oocysts in mosquitoes according to hemoglobin genotype of the gametocyte source.ResultsAmong the 1557 children enrolled in the study, 314 (20.16%) were of the HbAS genotype. The prevalence of children with P. falciparum gametocytes was 18.47% in HbAS individuals and 13.57% in HbAA, and the difference is significant (χ2 = 4.61, P = 0.032). Multiplicity of infection was lower in HbAS gametocyte carriers (median = 2 genotypes/carrier in HbAS versus 3.5 genotypes/carrier in HbAA, Wilcoxon sum rank test = 188, P = 0.032). Gametocyte densities in the blood donor significantly influenced mosquito infection prevalence in both HbAS and HbAA individuals. The HbAS genotype had no significant effect on mosquito infection outcomes when using immune or naïve serum in feeding assays. In AB replacement feeding experiments, the odds ratio of mosquito infection for HbAA blood as compared to HbAS was 0.56 (95% CI 0.29–1.10), indicating a twice higher risk of infection in mosquitoes fed on gametocyte-containing blood of HbAS genotype.ConclusionPlasmodium transmission stages were more prevalent in SCT individuals. This may reflect the parasite’s enhanced investment in the sexual stage to increase their survival rate when asexual replication is impeded. The public health impact of our results points the need for intensive malaria control interventions in areas with high prevalence of HbAS. The similar infection parameters in feeding experiments where mosquitoes received the original serum from the blood donor indicated that immune responses to gametocyte surface proteins occur in both HbAS and HbAA individuals. The higher risk of infection in mosquitoes fed on HbAS blood depleted of immune factors suggests that changes in the membrane properties in HbAS erythrocytes may impact on the maturation process of gametocytes within circulating red blood cells.

Effect of iron fortification on anaemia and risk of malaria among Ghanaian pre-school children with haemoglobinopathies and different ABO blood groups

BackgroundHaemoglobinopathies such as sickle cell disorder and glucose-6-phosphate dehydrogenase (G6PD) deficiency as well as differences in ABO blood groups have been shown to influence the risk of malaria and/or anaemia in malaria-endemic areas. This study assessed the effect of adding MNP containing iron to home-made weaning meals on anaemia and the risk of malaria in Ghanaian pre-school children with haemoglobinopathies and different ABO blood groups.MethodsThis study was a double-blind, randomly clustered trial conducted within six months among infants and young children aged 6 to 35 months in rural Ghana (775 clusters, n = 860). Participants were randomly selected into clusters to receive daily semiliquid home-prepared meals mixed with either micronutrient powder without iron (noniron group) or with iron (iron group; 12.5 mg of iron daily) for 5 months. Malaria infection was detected by microscopy, blood haemoglobin (Hb) levels were measured with a HemoCue Hb analyzer, the reversed ABO blood grouping microtube assay was performed, and genotyping was performed by PCR–RFLP analysis.ResultsThe prevalence of G6PD deficiency among the study participants was 11.2%. However, the prevalence of G6PD deficiency in hemizygous males (8.5%) was significantly higher than that in homozygous females (2.7%) (p = 0.005). The prevalence rates of sickle cell traits (HbAS and HbSC) and sickle cell disorder (HbSS) were 17.5% and 0.5%, respectively. Blood group O was dominant (41.4%), followed by blood group A (29.6%) and blood group B (23.3%), while blood group AB (5.7%) had the least frequency among the study participants. We observed that children on an iron supplement with HbAS had significantly moderate anaemia at the endline (EL) compared to the baseline level (BL) (p = 0.004). However, subjects with HbAS and HbAC and blood groups A and O in the iron group had a significantly increased number of malaria episodes at EL than at BL (p < 0.05). Furthermore, children in the iron group with HbSS (p < 0.001) and the noniron group with HbCC (p = 0.010) were significantly less likely to develop malaria.ConclusionsIron supplementation increased anaemia in children with HbAS genotypes and provided less protection against malaria in children with HbAC and AS and blood groups A and O.Trial registrationclinicaltrials.gov Identifier: NCT01001871. Registered 27/10/2009.Registration number: https://clinicaltrials.gov/ct2/show/record/NCT01001871.

