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Haemoglobin (Hb) H disease and HbH disease with co-inherited HbE mutation are the most prevalent forms of α-thalassaemia in Southeast Asia. Data were limited when comparing clinical phenotypes between these two patient groups. We conducted a Thai multicentre study and enrolled 588 patients [median (IQR) age 13.0 (6.7-20.3) years], including those with deletional HbH disease with (n = 47) and without (n = 187) co-inherited HbE mutation and non-deletional HbH disease with (n = 101) and without (n = 253) co-inherited HbE mutation. Patients with HbH disease with co-inherited HbE mutation suffered more severe manifestations than those without. This observation was more pronounced in patients with non-deletional HbH disease. A greater proportion of patients with non-deletional HbH disease with co-inherited HbE mutation (43.6%) eventually required regular transfusions compared to those without (30.4%, p = 0.019). Among those with non-deletional HbH disease who did not require regular transfusions, Hb levels were lower in patients with co-inherited HbE mutation [8.1 (7.2-8.6) vs. 8.8 (8.2-9.5) g/dL, p < 0.001]. Among patients requiring regular transfusions who underwent splenectomy, 11/12 patients with non-deletional HbH disease stopped transfusion compared with 1/3 in non-deletional HbH disease with co-inherited HbE mutation group (p = 0.024). These findings provide insights for the clinical monitoring and management of HbH disease in the region., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Hemoglobin (Hb) H disease presents a wide range of clinical phenotypes, from asymptomatic to severe forms, depending on significant genetic heterogeneity. This is the first report of clinical and hematological features of the nondeletional HbH disease caused by -- CR /α CS α. A baby was born to a father and a mother with -- CR and α CS α carriers, respectively. She had severe symptomatic hypochromic microcytic anemia at 2 months of age with Hb 7.8 g/dL, packed cell volume (PCV) 0.27 L/L, mean corpuscular volume (MCV) 64.3 fL, and mean corpuscular Hb (MCH) 18.3 pg. The Hb analysis using capillary electrophoresis (CE) showed Hb Bart's, HbH, and Hb CS peaks at 17.1%, 2.2%, and 1.6%, respectively. A better understanding of a patient's clinical and hematological features with -- CR /α CS α is useful for hemoglobinopathy counseling for the national thalassemia controlling program.

Introduction: Hereditary pyropoikilocytosis (HPP) is the most common cause of non-thalassemic severe inherited hemolytic anemia in Thai population. Up to 90% of affected patients harbor biallelic mutations of SPTB Providence (SPTB c.6055T>C), SPTB Buffalo (SPTB c.6074T>G), and SPTB Chiang Mai (SPTB c.6224A>G). This study aimed to develop a simple assay for mass screening of the three common SPTB mutations and to study their carrier frequencies in a healthy Thai population., Methods: We combined multiplex amplification refractory mutation system-PCR (ARMS-PCR) and high-resolution melting (HRM) curve analysis to create a one-step single-tube assay. The primers were designed to generate products with different melting temperatures in the presence of 6055C, 6074G, and 6224G. Internal control primers were added for quality control. Residual samples from blood donors and healthy adolescents were collected and tested for the three common SPTB mutations using the newly developed assay., Results: Optimized multiplex ARMS-PCR/HRM curve assay yielded well-separated melt curves to detect the three SPTB mutations with 4-h turnaround time. The assay was validated in screening of 2261 non-repetitive blood donors and 89 adolescents, in which 10 (0.43%), 2 (0.09%), and 3 (0.13%) individuals were identified as carriers of SPTB Providence, SPTB Buffalo, and SPTB Chiang Mai, respectively. All mutated SPTB and 20 random wild-type samples were confirmed using Sanger sequencing with 100% accuracy., Conclusion: The novel ARMS-PCR/HRM curve assay is simple, accurate, and time-effective for mass screening of the common SPTB mutations. This can be employed to prevent HPP birth in a Thai population., (© 2023 John Wiley & Sons Ltd.)

