Proteomic profiling of circulating ß-thalassaemia/haemoglobin E extra-cellular vesicles reveals that association with immunoglobulin induces membrane vesiculation.

Splenectomised ß-thalassaemia/haemoglobin E (HbE) patients have increased levels of circulating microparticles or medium extra-cellular vesicles (mEVs). The splenectomised mEVs play important roles in thromboembolic complications in patients since they can induce platelet activation and endothelial cell dysfunction. However, a comprehensive understanding of the mechanism of mEV generation in thalassaemia disease has still not been reached. Thalassaemic mEVs are hypothesised to be generated from cellular oxidative stress in red blood cells (RBCs) and platelets. Therefore, a proteomic analysis of mEVs from splenectomised and non-splenectomised ß-thalassaemia/HbE patients was performed by liquid chromatography with tandem mass spectrometry. A total of 171 proteins were identified among mEVs. Interestingly, 72 proteins were uniquely found in splenectomised mEVs including immunoglobulin subunits and cytoskeleton proteins. Immunoglobulin G (IgG)-bearing mEVs in splenectomised patients were significantly increased. Furthermore, complement C1q was detected in both mEVs with IgG binding and mEVs without IgG binding. Interestingly, the percentage of mEVs generated from RBCs with IgG binding was approximately 15-20 times higher than the percentage of RBCs binding with IgG. This suggested that the vesiculation of thalassaemia mEVs could be a mechanism of RBCs to eliminate membrane patches harbouring immune complex and may consequently prevent cells from phagocytosis and lysis.

A comparative evaluation of the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC) with capillary zone electrophoresis (CZE) assays for the detection of hemoglobin variants and the quantitation of HbA0, A2, E, and F.

OBJECTIVES: Hemoglobinopathies, including thalassemia and hemoglobin (Hb) variants, are common hematological disorders in tropical countries. Accurate and precise separation of hemoglobin types and reliable quantitation are necessary for differential diagnosis of these disorders. METHODS: We have evaluated the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC; Trinity Biotech, Co. Wicklow, Ireland) to assist in the presumptive diagnosis of thalassemia and Hb variants commonly found in Southeast Asian countries. HbA0, HbA2, HbE, and HbF levels were separated and quantified in 120 blood samples from unrelated adult subjects and compared with those analyzed by capillary zone electrophoresis (CZE; CAPILLARYS™ 2, Sebia, Norcross, GA, US). The Hb analysis patterns of Hb variants obtained from the PR-HPLC system were also compared to those obtained from HPLC (VARIANT II, ß-thalassemia Short Program, Bio-Rad, Laboratories, Hercules, CA, US) and CZE systems. RESULTS: The PR-HPLC had excellent precision with a coefficient of variation (CV) for HbA2 quantitation of 3.8 % within-run and 5.2 % between-run. The levels of HbA2/E quantified by the PR-HPLC system correlated well with those of the CZE system (r=0.997). In addition, thalassemia interpretation results obtained from the PR-HPLC and the CZE showed 100 % agreement. Moreover, chromatograms of the PR-HPLC were also comparable to those of VII-HPLC and CAP2-CZE electropherograms. CONCLUSIONS: The PR-HPLC system would be applicable to diagnose common forms of thalassemia and Hb variants in Southeast Asia.

Identification of a Novel Variant c.163delG in HBB Gene Resulting in a Beta Null Phenotype in a Proband with Thalassemia Intermedia.

A 21-year-old patient presented with a previous medical history of pallor, mild icterus, increased fatigue, low hemoglobin, and abnormal hemoglobin variant analysis with more than 70 transfusions. He was referred for genetic analysis to identify the pathogenic variations in the ß-globin gene. Sanger's sequencing of the proband and his family revealed the presence of a novel frame shift variant HBB:c.163delG in a compound heterozygous state with hemoglobin E (HbE) (HBB:c.79G > A) variant. The father and the sibling of the patient were found to be normal for the HBB gene. Mother was found to be heterozygous for HbE (HBB:c.79G > A) variant. In silico analysis by Mutalyzer predicted that c.163delG variant generated a premature stop codon after seven codons, leading to a truncated protein. FoldX protein stability analysis showed a positive ΔΔG value of 45.27 kcal/mol suggesting a decrease in protein stability. HBB:c.79G > A is a known variant coding for HbE variant, which results in the reduced synthesis of ß-globin chain and shows mild thalassemia. Combined effect of HBB:c.163delG and HBB:c.79G > A variants in the proband might have led to the reduced synthesis of ß-globin chains resulting in a thalassemia intermedia type of clinical manifestation.