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.

Effect of α+ Thalassemia on the Severity of Plasmodium falciparum Malaria in Different Sickle Cell Genotypes in Indian Adults: A Hospital-Based Study

There is a paucity of literature on the association of α+-thalassemia, sickle-cell hemoglobin disorders, and malaria in India. This study aimed to understand the effect of α+-thalassemia on the severity of Plasmodium falciparum malaria in adults with respect to sickle-cell genotypes. The study subjects were categorized into ‘severe-malaria’ and ‘uncomplicated-malaria’ and age-gender matched ‘control’ groups. Sickle-cell and α+-thalassemia were investigated in all the recruited subjects. The effect of α+-thalassemia on the severity of malaria was analyzed in HbAA and sickle-cell genotypes (HbAS and HbSS) separately. The prevalence of α+-thalassemia in various groups ranged from 41.5% to 81.8%. The prevalence of α+-thalassemia was lower (OR = 1.64; p = 0.0013) in severe malaria (41.5%) as compared to healthy controls (53.8%) with HbAA genotype. In contrast, in HbAS genotype, the prevalence of α+-thalassemia was higher (OR = 4.11; p = 0.0002) in severe malaria (81.8%) compared to controls (52.2%). In severe malaria with HbAA genotype, there was a significantly higher hemoglobin level and low MCV and MCH level in patients with α+-thalassemia compared to the normal α-globin genotype. Further, the incidence of cerebral malaria, hepatopathy, and mortality was lower in patients (HbAA) with α+-thalassemia as compared to normal α-globin genotype (HbAA). In severe malaria with either HbAS or HbSS genotype, only a few parameters showed statistical differences with respect to α+-thalassemia. Low prevalence of α+-thalassemia in severe malaria with HbAA genotype compared to healthy controls with HbAA genotype indicates the protective effect of α+-thalassemia against severe malaria. However, the high prevalence of α+-thalassemia in patients with HbAS genotype depicts its interference in the protective effect of sickle-cell against severe malaria.

Pattern of Hb genotype in surgical patients at a tertiary hospital in Southern, Nigeria

Background: Haemoglobin (Hb) genotype is not a routine test in adult pre-operative investigations in majority of clinical settings, though in the obstetric and gynaecological patients already have their Hb genotype as a part of the routine antenatal investigations. However, in paediatric work up for surgical procedures Hb genotype is a routine investigation. This is due to sickle cell endemicity in our locality. Aim: To determine the pattern of HB Genotype in surgical patients at the Rivers State University Teaching Hospital (RSUTH). Method: This was a one-year retrospective study of surgical patients (Surgery and Obstetrics/Gynaecology departments) of the RSUTH. The patients comprised of all the consecutive cases of the surgeries in these departments for the period under review. Ethical clearance was obtained from ethical committee of the Rivers State Hospital Management Board. Structured profoma was used to extract information from patients’ case notes and analyzed using SPSS version 25. Result: There were a total of 370 subjects that were recruited for the study under review. There were 146 (39.5%) males and 224 (60.5%) females. The mean age was 31 years. The age range was 22 years to 56 years. One hundred and ninety four (52.4%) were obstetrics and gynaecological surgeries while 176 (47.6 %) were non-gynaecological surgeries. The commonest indication for surgery was caesarean representing 126 (34.1%) of the subject. The most common Hb genotype was AA 246 (66.5%)of which females contributed to 148 (40%) and males 98(26.5%)had Hb AA while 124 (33.5%)of the subjects were Hb AS. Females that were Hb As were 74 (20%) while males were 50 (13.5%). There were no subjects that were Hb SS. Conclusion: This study showed the most prevalent Hb Genotype as AA (66.5%) in surgical patients at the Rivers State University Teaching Hospital (RSUTH), while 33.5% had Hb AS genotype. None of these subjects had sickle cell (HbSS) homozygous trait

Spleen size in homozygous sickle cell disease: trends in a birth cohort using ultrasound.