Background: It was hypothesized that glucose-6-phosphate dehydrogenase (G6PD) deficiency confers a protective effect against malaria infection, however, safety concerns have been raised regarding haemolytic toxicity caused by radical cure with 8-aminoquinolines in G6PD-deficient individuals. Malaria elimination and control are also complicated by the high prevalence of G6PD deficiency in malaria-endemic areas. Hence, accurate identification of G6PD deficiency is required to identify those who are eligible for malaria treatment using 8-aminoquinolines., Methods: The prevalence of G6PD deficiency among 408 Thai participants diagnosed with malaria by microscopy (71), and malaria-negative controls (337), was assessed using a phenotypic test based on water-soluble tetrazolium salts. High-resolution melting (HRM) curve analysis was developed from a previous study to enable the detection of 15 common missense, synonymous and intronic G6PD mutations in Asian populations. The identified mutations were subjected to biochemical and structural characterisation to understand the molecular mechanisms underlying enzyme deficiency., Results: Based on phenotypic testing, the prevalence of G6PD deficiency (< 30% activity) was 6.13% (25/408) and intermediate deficiency (30-70% activity) was found in 15.20% (62/408) of participants. Several G6PD genotypes with newly discovered double missense variants were identified by HRM assays, including G6PD Gaohe + Viangchan, G6PD Valladolid + Viangchan and G6PD Canton + Viangchan. A significantly high frequency of synonymous (c.1311C>T) and intronic (c.1365-13T>C and c.486-34delT) mutations was detected with intermediate to normal enzyme activity. The double missense mutations were less catalytically active than their corresponding single missense mutations, resulting in severe enzyme deficiency. While the mutations had a minor effect on binding affinity, structural instability was a key contributor to the enzyme deficiency observed in G6PD-deficient individuals., Conclusions: With varying degrees of enzyme deficiency, G6PD genotyping can be used as a complement to phenotypic screening to identify those who are eligible for 8-aminoquinolines. The information gained from this study could be useful for management and treatment of malaria, as well as for the prevention of unanticipated reactions to certain medications and foods in the studied population., (© 2024. The Author(s).)

Red blood cell (RBC) membrane disorders represent a significant category of hereditary hemolytic anemia; however, information from Southeast Asia is limited. We established a national registry aiming to characterize RBC membrane disorders and their molecular features in Thailand. A total of 100 patients (99 kindreds) diagnosed with RBC membrane disorders between 2011 and 2020 from seven university hospitals were enrolled. The most prevalent disorders observed were hereditary elliptocytosis (HE; n=33), hereditary pyropoikilocytosis (HPP; n=28), hereditary spherocytosis (HS; n=19), Southeast Asian ovalocytosis (SAO; n=10 of 9 kindreds), and two cases of homozygous SAO. The remaining cases were grouped as unclassified membrane disorder. Seventy-six patients (76%) were molecularly confirmed by PCR, direct DNA sequencing, or hi-throughput sequencing. The primary causative gene for HE and HPP was SPTB, accounting for 28 out of 29 studied alleles for HE and 56 of 56 studied alleles for HPP. In the case of HS, dominant sporadic mutations in the ANK1 gene (n=4) and SPTB gene (n=3) were identified as the underlying cause. Notably, the four most common variants causing HE and HPP were SPTB Providence (c.6055 T>C), SPTB Buffalo (c.6074 T>G), SPTB Chiang Mai (c.6224 A>G), and SPTB c.6171&#95;&#95;82delins TGCCCAGCT. These recurrent SPTB mutations accounted for 79 out of 84 mutated SPTB alleles (94%). In summary, HE and hereditary HPP associated with recurrent SPTB mutations are the predominant types of RBC membrane disorders observed in Thailand. These findings have significant implications for the clinical management and future research of RBC membrane disorders in the region., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The study aimed to determine efficacy and safety of generic deferasirox monotherapy. Deferasirox was administered in transfusion-induced iron overloaded thalassemia. Efficacy was defined as responders and nonresponders by ≤ 15 reduced serum ferritin from baseline. Adverse events were also monitored. Fifty-two patients with mainly Hb E/β-thalassemia at the mean (SD) age of 8.7 (4.1) years, were enrolled. The mean (SD) daily transfusion iron load was 0.47 (0.1) mg/kg and maximum daily deferasirox was 35.0 (6.2) mg/kg. Altogether, 52, 40 and 18 patients completed the first, second and third years of study, respectively. The median baseline serum ferritin 2,383 ng/mL decreased to 1,478, 1,038 and 1,268 ng/mL at the end of first, second and third years, respectively, with overall response rate at 73.1% (38/52). Patients with baseline serum ferritin >2,500 ng/mL showed a change in serum ferritin higher than those ≤2,500 ng/mL starting from the 9th month of chelation. Adverse events were found in 5 of 52 patients (9.6%) including transaminitis (n = 2), one each of proteinuria, rash and proximal tubular dysfunction which resolved after transient stopping or decreasing the chelation dose. Generic deferasirox was effective and safe among pediatric patients with transfusion-induced iron overloaded thalassemia.