Spectrum of Adrenal Dysfunction in Hemoglobin E/Beta Thalassemia.

BACKGROUND: Adrenal insufficiency (AI) in hemoglobin E (HbE)/beta thalassemia, including evaluation of mineralocorticoid axis, had not been studied. AIMS AND OBJECTIVES: In this study, we attempted to evaluate the prevalence of AI in HbE/beta thalassemia and wanted to determine if the prevalence of AI varied according to severity of HbE/beta thalassemia and transfusion requirements. METHODS: In this observational, cross-sectional study, 104 patients with HbE/beta thalassemia were evaluated. Among them, 57 and 47 were transfusion dependent and non-transfusion dependent. According to Mahidol criteria, patients were classified into mild (n = 39), moderate (n = 39), and severe (n = 26) disease. Early morning (8 Am) serum cortisol, plasma ACTH, and plasma aldosterone, renin were measured. Patients with baseline cortisol of 5 to 18 µg/dL underwent both 1 µg and 250 µg short Synacthen test. According to these results, patients were classified as having either normal, subclinical, or overt (primary/secondary) adrenal dysfunction. RESULTS: Adrenal insufficiency was found in 41% (n = 43). Among them 83.7% (n = 34) had primary AI and 16.3% (n = 9) had secondary AI. Thirty-three patients (31%) with normal or elevated ACTH and with low or normal aldosterone with high renin were diagnosed as having subclinical AI. There was no difference in prevalence of AI between transfusion dependent and non-transfusion dependent (P = .56) nor was there was any difference in prevalence of AI according to disease severity (P = .52). CONCLUSION: Adrenal insufficiency is common in HbE/beta thalassemia and is independent of transfusion dependency and disease severity.

Molecular Characterization of Haemoglobin E.

OBJECTIVE: To detect mutation in cases having haemoglobin A2 level >7% on high performance liquid chromatography. METHODS: 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 ≥7%. 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 variant-II 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(38.5%) were males and 24(61.5%) were females. The overall median age was 14 (23) years. There were 29 (74.4%) patients with thalassemia family history, and 22(56.4%) had a positive family history of transfusion related to thalassemia, while no patient had a family history of iron therapy. The median haemoglobin A, haemoglobin A2 and haemoglobin F levels were 72.2 (65.2-79.1) %, 26.6 (19.1-34.0) % and 0.9 (-0.8-2.6) %, respectively. After molecular investigation, HbAE mutation was found in 23(59%) patients, while wild type HbAA genotype was found in 16(41%). The heterozygous HbE mutation was present in 23(59%) 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 ≥7%.

Molecular understanding of unusual HbE-ß+-thalassemia with Hb phenotype similar to HbE heterozygote: simple and rapid differentiation using HbE levels.

BACKGROUND: Low HbF expression in HbE-ß+-thalassemia may lead to misdiagnosis of HbE heterozygosity. We aimed to characterize the ß- and α-globin genes and the modifying factors related to HbF expression in patients with an Hb phenotype similar to that of HbE heterozygotes. Furthermore, screening tools for differentiating HbE-ß+-thalassemia from HbE heterozygotes have been investigated. PARTICIPANTS AND METHODS: A total of 2133 participants with HbE and HbA with varying HbF levels were recruited. Polymerase chain reaction-based DNA analysis and sequencing were performed to characterize ß- and α-globin genes. DNA polymorphism at position -158 nt 5' to Gγ-globin was performed by XmnI restriction digestion. Receiver operating characteristic (ROC) curves were constructed using the area under the curve (AUC). Cutoff values of HbA2, HbE, and HbF levels for the differentiation of HbE-ß+-thalassemia from HbE heterozygotes were determined. RESULTS: Five ß+-thalassemia mutations trans to ßE-gene (ß-87(C>A), ß-31(A>G), ß-28(A>G), ß19(A>G), and ß126(T>G)) were identified in 79 patients. Among these, 54 presented with low HbF levels, and 25 presented with high HbF levels. ROC curve analysis revealed an excellent AUC of 1.000 (95% confidence interval:1.000-1.000) for HbE levels, and a cut-off point of ≥35.0% had 100.0% sensitivity, specificity, and Youden's index for differentiating HbE-ß+-thalassemia from HbE heterozygotes. The proportion of α-thalassemia mutations was 46.3 and 8.0% among HbE-ß+-thalassemia patients with low and high HbF levels, respectively. Two rare α-thalassemia mutations (Cap +14(C>G) and initiation codon (ATG>-TG)) of α2-globin genes were identified. The genotype and allele of the polymorphism at -158 nt 5' to Gγ-globin was found to be negatively associated with HbF expression. CONCLUSIONS: HbE-ß+-thalassemia cannot be disregarded until appropriate DNA analysis is performed, and the detection of α-thalassemia mutations should always be performed under these conditions. An HbE level ≥35.0% may indicate screening of samples for DNA analysis for HbE-ß+-thalassemia diagnosis.