To provide ultrasound baselines for spleen length in homozygous sickle cell disease (HbSS) and in normal controls with a HbAA genotype. The Jamaican cohort study identified 311 babies with HbSS and 246 matched HbAA controls during the screening of 100,000 consecutive deliveries in Kingston, Jamaica from 1973 to 1981. Ultrasonography commenced in 1988 when the youngest patients were aged 6 years at which time deaths, emigrations and default had reduced the numbers to 206 HbSS and 89 controls. It continued annually until 2000. The spleen was visualized in all HbAA controls but in only 1103/2138 (52%) scans in HbSS. Where available, mean splenic lengths were significantly lower in HbSS (77-103 mm in males, 70-83 mm in females) compared to normal controls (89-101 mm in males, 86-95 mm in females). Assessed by statistical modelling after adjusting for body height, the splenic ratio (splenic length/body height) declined over the age range 12-20 years in HbSS, consistent with progressive splenic fibrosis. Genetic factors known to inhibit sickling, α thalassemia and fetal hemoglobin level (HbF) significantly reduced the decline in splenic ratio. Clinical splenomegaly was an insensitive measure of splenic enlargement as only 50% of patients aged 18 years and above with spleens measuring ≥150 mm on ultrasonography had palpable spleens. An age-related decline in splenic length occurred in HbSS and occurred more slowly with genetic factors known to inhibit sickling. The standards provided may be of value in assessing minor degrees of subclinical acute splenic sequestration. These are the first standards available for splenic length in HbSS. They may be useful in detecting red cell sequestration, not apparent from clinical splenomegaly and also provide a model for identifying factors inhibiting vaso-occlusion.

Epigenetic analysis in placentas from sickle cell disease patients reveals a hypermethylation profile.

Pregnancy in Sickle Cell Disease (SCD) women is associated to increased risk of clinical and obstetrical complications. Placentas from SCD pregnancies can present increased abnormal findings, which may lead to placental insufficiency, favoring adverse perinatal outcome. These placental abnormalities are well known and reported, however little is known about the molecular mechanisms, such as epigenetics. Thus, our aim was to evaluate the DNA methylation profile in placentas from women with SCD (HbSS and HbSC genotypes), compared to uncomplicated controls (HbAA). We included in this study 11 pregnant women with HbSS, 11 with HbSC and 21 with HbAA genotypes. Illumina Methylation EPIC BeadChip was used to assess the whole placental DNA methylation. Pyrosequencing was used for array data validation and qRT-PCR was applied for gene expression analysis. Our results showed high frequency of hypermethylated CpGs sites in HbSS and HbSC groups with 73.5% and 76.2% respectively, when compared with the control group. Differentially methylated regions (DMRs) also showed an increased hypermethylation status for the HbSS (89%) and HbSC (86%) groups, when compared with the control group methylation data. DMRs were selected for methylation validation (4 DMRs-HbSS and 3 DMRs the HbSC groups) and after analyses three were validated in the HbSS group, and none in the HbSC group. The gene expression analysis showed differential expression for the PTGFR (-2.97-fold) and GPR56 (3.0-fold) genes in the HbSS group, and for the SPOCK1 (-2.40-fold) and ADCY4 (1.80-fold) genes in the HbSC group. Taken together, these data strongly suggest that SCD (HbSS and HbSC genotypes) can alter placental DNA methylation and lead to gene expression changes. These changes possibly contribute to abnormal placental development and could impact in the clinical course, especially for the fetus, possibly leading to increased risk of abortion, fetal growth restriction (FGR), stillbirth, small for gestational age newborns and prematurity.