Background: Congenital neutropenia is a rare disease. Recurrent infections since young age are the presentation. The most common mutation causing severe congenital neutropenia (SCN) and cyclic neutropenia (CyN) is the ELANE gene. The objectives of this study were to screen the three common genetic mutations of ELANE, HAX1 and GFI1 in children with chronic neutropenia and to describe the clinical characteristics of children who had the mutations., Methods: Infants having ANC < 1,000/cu mm or children aged > 1 year having ANC < 1,500/cu mm at least 3 times in 3 months were enrolled in the study. Patients who had acquired neutropenia due to infection, immune deficiency, or drugs were excluded. The ELANE gene was first studied; and if mutations were not identified, the HAX1 and GFI1 genes were further examined., Results: A total of 60 patients were enrolled in the study. The median (range) age, ratio of female to male, ANC, and last follow-up age were 9.2 (0.5-45.2) months, 1:1.2, 248 (0-1,101) /cu mm, and 19.9 (3.5-202.3) months, respectively. Infections were noted in 67.3% of all patients. ELANE gene mutation was found in only four patients (6.7%), and the rest (56 patients) showed no mutations in the HAX1 and GFI1 genes. In patients without mutations, 66.0% had normal ANC during the follow-up, with a median (range) age for normal ANC of 19.8 (4.0-60.0) months. Two novel mutations p. Ala79del (c.234&#95;236del) and p. Val197GlufsTer18 (c.589&#95;590insAGGCCGGC) were identified, and they respectively cause SCN and CyN. Patients with the two novel mutations presented with several episodes of infection, including pneumonia, sepsis, abscess, otitis media, and gum infection., Conclusion: The genetic screening for ELANE, HAX1, and GFI1 gene mutations in 60 patients with chronic neutropenia could identify four patients (6.7%) with ELANE gene mutation and two novel mutations, p. Ala79del in exon 3 and p. Val197GlufsTer18 in exon 4 causing SCN; and CyN, respectively., (© 2023. The Author(s).)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzymopathy caused by mutations in the G6PD gene. A medical concern associated with G6PD deficiency is acute hemolytic anemia induced by certain foods, drugs, and infections. Although phenotypic tests can correctly identify hemizygous males, as well as homozygous and compound heterozygous females, heterozygous females with a wide range of G6PD activity may be misclassified as normal. This study aimed to develop multiplex high-resolution melting (HRM) analyses to enable the accurate detection of G6PD mutations, especially among females with heterozygous deficiency. Multiplex HRM assays were developed to detect six G6PD variants, i.e., G6PD Gaohe (c.95A>G), G6PD Chinese-4 (c.392G>T), G6PD Mahidol (c.487G>A), G6PD Viangchan (c.871G>A), G6PD Chinese-5 (c.1024C>T), and G6PD Union (c.1360C>T) in two reactions. The assays were validated and then applied to genotype G6PD mutations in 248 Thai females. The sensitivity of the HRM assays developed was 100% [95% confidence interval (CI): 94.40%-100%] with a specificity of 100% (95% CI: 88.78%-100%) for detecting these six mutations. The prevalence of G6PD deficiency was estimated as 3.63% (9/248) for G6PD deficiency and 31.05% (77/248) for intermediate deficiency by phenotypic assay. The developed HRM assays identified three participants with normal enzyme activity as heterozygous for G6PD Viangchan. Interestingly, a deletion in intron 5 nucleotide position 637/638 (c.486-34delT) was also detected by the developed HRM assays. G6PD genotyping revealed a total of 12 G6PD genotypes, with a high prevalence of intronic variants. Our results suggested that HRM analysis-based genotyping is a simple and reliable approach for detecting G6PD mutations, and could be used to prevent the misdiagnosis of heterozygous females by phenotypic assay. This study also sheds light on the possibility of overlooking intronic variants, which could affect G6PD expression and contribute to enzyme deficiency., Competing Interests: : The authors have declared that no competing interests exist., (Copyright: © 2023 Chamchoy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Management of transfusion-dependent thalassemia (TDT) can be challenging due to numerous potential disease-related complications and comorbidities in particular age groups. The objective of this study was to report thalassemia-related complications and risk factors in pediatric, adolescent, and young adult patients with TDT., Methods: A multicenter web-based registry was conducted in patients with TDT aged 25 years and younger from eight university hospitals covering all parts of Thailand. Factors significantly associated with each complication were analyzed by logistic regression methods., Results: Of 605 patients, 267 thalassemia-related complications were reported from 231 pediatric, adolescent, and young adult patients with TDT patients (38.2%). The most common complications were infections, followed by cholelithiasis and growth failure. Splenectomy and elevated pre-transfusion hemoglobin were statistically significant risk factors for infections (adjusted odds ratio [AOR] = 2.3, 95% confidence interval [CI]: 1.2-4.5, p-value = .01 and AOR = 1.5, 95% CI: 1.2-1.7, p-value < .005, respectively). There were two statistically significant risk factors conferred endocrinopathies, including older age (AOR = 1.06, 95% CI: 1.01-1.1, p-value = .01) and being male (AOR = 2.4, 95% CI: 1.4-4.0, p-value = .002)., Conclusion: Nearly 40% of the patients in this cohort had thalassemia-related complications. Periodic surveillance and optimal care for respective complications may minimize comorbidities in pediatric, adolescent, and young adult patients with TDT., (© 2023 Wiley Periodicals LLC.)