HbE-ß⁺-thalassemia displays a wide range of HbF expression, which may lead to the misdiagnosis of HbE heterozygosity in patients whose Hb analysis shows HbE and HbA. α-Thalassemia may be a major factor associated with decreased secondary activation of HbF expression in the disease.HbE may be a potential indicator for effectively differentiating HbE-ß⁺-thalassemia from HbE heterozygotes.The high proportion and heterogeneity of α-thalassemia mutations found in patients with HbE-ß⁺-thalassemia evoke a complex thalassemia syndrome, requiring complete DNA analysis.

Undetectable high-performance liquid chromatography haemoglobin A1c on variant haemoglobin E phenotype: a case report.

The gold standard for long-term monitoring of diabetic patients is glycated haemoglobin (HbA1c), which is routinely tested for glycaemic control. Furthermore, the National glycohemoglobin standardization program (NGSP) has designated high-performance liquid chromatography (HPLC) as the reference method for HbA1c measurement. A woman from the Sumba tribe, Indonesia, aged 52, visited the Internal Medicine Clinic for a routine check-up. She had been taking diabetic and hypertension medicines on a regular basis for over 10 years. The HPLC procedure yielded "no result" for the patient's HbA1c assessment and there was no peak on the HPLC graphic. However, there was a discrepancy between the data history of HbA1c measured by turbidimetric method (average of 51 mmol/mol, reference range < 48 mmol/mol), fasting blood glucose (average of 7.7 mmol/L, reference range < 7.0 mmol/L) and 2-hour plasma glucose (average of 13 mmol/L, reference range < 11.1 mmol/L). Glycated albumin was 3.1 mmol/L (reference range 1.8-2.4 mmol/L). Haemoglobin electrophoresis identified homozygote haemoglobinopathy E (HbE). Patients with haemoglobin variants are proposed to utilize glycated albumin.

Generation of human induced pluripotent stem cell line (MUi033-A) from a male with homozygous for Hemoglobin E.

Hemoglobin E (HbE), a common variant in Southeast Asian populations, results from a G to A substitution at codon 26 of the HBB gene, causing abnormal Hb and mild ß-thalassemia-like symptoms. Here, we derived an induced pluripotent stem cell (iPSC) line, named MUi033-A, from a male homozygous for HbE. The iPSC line demonstrates a normal karyotype and embryonic stem cell-like properties including pluripotency gene expression, and tri-lineage differentiation potential. This iPSC resource holds the potential for investigating gene therapy targeting HbE mutation.

Hemoglobin profile and molecular characteristics of the complex interaction of hemoglobin Doi-Saket [α9(A7) asn > lys, HBA2:c.30C > a], a novel α2α1 hybrid globin variant, with hemoglobin E [ß26(B8) Glu > lys, HBB:c.79G > A] and deletional α+-thalassemia in a Thai family.