Sociodemographic Determinants of Nutritional Status in Children with Sickle Cell Anaemia in Benin City Edo State, Nigeria

Background: Nigeria has the highest burden of sickle cell anaemia (SCA) in Africa. Patients with SCA present with life-long complications in addition to growth failure. With comprehensive care, patients with SCA have improved survival and well-being, with some studies reporting overnutrition. This study aims to assess nutritional status and sociodemographic determinants in children with SCA.&#x0D; Methods: A descriptive cross-sectional study was carried out among children aged 1-16years attending the paediatric SCA clinics and their sex and age-matched controls. Anthropometric measurements were carried out to determine their nutritional status, and the data obtained were analyzed using SPSS software. The sociodemographic and relevant clinical information was obtained using a semi-structured questionnaire.&#x0D; Results: The majority (86%) of the children with SCA had normal height and weight, but the mean weight, weight for age z-score (WAZ), body mass index z-score (BMIZ) and height for age z-score (HAZ) were significantly lower than the HbAA controls. The mean WAZ was significantly higher in children from high socioeconomic class (SEC) and those whose mothers had tertiary education than those from lower educational statuses. Similarly, the mean HAZ was significantly higher in children from high SEC, tertiary maternal education and younger children.&#x0D; Conclusion: Most children with SCA have normal nutritional status despite significantly lower nutritional indicators compared to children with HbAA genotype. Higher educational status of mothers and SEC were associated with a significantly higher nutritional status among the subjects.

P-062: MATRIX COMPARISON OF HEMOTYPESC AND SICKLESCAN POINT-OF-CARE DIAGNOSTICS FOR EARLY DIAGNOSIS OF SICKLE CELL DISEASE IN RURAL SUB-SAHARAN AFRICA

Purpose: In sub-Saharan Africa, universal sickle cell disease-newborn screening (SCD-NBS) programs are nonexistent due to high costs associated with conventional laboratory technologies, competing for national healthcare priorities, and budgetary constraints. However, the recent emergence of lateral-flow immunoassay microtechnology point-of-care tests (POCT) such the SickleSCAN™ and HemoTypeSC™ present exciting opportunities to change the status quo and potentially revolutionize healthcare for children as best practice for early diagnosis of SCD. Materials and methods: We perform a matrix comparison of diagnostic accuracy in field conditions for the SickleSCAN™ and HemoTypeSC™ tests. We assessed the performance characteristics of SickleSCAN™ and HemoTypeSC™ by evaluating children with positive Emmel test for HbSS, HbSC, and HbAS; and children, including newborns, without known coagulopathy in a rural district in Sierra Leone. Results: A total of seventy-four (33 females/41 males) children from three participating community health centers were analyzed by both methods. The ages ranged from twenty-four hours old to seventeen years old. HemoTypeSC™ and SickleSCAN™ correctly identified hemoglobinopathy phenotypes in 26 children with positive Emmel test as follows: twenty HbSS and four HbAS. Two positive Emmel test results (HbAS) were identified as HbAC and HbAA by the two tests indicating that the two tests have excellent sensitivity and specificity for the identification of the HbS pattern, consistent with SCD. Of the 74 samples that had two SCD POCT scores, there was no discordant result between the HemoTypeSC™ and the SickleSCAN™. Results for both tests were as follows: HbAA 36 (46.8%), HbAS 17 (22.1%), HbSC 1 (1.3%), and HbSS (26.0%). No newborns were identified with sickle cell disease (HbSS). The two tests identified seven newborns with sickle cell trait (HbAS). Twenty-one newborns had the normal HbAA genotype. Overall, HemoTypeSC™ and SickleSCAN™ exhibited sensitivity and specificity of 100% and 100% respectively for all possible phenotypes (HbAA, HbAS, and HbSS) detected. Conclusion: The HemoTypeSC and SickleSCAN tests demonstrated 100% concordance for identifying homozygote healthy people, sickle cell trait, and disease patients in this study. Our matrix comparison study highlighted the potential use of POCTs as both first?tier and second-tier screening and confirmation tests when presumptive SCD or trait is indicated. This is an acceptable practice for SCD screening as the current consensus is to use the same method on the same sample if no second?tier screening method is available and to confirm the screening result with a second method on a second sample to make a diagnosis. We, therefore, conclude that POCTs are suitable for population and newborn screening for the HbS phenotype in limited-resource settings where the disease burden is greatest and conventional laboratory methods are nonexistent. The authors do not declare any conflict of interest