Many therapeutic proteins are expressed in Escherichia coli bacteria for the low cost and high yield obtained. However, these gram-negative bacteria also generate undesirable endotoxin byproducts such as lipopolysaccharides (LPS). These endotoxins can induce a human immune response and cause severe inflammation. To mitigate this problem, we have employed the ClearColi BL21 (DE3) endotoxin-free cells as an expression host for Cas9 protein production. Cas9 is an endonuclease enzyme that plays a key role in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (CRISPR/Cas9) genome editing technique. This technology is very promising for use in diagnostics as well as treatment of diseases, especially for genetic diseases such as thalassemia. The potential uses for this technology thus generate a considerable interest for Cas9 utilization as a therapeutic protein in clinical treatment. Therefore, special care in protein production should be a major concern. Accordingly, we expressed the Cas9 protein in endotoxin-free bacterial cells achieving 99% purity with activity comparable to commercially available Cas9. Our protocol therefore yields a cost-effective product suitable for invitro experiments with stem cells., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objectives: Patients with high-risk hematologic diseases require intensive modalities, including high-dose chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT). Haploidentical T-cell-replete transplantation is a logical choice because of the limited availability of matched sibling donors and the prolonged time needed to identify matched unrelated donors in Thailand., Methods: The clinical outcomes data of 43 patients undergoing allo-HSCT were reviewed. All patients had high-risk hematologic malignancies, were younger than 20 years, and were in complete cytological remission at the time of allo-HSCT. We used two different conditioning regimens: total body irradiation (TBI) combined with cyclophosphamide, fludarabine, and melphalan (n = 23) and thiotepa combined with fludarabine and busulfan (n = 20). All patients received a graft-versus-host disease prophylaxis regimen consisting of cyclophosphamide, mycophenolate mofetil, and a calcineurin inhibitor or sirolimus., Results: There was no difference in engraftment between patients receiving either of the regimens. After a median follow-up of 35.8 (range, 0.6-106.2) months, the overall survival (OS) and event-free survival (EFS) rates were 62.4% and 54.7%, respectively. OS and EFS were comparable between the respective regimens., Conclusions: We conclude that thiotepa-based conditioning has similar efficacy and tolerability as TBI-based conditioning for haploidentical HSCT with post-transplant cyclophosphamide., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Hemoglobin (Hb) H is generally recognized as mild thalassemia, despite its actual phenotypic diversity. A disease severity scoring system to guide initiation of regular transfusion among severely affected pediatric patients has not previously been reported., Methods: Patients with HbH were classified into transfusion-dependent thalassemia (TDT) and non-transfusion-dependent thalassemia (NTDT) as a surrogate for disease severity. Alpha-globin genotypes and relevant clinical parameters associated with TDT were identified. Univariate and multiple logistic regression analyses were performed to yield the most suitable severity scoring system., Results: From 246 patients with a median age of 14.3 (interquartile range 9.9-18.4) years initially enrolled into the study, the chance of having severe disease and developing TDT was remarkable only among patients with non-deletional HbH, for whom the scoring system was developed. Univariate and multiple logistic regression analyses resulted in three retained parameters associated with TDT, β-coefficients of which were used to develop the score. The final scoring system comprised age at diagnosis <2 years (score = 1), spleen size ≥3 cm (score = 1) and Hb at steady-state <7 (score = 4) or 7-8 g/dL (score = 3). A cutoff score ≥4 was associated with severe disease likely requiring regular transfusion (sensitivity 89.3%, specificity 81.4%), given regular transfusion resulted in maintained growth. The scoring system was validated in the second cohort of 77 non-deletional HbH, from which comparable sensitivity and specificity were obtained., Conclusion: The newly developed scoring system was practical and helpful to highlight severely affected pediatric non-deletional HbH patients with potential needs of regular transfusion. This can be used as a guide for optimal treatment and disease monitoring in the future., (© 2023 Japan Pediatric Society.)