BACKGROUND: An increasing number of α-hemoglobin (Hb) variants is causing various clinical symptoms; therefore, accurate identification of these Hb variants is important. OBJECTIVE: This study aimed to describe the molecular and hematological characteristics of novel Hb Doi-Saket that gives rise to a typical α+-thalassemia phenotype in carriers with and without other hemoglobinopathies. MATERIALS AND METHODS: Biological samples from a proband and his family members were analyzed. Hematological profiles were analyzed using a standard automated cell counter. Hb was analyzed by capillary electrophoresis and high-performance liquid chromatography. Mutations and globin haplotype were identified by DNA analysis. Novel diagnostic tools based on allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism were developed. RESULTS: Hb analysis showed a major abnormal Hb fraction, moving slower than HbA, and a minor Hb fraction alongside HbA2 in the proband, his father, and son. DNA analysis of the α-globin gene identified the -α3.7 deletion and in cis the C > A mutation on codon 9 of the α2α1 gene, corresponding to Hb Doi-Saket [α9(A7) Asn > Lys]. This mutation could be identified using newly developed allele-specific PCR-based assays. The Hb Doi-Saket al.lele was significantly associated with haplotype [- + M + + 0 -]. Interaction of αDoi-Saket with ßE globin chains led to a new Hb variant (HbE Doi-Saket). Phenotypic expression was clinically silent in heterozygotes and might present slight microcytosis. CONCLUSIONS: Hb Doi-Saket emphasizes a great diversity present in α-globin gene. The mutation in this family from Thailand was linked to -α3.7 and caused mild microcytosis in the carriers. The combination of this variant with deletions in α genes might cause a severe clinical phenotype. Different methods of separation can provide useful information in diagnosis, and a complete molecular approach is needed for confirmation before considering patient management.

The Hb Doi-Saket is a novel α-globin variant mutation occurring in the α2-globin gene in cis to the -α^3.7 kb chromosome.The carrier of Hb Doi-Saket may present slight microcytosis and have severe clinical entities when it interacts with deletions in α-globin genes.Hb analysis with the HPLC system could completely separate Hb Doi-Saket and its derivative from other Hbs.

Up-regulation of microRNA 101-3p during erythropoiesis in ß-thalassemia/HbE.

Ineffective erythropoiesis is the main cause of anemia in ß-thalassemia. The crucial hallmark of ineffective erythropoiesis is the high proliferation of erythroblast. microRNA (miR/miRNA) involves several biological processes, including cell proliferation and erythropoiesis. miR-101 was widely studied and associated with proliferation in several types of cancer. However, the miR-101-3p has not been studied in ß-thalassemia/HbE. Therefore, this study aims to investigate the expression of miR-101-3p during erythropoiesis in ß-thalassemia/HbE. The results showed that miR-101-3p was upregulated in the erythroblast of ß-thalassemia/HbE patients on day 7, indicating that miR-101-3p may be involved with high proliferation in ß-thalassemia/HbE. Therefore, the mRNA targets of miR-101-3p including Rac1, SUB1, TET2, and TRIM44 were investigated to determine the mechanisms involved with high proliferation of ß-thalassemia/HbE erythroblasts. Rac1 expression was significantly reduced at day 11 in severe ß-thalassemia/HbE compared to normal controls and mild ß-thalassemia/HbE. SUB1 gene expression was significantly lower in severe ß-thalassemia/HbE compared to normal controls at day 9 of culture. For TET2 and TRIM44 expression, a significant difference was not observed among normal and ß-thalassemia/HbE. However, the high expression of miR-101-3p at day 7 and these target genes was not correlated, suggesting that this miRNA may regulate ineffective erythropoiesis in ß-thalassemia/HbE via other target genes.

Essential genetic modifiers and their measurable impact in a community-recruited population analysis for non-severe hemoglobin E/ß-thalassemia prenatal genetic counseling.

The study aimed to identify essential phenotype-modulating factors among the pre-existence of several important ones and clarify their measurable impact on the clinical severity of hemoglobin (Hb) E/ß-thalassemia in a community-recruited population analysis. This prospective study was designed to compare modifiers between community- (less or no symptoms) and hospital-recruited individuals with Hb E/ß-thalassemia. The formerly included couples previously assessed for prenatal thalassemia at-risk status at 42 community and 7 referral hospitals in Thailand through on-site investigations between June 2020 and December 2021. The control included Hb E/ß-thalassemia patients undergoing transfusions. The Mahidol score classified disease severity. Beta-globin, α0-thalassemia (-SEA, -THAI), α+-thalassemia (-α3.7, -α4.2), Hb Constant Spring (αCS) alleles, rs766432 in BCL11A, rs9399137 in HBS1L-MYB, and rs7482144-XmnI were evaluated. Modifiers were compared between 102 community- and 104 hospital-recruited cases. Alleles of ß+, -SEA, -α3.7, αCS, and a minor allele of rs9399137 were prevalent in the community and mild severity groups (p < 0.05). Multiple linear regression analysis associated modulating alleles with -4.299 (-SEA), -3.654 (ß+), -3.065 (rs9399137, C/C), -2.888 (αCS), -2.623 (-α3.7), -2.361 (rs7482144, A/A), -1.258 (rs9399137, C/T), and -1.174 (rs7482144, A/G) severity score reductions (p < 0.05). Certain modifiers must be considered in routine prenatal genetic counseling for Hb E/ß-thalassemia.