P-086: POINT-OF-CARE SCREENING FOR SICKLE CELL DISEASE IN RURAL AFRICAN COMMUNITY HEALTHCARE CENTERS

Purpose: Over 200 000 babies are born with sickle cell disease (SCD) annually in sub-Saharan Africa. In many African nations, however, 50% to 90% of children with SCD will not survive until their fifth birthday and will die due to susceptibility to malaria, sepsis, anemia, and infection without being diagnosed with SCD. The incidence of SCD and trait in newborns in Sierra Leone is unknown. Early diagnosis of SCD and treatment can greatly improve patient outcomes. The purpose of this study was to assess the feasibility of a Nurse Champion sickle cell disease point of care testing (SCD-POCT) model in community healthcare centers using a novel microtechnology diagnostic device. Materials and methods: This cross-sectional study was conducted at a regional government hospital in a rural district in Sierra Leone. Immediate, free screening was offered to newborns over 24 hours old delivered at the maternity ward. We used a validated, low-cost, point-of-care, lateral flow immunoassay (SickleSCAN®) device to screen for SCD and trait. The screening tests were conducted by 12 nurse-midwives. A brief questionnaire was used to record the test evaluators’ experience with the device. Results: Fifty-two (30 males/22 females) newborns screened within 24-48 hours of birth over four weeks. Two newborns were identified with sickle cell disease (HbSS). The test identified 13 newborns with sickle cell trait (HbAS). Thirty-seven newborns had the normal HbAA genotype. The heterozygous HbSC genotype was not observed in the study cohort. 12 nurse-midwives evaluators rated the acceptability and feasibility of screening for SCD at the bedside as very good. None of the evaluators deemed the device to be inappropriate for SCD screening. Conclusion: Our study demonstrates a practical method of instituting SCD screening in maternity wards with nurses and midwives as frontline implementers. Our study is a “real world” confirmation that the SickleSCAN device produces reliable results; that nurses are uniquely positioned to educate and advocate for newborn screening, and serves as a “proof of concept” for service delivery, particularly in settings where access is limited and point-of-care testing is less well studied. The authors do not declare any conflict of interest

ՀՆԴԿԱՀԱՎԵՐԻ ԱՐՅԱՆ ՊՈԼԻՄՈՐՖ ՍՊԻՏԱԿՈՒՑՆԵՐԻ ԿԻՐԱՌՈՒՄԸ ՈՐՊԵՍ ՍԵՌԵՐԻ ԱՌԱՋԱՑՈՒՄԸ ԿԱՐԳԱՎՈՐՈՂ ՄՈԼԵԿՈՒԼԱՅԻՆ ՄԱՐԿԵՐՆԵՐ

The conducted investigations testify that due to the genotypes and their occurrence frequency in mother turkeys, the numerical sex ratio undergoes certain changes in the generation. Males emerged from the mothers with TfAA CpCD HbAB and TfBD CpCD HbAA genotypes make up about 78 and 69 %, whereas females produced from the mothers with TfAA CpCD HbAB and TfBD CpCD HbAA genotypes are 65 %. The number of males and females produced from the mothers with TfAB CpBD HbCC, TfBC CpAB HbBD and TfCD CpCC HbCD genotypes is equal (P&gt;0,999). The obtained outcomes can be applied in the turkey breeding activities as genetic markers.