Background: Plasmodium vivax remains the malaria species posing a major threat to human health worldwide owing to its relapse mechanism. Currently, the only drugs of choice for radical cure are the 8-aminoquinolines (primaquine and tafenoquine), which are capable of killing hypnozoites and thus preventing P. vivax relapse. However, the therapeutic use of primaquine and tafenoquine is restricted because these drugs can cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. This study aimed to assess and understand the hemolytic risk of using 8-aminoquinolines for radical treatment in a malaria endemic area of Thailand. Methods: The prevalence of G6PD deficiency was determined using a quantitative test in 1,125 individuals. Multiplexed high-resolution meltinging (HRM) assays were developed and applied to detect 12 G6PD mutations. Furthermore, biochemical and structural characterization of G6PD variants was carried out to understand the molecular basis of enzyme deficiency. Results: The prevalence of G6PD deficiency was 6.76% (76/1,125), as assessed by a phenotypic test. Multiplexed HRM assays revealed G6PD Mahidol in 15.04% (77/512) of males and 28.38% (174/613) of females, as well as G6PD Aures in one female. G6PD activity above the 30% cut-off was detected in those carrying G6PD Mahidol, even in hemizygous male individuals. Two variants, G6PD Murcia Oristano and G6PD Songklanagarind + Viangchan, were identified for the first time in Thailand. Biochemical characterization revealed that structural instability is the primary cause of enzyme deficiency in G6PD Aures, G6PD Murcia Oristano, G6PD Songklanagarind + Viangchan, and G6PD Chinese 4 + Viangchan, with double G6PD mutations causing more severe enzyme deficiency. Conclusion: In western Thailand, up to 22% of people may be ineligible for radical cure. Routine qualitative tests may be insufficient for G6PD testing, so quantitative tests should be implemented. G6PD genotyping should also be used to confirm G6PD status, especially in female individuals suspected of having G6PD deficiency. People with double G6PD mutations are more likely to have hemolysis than are those with single G6PD mutations because the double mutations significantly reduce the catalytic activity as well as the structural stability of the protein., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sudsumrit, Chamchoy, Songdej, Adisakwattana, Krudsood, Adams, Imwong, Leartsakulpanich and Boonyuen.)

Background: Thalassemia is a common genetic disease in Southeast Asia. Red blood cell (RBC) transfusion is an essential treatment for severe forms of thalassemia. We performed a study to demonstrate RBC alloimmunization and other transfusion-related complications in patients with transfusion-dependent thalassemia (TDT)., Study Design and Methods: A multi-center web-based registry of TDT was conducted in eight medical centers across Thailand. Thalassemia information, transfusion therapy, and transfusion-related complications were collected. Factors associated with each complication were demonstrated using the logistic regression analysis., Results: Of 1000 patients recruited for the study, 449 were males (44.9%). The mean age was 23.9 ± 15.4 years. The majority of patients, 738 (73.8%) had hemoglobin E/beta-thalassemia. In the study, 421 transfusion-related complications were reported from 357 patients (35.7%). Alloimmunization was the most common complication which was found in 156 patients (15.6%) with 284 positive antibody tests. The most frequent antibodies against RBC were anti-E (80/284, 28.2%) followed by anti-Mi a (45/284, 15.8%) and anti-c (32/284, 11.3%). Age ≥3 years at initial blood transfusion, splenomegaly, higher frequencies, and volumes of transfusion were significant factors associated with alloimmunization. None of the patients had to terminate blood transfusion due to multiple alloantibodies. Other commonly seen complications were allergic reactions (130, 13.0%), autoimmune hemolytic anemia (70, 7.0%) and febrile non-hemolytic transfusion reaction (54, 5.4%)., Conclusions: Transfusion-related complications, especially alloimmunization, were common among Thai patients with TDT. Extended RBC antigen-matching for the Rh system and Mi a should be implemented to prevent the development of alloantibodies in multi-transfused patients., (© 2022 AABB.)