Direct correction of haemoglobin E ß-thalassaemia using base editors.

Haemoglobin E (HbE) ß-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE ß-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamatic acid → AAG; lysine, E26K), and any mutation causing severe ß-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (ß-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 90% in primary human CD34 + cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations.

Clinical Classification, Screening, and Diagnosis in Beta-Thalassemia and Hemoglobin E/Beta-Thalassemia.

This article reviews the classification of beta-thalassemia syndromes, correlating clinical severity and genotype in the earlier classification, and broadening it recently based on clinical severity and transfusion status. The classification is dynamic, and individuals may progress from transfusion-independent to transfusion-dependent. Early and accurate diagnosis prevents delays in instituting treatment and comprehensive care, and precludes inappropriate and potentially harmful interventions. Screening can inform risk in an individual and subsequent generations when partners may be carriers as well. This article discusses the rationale for screening of the at-risk population. In the developed world, a more precise genetic diagnosis must be considered.

Whole exome sequencing and rare variant association study to identify genetic modifiers, KLF1 mutations, and a novel double mutation in Thai patients with hemoglobin E/beta-thalassemia.

OBJECTIVES: Clinical manifestations of patients with Hemoglobin E/beta-thalassemia vary from mild to severe phenotypes despite exhibiting the same genotype. Studies have partially identified genetic modifiers. We aimed to study the association between rare variants in protein-coding regions and clinical severity in Thai patients. METHODS: From April to November 2018, a case-control study was conducted based on clinical information and DNA samples collected from Thai patients with hemoglobin E/beta-thalassemia over the age of four years. Cases were patients with severe symptoms, while patients with mild symptoms acted as controls. Whole exome sequencing and rare variant association study were used to analyze the data. RESULTS: All 338 unrelated patients were classified into 165 severe and 173 mild cases. Genotypes comprised 81.4% of hemoglobin E/beta-thalassemia, 2.7% of homozygous or compound heterozygous beta-thalassemia, and 0.3% of (뫧)0 thalassemia Hb E while 15.7% of samples were not classified as beta-thalassemia. A novel cis heterozygotes of IVS I-7 (A > T) and codon 26 (G > A) was identified. Six genes (COL4A3, DLK1, FAM186A, PZP, THPO, and TRIM51) showed the strongest associations with severity (observed p-values of <0.05; significance lost after correction for multiplicity). Among known modifiers, KLF1 variants were found in four mild patients and one severe patient. CONCLUSION: No rare variants were identified as contributors to the clinical heterogeneity of hemoglobin E/beta-thalassemia. KLF1 mutations are potential genetic modifiers. Studies to identify genetic factors are still important and helpful for predicting severity and developing targeted therapy.

Successful outcome in a compound heterozygote haemoglobin E/beta-thalassaemia in pregnancy.

Haemoglobin E (HbE) affects at least 1 million people around the world. The carrier frequency of HbE/beta-thalassaemia (HbE/ß-thalassaemia) is highest in Southeast Asia. In India, the highest frequency is observed in the northeast region. Distinguishing between homozygous HbE disease and HbE/ß-thalassaemia is a challenge to the haematopathologist as well as to the treating obstetrician because both are clinically and haematologically similar, posing a difficulty in managing anaemia and assessing the fetal risk for the same disease. This article reports a case of compound heterozygote HbE/ß-thalassaemia in pregnancy and its successful outcome.

False hemoglobin A1c value as a result of compound heterozygotes of hemoglobin E and hemoglobin New York.

The presence of hemoglobin (Hb) variants might interfere with some glycated hemoglobin (HbA1c ) measurements. There have been a few reports of compound Hb variants affecting HbA1c testing. Here, we report a case of the coinheritance of two Hb variants in the ß-globin gene. High-performance liquid chromatography with the Hb program showed a high HbA2 level. Similarly, an E-window peak was separated on the high-performance liquid chromatography with a glycated Hb program. However, capillary electrophoresis showed two abnormal peaks and no HbA peak. Sanger sequencing confirmed the presence of Hb New York and HbE. This is the first report of a compound heterozygote for HbE and Hb New York. The double heterozygote caused erroneous results for HbA1c on high-performance liquid chromatography and enzyme assay.