Determination of Coagulation Profile among Children with Sickle Cell: Anemia in Steady-State and Crisis

Background: Patients with Sickle Cell Disease (SCD) have been found to have an aberrant coagulation profile. One of the primary elements hypothesized to contribute to the vaso-occlusive crisis that characterizes sickle cell disease is coagulopathy (SCD)&#x0D; Material and Methods: A total of 150 children were enrolled as follows, 50 children with Sickle Cell Anemia (SCA) in steady-state, 50 in crisis, and 50 with Hb AA genotype as control. 5 ml of Venous blood was collected. The platelets count was performed using Sysmex KX21N, the electrical impedance principle. The Mean Platelet Volume (MPV) is derived from the impedance platelet size distribution curve. STAGO PT31039352) semi-automated machine was used for estimation of PT, and APTT.&#x0D; Result: children with SCA have significantly a prolonged PT, and APTT compared with children with normal hemoglobin genotype (P. value &lt; 0.001) the mean of PT was (16.64, and 12.6) respectively, and APTT (41.45 and 37.94) consequently. A significant increase in platelet count between patients with SCA when compared with control (p. value 0.02), however a significant prolonged in APTT when compared to steady-state (P. value 0.005). MPV among children with crises when compared with steady-state revealed a significant result (p. value 0.006) the mean of MPV in steady-state = 6.79 while the mean of MPV in crisis = 7.09.&#x0D; Conclusion: children with sickle cell anemia had a longer coagulation profile and marked variation in platelet count, which may increase the risk of thrombosis or bleeding.

Increased Circulating Copeptin Levels Are Associated with Vaso-Occlusive Crisis and Right Ventricular Dysfunction in Sickle Cell Anemia

Objective: Vaso-occlusive crisis (VOC) is a common clinical manifestation of sickle cell anemia (SCA) and is associated with increased proinflammatory mediators. Copeptin is the C-terminal part of the prohormone for provasopressin and seems clinically relevant in various clinical conditions. Right ventricular (RV) dysfunction significantly appears in SCA patients due to pulmonary hypertension. This study aimed to investigate the association of copeptin levels in VOC patients and evaluate RV dysfunction. Materials and Methods: A total of 108 patients were enrolled in the study. Twenty-eight SCA patients in steady state (30.2 ± 0.9 years), 25 SCA patients in VOC (36.8 ± 11.8 years), and 55 healthy individuals (31.9 ± 9.4 years) with HbAA genotype were included. Clinical, echocardiographic, and laboratory data were recorded. ELISA was used for the determination of serum levels of copeptin. Results: VOC patients had significantly higher copeptin level compared both with controls and SCA subjects in steady state (22.6 ± 13.0 vs. 11.3 ± 5.7 pmol/L, 22.6 ± 13.0 vs. 12.4 ± 5.8 pmol/L, p = 0.009 for both). Additionally, the copeptin level was significantly higher in SCA patients with RV dysfunction than those without RV dysfunction (23.2 ± 12.2 vs. 15.3 ± 9.5 pmol/L, p = 0.024). Multiple logistic regression analysis revealed that high-sensitivity C-reactive protein and copeptin levels were found to be associated with VOC. Conclusion: This study showed that copeptin and hs-CRP levels were increased in patients with VOC, and it was found that RV dysfunction was more common in SCA patients with VOC than in the control group. Copeptin can be considered for use as a potential biomarker in predicting VOC crisis in SCA patients and in the early detection of patients with SCA who have the potential to develop RV dysfunction.

Individuals with Sickle Cell Disease Have a Higher Burden of Mitochondrial DNA Heteroplasmy