Next-generation sequencing has shed light on the diagnosis of previously unsolved cases of inherited haemolytic anaemia (IHA). We employed whole-exome sequencing to explore the molecular diagnostic spectrum of 21 unrelated Thai paediatric patients with non-thalassemic IHA, presenting hydrops fetalis and/or becoming transfusion-dependent for 1 year or more or throughout their lifespan. Anaemia was detected prenatally, within the first month and the fifth year of life in three, 12 and six patients respectively. Molecular diagnosis obtained from all patients revealed SPTB as the most frequently mutated gene (four reported, three novel), found in 31 of 42 studied alleles. The other two mutated genes identified were ANK1 (three novel) and KLF1 (two reported). Four recurring mutations within exon 29/30 (NM&#95;001024858.2) accounted for the vast majority (90%) of mutated SPTB alleles, biallelic inheritance of which resulted in the most severe phenotypes: hydrops fetalis and life-long transfusion dependency. Dominant ANK1 (n = 3) and SPTB (n = 2) mutations and biallelic class 2 KLF1 mutations (n = 1) led to a shorter period of transfusion dependency. Our study demonstrated that mutated SPTB causing red-cell membranopathy is likely the most common cause of severe non-thalassemic IHA among Thai patients. This urges carrier screening in the population to prevent subsequent, severely affected births., (© 2022 British Society for Haematology and John Wiley & Sons Ltd.)

The accumulation of unbound α-globin chains in red blood cells is a crucial pathophysiology of β-thalassemia. IOX1 (5-carboxy-8-hydroxyquinoline) is a broad-spectrum 2-oxoglutarate (2OG)-dependent oxygenase inhibitor that can reduce α-globin mRNA expression in human cord blood erythroid progenitor cells. Therefore, IOX1 has been proposed as a potential compound for β-thalassemia treatment through the decrease in α-globin chain synthesis. However, there is no empirical evidence regarding the consequences of IOX1 in β-thalassemia. In this study, the therapeutic effects of IOX1 were investigated in β 0 -thalassemia/hemoglobin E (HbE) erythroid progenitor cells during in vitro erythropoiesis. The results indicated that IOX1 had no impact on α-globin gene expression, but it led instead to significant decreases in γ-globin and fetal hemoglobin (HbF, α 2 γ 2 ) production without affecting well-known globin regulators: KLF1, BCL11A, LRF, and GATA1. In addition, differential mRNA expression of several genes in the hypoxia response pathway revealed the induction of EGLN1, the PHD2-encoding gene, as a result of IOX1 treatment. These findings suggested that IOX1 fails to lower α-globin gene expression; on the contrary, it mediates γ-globin and HbF silencing in β 0 -thalassemia/HbE erythroid progenitor cells. Because of the negative correlation of EGLN1 and γ-globin gene expression after IOX1 treatment, repurposing IOX1 to study the hypoxia response pathway and γ-globin regulation may provide beneficial information for β-thalassemia., Competing Interests: Conflict of Interest Disclosure The authors declare no competing interests., (Copyright © 2022 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: Transcobalamin deficiency is a rare inborn metabolic disorder, characterized by pancytopenia, megaloblastic anemia, failure to thrive, diarrhea, and psychomotor retardation., Case Presentation: We describe a patient who first presented at 3 months of age, with pancytopenia, hepatosplenomegaly, recurrent infection, metabolic acidosis, and acute hemolytic crisis. Extensive hematologic and immunologic investigations did not identify inherited bone marrow failure syndrome, acute leukemia or its related disorders. Whole exome sequencing identified a novel homozygous TCN2 mutation, c.428-2A > G and mRNA study confirmed an aberrant transcription of exon 4 skipping. The mutant protein is predicted to have an in-fame 51 amino acids deletion (NP&#95;000346:p.Gly143&#95;Val193del). The patient exhibited marked clinical improvement following hydroxocobalamin treatment., Conclusions: Transcobalamin deficiency should be investigated in infants with unexplained pancytopenia and acute hemolytic crisis with or without typical evidence of vitamin B12 deficiency., (© 2022. The Author(s).)