Non-Transfusion-Dependent Thalassemia: A Panoramic Review.

Non-transfusion-dependent thalassemia (NTDT) has been considered less severe than its transfusion-dependent variants. The most common forms of NTDT include ß-thalassemia intermedia, hemoglobin E/beta thalassemia, and hemoglobin H disease. Patients with NTDT develop several clinical complications, despite their regular transfusion independence. Ineffective erythropoiesis, iron overload, and hypercoagulability are pathophysiological factors that lead to morbidities in these patients. Therefore, an early and accurate diagnosis of NTDT is essential to ascertaining early interventions. Currently, several conventional management options are available, with guidelines suggested by the Thalassemia International Federation, and novel therapies are being developed in light of the advancement of the understanding of this disease. This review aimed to increase clinicians' awareness of NTDT, from its basic medical definition and genetics to its pathophysiology. Specific complications to NTDT were reviewed, along with the risk factors for its development. The indications of different therapeutic options were outlined, and recent advancements were reviewed.

Genetic correction of haemoglobin E in an immortalised haemoglobin E/beta-thalassaemia cell line using the CRISPR/Cas9 system.

ß-thalassaemia is one of the most common genetic blood diseases worldwide with over 300 mutations in the HBB gene affecting red blood cell functions. Recently, advances in genome editing technology have provided a powerful tool for precise genetic correction. Generation of patient-derived induced pluripotent stem cells (iPSCs) followed by genetic correction of HBB mutations and differentiation into haematopoietic stem/progenitor cells (HSPCs) offers a potential therapy to cure the disease. However, the biggest challenge is to generate functional HSPCs that are capable of self-renewal and transplantable. In addition, functional analyses of iPSC-derived erythroid cells are hampered by poor erythroid expansion and incomplete erythroid differentiation. Previously, we generated an immortalised erythroid cell line (SiBBE) with unique properties, including unlimited expansion and the ability to differentiate into mature erythrocytes. In this study, we report a highly efficient genetic correction of HbE mutation in the SiBBE cells using the CRISPR/Cas9 system. The HbE-corrected clones restored ß-globin production with reduced levels of HbE upon erythroid differentiation. Our approach provides a sustainable supply of corrected erythroid cells and represents a valuable model for validating the therapeutic efficacy of gene editing systems.

Performance analysis of machine learning algorithms and screening formulae for ß-thalassemia trait screening of Indian antenatal women.

BACKGROUND: Currently, more than forty discrimination formulae based on red blood cell (RBC) parameters and some supervised machine learning algorithms (MLAs) have been recommended for ß-thalassemia trait (BTT) screening. The present study was aimed to evaluate and compare the performance of 26 such formulae and 13 MLAs on antenatal woman data with a recently developed formula SCSBTT, which is available for evaluation in over seventy countries as an Android app, called SUSOKA[16]. METHODS: A diagnostic database of 2942 antenatal females were collected from PGIMER, Chandigarh, India and was used for this analysis. The data set consists of hypochromic microcytic anemia, BTT, Hemoglobin E trait, double heterozygote for Hemoglobin S and BTT, heterozygote for Hemoglobin D Punjab and normal subjects. Performance of the formulae and the MLAs were assessed by Sensitivity, Specificity, Youden's Index, and AUC-ROC measures. A final recommendation was made from the ranking obtained through two Multiple Criteria Decision-Making (MCDM) techniques, namely, Simultaneous Evaluation of Criteria and Alternatives (SECA) and TOPSIS. RESULTS: It was observed that Extreme Learning Machine (ELM) and Gradient Boosting Classifier (GBC) showed maximum Youden's index and AUC-ROC measures compared to all discriminating formulae. Sensitivity remains maximum for SCSBTT. K-means clustering and the ranking from MCDM methods show that SCSBTT, Shine & Lal and Ravanbakhsh-F4 formula ensures higher performance among all formulae. The discriminant power of some MLAs and formulae was found considerably lower than that reported in original studies. CONCLUSION: Comparative information on MLAs can aid researchers in developing new discriminating formulae that simultaneously ensure higher sensitivity and specificity. More multi-centric verification of the formulae on heterogeneous data is indispensable. SCSBTT and Shine & Lal formula, and ELM and GBC are recommended for screening BTT based on MCDM. SCSBTT can be used with certainty as a tangible cost-saving screening tool for mass screening for antenatal women in India and other countries.