Background: The simple point mutation that causes sickle cell disease (SCD) belies the extensive systemic damage it can cause. While the sickle pathology is initiated by polymerization of HbS, the multiple end-organ damage is inflicted by years of on-going inflammation and vasculopathy. An emerging marker of inflammation is the accumulation of acquired heteroplasmy mutations in mitochondrial DNA (mtDNA). Given the underlying chronic inflammation in SCD, we hypothesized that SCD patients display increased rates of mtDNA mutations, and previously confirmed (1). Here, we further performed indepth analyses in an ethnically matched normal (HbAA) as well as sickle trait (HbAS) subjects from another independent cohort, the Jackson Heart Study (JHS).Methods: We analyzed and compared whole genome sequencing (WGS) data from the from NIH cohort of 676 SCD patients of African ancestry with that of 621 ethnic-matched indviduals from the 1000 Genomes Project (1KG), and 3,580 individuals from the JHS cohort. The NIH SCD cohort included 561 HbSS & HbSβ 0thalassemia (combined), 90 HbSC, and 25 HbSβ + thalassemia genotypes, the 1KG cohort - 516 HbAA and 105 HbAS and JHS cohort - 3,200 HbAA, 89 HbAC (hemoglobin C trait), and 291 HbAS. Additionally, to further understand any potential sequencing depth bias, as well as to compare between two patient cohorts (NIH SCD & JHS cohorts) with underlying conditons that may influence the heteroplasmy bias, we downsampled 300 NIH cohort HbSS samples to a sequencing depth similar to JHS cohort, and compared their heteroplasmy burden. Mitochondrial sequences extracted from the cleaned WGS data of these 3 cohorts were analyzed for heteroplasmic and homoplasmic variants using mitoCaller from the package mitoAnalyzer.Results: The average depth per locus was ~6,671X for the NIH SCD cohort , ~2,879X for the 1KG cohort, and ~2169X for JHS cohort. We compared the quantity of heteroplasmic variants across the different NIH SCD genotype with 1KG (HbAA & HbAS), and JHS (HbAA, HbAC and HbAS) genotypic groups. The median number of heteroplasmic variants per individual increased progressively from HbAA, HbAS, HbSβ +thalassemia, and HbSC with the highest median number of 118 in HbSS & HbSβ 0 (Fig 1A) in NIH SCD cohort. It is noteworthy that the median mtDNA heteroplasmy in HbAA individuals in 1KG cohort was significantly lower than those in JHS cohort (Table insert in Fig 1A) which may be related to the underlying cardiovascular disease in the JHS cohort; whereas similar heteroplasmy burden in HbAS individuals between these 2 cohorts may underscore the genotype (HbAS) as the driver of heteroplasmy in these cohorts. We compared the heteroplasmy burden of a downsampled subset (n=300) NIH HbSS with that of JHS HbAA, HbAC and HbAS genotypes (Fig 1B). Although, the 70% reduction in sequencing depth resulted in the slight reduction in heteroplasmy burden, we noticed higher heteroplasmic variability (standard deviation) in this subset of NIH HbSS patients. This variability may be attributable to extreme variation in SCD phenotypic severity. We then applied cumulative distribution function to this downsampled subset and compared with JHS genotypes. We found the NIH HbSS patients have disproportionately higher proportion of heteroplasmy variants (Fig 1D) when compared to the JHS genotypes (HbAA, HbAC, and HbAS).Conclusion: We conclude that there is an increased prevalence of heteroplasmic mtDNA variants in SCD compared to ethnic-matched normal (HbAA) populations. Normal individuals with HbAA in JHS cohort have significantly higher heteroplasmic burden compared to those in 1KG cohort, suggesting an underlying cardiovascular disease in JHS cohort as a driving factor. Within each 1KG and JHS cohorts, individuals with sickle cell trait (HbAS) have similar heteroplasmy burden and also higher than those with HbAA, highlighting the potential significance of this genotype. Reducing the sequencing depth by > 70% (downsampling) led to the filtering out of heteroplasmy variants that would have been discovered with the original deeper sequencing depth of ~7300X. Nonetheless, downsampled HbSS samples still retained disproportionately higher heteroplasmy burden compared to non-SCD subjects. We are currently investigating if there is any correlation between mtDNA heteroplasmy burden and severity of clinical phenotypes among the SCD patients.1. Ahmad, MM et al, Blood 136 (1):11-11 (2020) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.