Reactivating of fetal hemoglobin (HbF; α2γ2) can ameliorate the severity of β-thalassemia disease by compensating for adult hemoglobin deficiency in patients. Previously, microarray analysis revealed that zinc finger protein (ZNF)802 (also known as Juxta-posed with another zinc finger gene-1 (JAZF1)) was upregulated in human erythroblasts derived from adult peripheral blood compared with fetal liver-derived cells, implying a potential role as a HbF repressor. However, deficiency in ZNF802 induced by lentiviral shRNA in β 0 -thalassemia/hemoglobinE erythroblasts had no effect on erythroblast proliferation and differentiation. Remarkably, the induction of HBG expression was observed at the transcriptional and translational levels resulting in an increase of HbF to 35.0 ± 3.5%. Interestingly, the embryonic globin transcripts were also upregulated but the translation of embryonic globin was not detected. These results suggest ZNF802 might be a transcriptional repressor of the γ-globin gene in adult erythroid cells., (© 2022. The Author(s).)

Aims: The purpose of this study was to design and manufacture CD19 chimeric antigen receptor (CAR)-modified T cells for clinical use in Thailand, as a model for how this technology can be directly applied at individual institutions treating high-risk leukemia patients., Methods: We constructed second-generation CAR T cells expressing CD19 scFV-CD28-CD3ζ with different lengths of the spacer region: full, intermediate, and short length, by using a lentiviral vector. We wanted to determine whether the difference in length of the spacer would affect the cytotoxic potential of the CD19 CAR T cells against the leukemic cells., Results: We found that all constructs of CD19 CAR T cells exhibited a similar level of cytotoxicity against several human lymphoma and leukemia cell lines. For the clinical application, we chose the intermediate length spacer construct CD19 CAR T cells, hypothesizing that the highest transduction efficiency coupled with a slower initial proliferation in vitro might lead to effective leukemic cell kill, yet a lower probability for serious clinical side effects. We then tested the clinical efficacy of our CD19 CAR T cells in one patient with refractory/relapsed acute B-cell lymphoblastic leukemia. This patient indeed had minimal clinical side effects after the CAR T-cell infusion, and he remains in an unmaintained, ongoing complete remission 10+ months after his T-cell treatment., Conclusion: Our CD19 CAR T cells demonstrated efficacies in acute lymphoblastic B-cell leukemia, and will be used to establish an immunotherapeutic program for high-risk B-cell acute lymphoblastic leukemia in Thailand. We propose that this approach can be used as a model for how this new exciting technology can be applied directly at individual institutions that treat (a large number of) patients with high-risk leukemia., (© 2020 John Wiley & Sons Australia, Ltd.)

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only established treatment that is potentially curative, but it is limited by the availability of donors and the medical condition of the patient. To expand the donor pool to include haploidentical related donors, we introduced a program consisting of a pharmacologic pre transplant immune suppression phase (PTIS) and two courses of dexamethasone (DXM) and fludarabine (FLU) followed by pre transplant conditioning with intravenous FLU busulfan (BU) and post transplant graft- vs. -host disease (GvHD) prophylaxis with cyclophosphamide (CPM), tacrolimus, and mycophenolate mofetil. We transplanted 83 consecutive transfusion-dependent patients with thalassemia; the 3-year projected overall and event-free survival is over 96.0%, and there have been no secondary graft failures. Of the first 31 patients, we had two graft failures, both of them occurring in patients with extremely high titers of anti-donor-specific human leukocyte antigen (HLA) antibodies [anti-donor specific antibodies (DSAs)], but after adjusting the PTIS to include bortezomib (BORT) and rituximab (RIX) for patients with high titers of anti-DSAs and using pharmacologic dose guidance for BU, we had no graft failures in the last 52 patients. Six (7.0%) of 83 patients developed severe GvHD. We conclude that this is a safe and efficacious approach to allogeneic HSCT in thalassemia.

Background: Patients with thalassemia encounter increased consumption of zinc (Zn) and copper (Cu) from chronic hemolysis and increased excretion from iron chelation. Iron-enriched diet restriction may result in low Zn and Cu intakes. Recent data on Zn and Cu status among Thai pediatric patients with thalassemia are lacking. This study aimed to identify frequencies and determine risk factors of Zn and Cu deficiencies among patients with thalassemia., Methods: Patients with transfusion-dependent thalassemia (TDT) receiving iron chelation ≥12 months and nonTDT (NTDT) aged 2-20 years were recruited. Serum Zn and Cu were measured. Dietary intakes were ascertained by interviews., Results: A total of 209 patients (TDT = 126, NTDT = 83) were enrolled. Zn deficiency seemed to be associated with disease severity as median (IQR) Zn level of TDT was lower than that of NTDT [77 (69-85) vs. 80 (72-88) mcg/dL, p = 0.05], while higher frequency of Zn deficiency was identified in the former (24 % vs. 14 %). In TDT, Zn deficiency was associated with patients >10 years (OR 4.6; 95 %CI 1.1-6.4, p = 0.03), which likely resulted from combined low dietary Zn intake, prolonged exposures to hemolysis and iron chelators. Frequencies of Cu deficiency were similarly low in TDT and NTDT (8% and 7%) with comparable median (IQR) Cu levels of 103 (90-124) and 110 (92-132) mcg/dL, respectively (p = 0.13). Cu levels were inversely associated with age (r=-0.65 and r=-0.62 in TDT and NTDT, respectively; p < 0.001)., Conclusion: Compared with younger patients, Zn and Cu deficiencies were more common among patients with thalassemia >10 years. Age was a major factor associated with both Zn and Cu deficiencies., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues., (© 2021. The Author(s).)

Betibeglogene autotemcel (beti-cel) gene therapy (GT) for patients with transfusion-dependent β-thalassemia uses autologous CD34+ cells transduced with BB305 lentiviral vector (LVV), which encodes a modified β-globin gene. BB305 LVV also contains select HIV sequences for viral packaging, reverse transcription, and integration. This case report describes a patient successfully treated with beti-cel in a phase 1/2 study (HGB-204; #NCT01745120) and subsequently diagnosed with wild-type (WT) HIV infection. From 3.5 to 21 months postinfusion, the patient stopped chronic red blood cell transfusions; total hemoglobin (Hb) and GT-derived HbAT87Q levels were 6.6 to 9.5 and 2.8 to 3.8 g/dL, respectively. At 21 months postinfusion, the patient resumed transfusions for anemia that coincided with an HIV-1 infection diagnosis. Quantitative polymerase chain reaction assays detected no replication-competent lentivirus. Next-generation sequencing confirmed WT HIV sequences. Six months after starting antiretroviral therapy, total Hb and HbAT87Q levels recovered to 8.6 and 3.6 g/dL, respectively, and 3.5 years postinfusion, 13.4 months had elapsed since the patient's last transfusion. To our knowledge, this is the first report of WT HIV infection in an LVV-based GT recipient and demonstrates persistent long-term hematopoiesis after treatment with beti-cel and the ability to differentiate between WT HIV and BB305-derived sequences., (© 2021 by The American Society of Hematology.)

Accurate measurement of glucose-6-phosphate dehydrogenase (G6PD) activity is critical for malaria treatment as misclassification of G6PD deficiency could cause serious harm to patients. G6PD activity should be assessed in blood samples on the day of collection. Otherwise, specimens should be stored under suitable conditions to prevent loss of G6PD activity. Here, we assessed stability and integrity of G6PD testing in samples from normal controls, heterozygous females, and G6PD deficient individuals using water-soluble tetrazolium salts (WST-8) assay. Specimens were stored as ethylenediaminetetraacetic acid (EDTA) whole blood and dried blood spots (DBS) at various temperatures (37 °C, room temperature, 4 °C and -20 °C) and under different humidity conditions (with and without desiccant). G6PD normal samples were stable for up to 1 year when stored at -20 °C under controlled conditions, with 85% and 91% G6PD activity in EDTA whole blood and DBS in the presence of desiccant, respectively. Specimens from heterozygous females showed greater G6PD activity when stored as DBS, with 85% enzyme activity after 1 year of storage at -20 °C under controlled conditions in the presence of desiccant. G6PD deficient samples rapidly lost enzyme activity in all storage conditions tested. However, the reduction in G6PD enzyme activity in G6PD deficient samples did not interfere with G6PD classification. Samples stored under suitable conditions for G6PD testing will allow accurate measurement of enzyme activity, prevent misclassification of G6PD deficiency and enable safe and effective use of antimalarial drugs such as primaquine and tafenoquine., (Copyright © 2021. Published by Elsevier B.V.)

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymopathy in humans, is prevalent in tropical and subtropical areas where malaria is endemic. Anti-malarial drugs, such as primaquine and tafenoquine, can cause haemolysis in G6PD-deficient individuals. Hence, G6PD testing is recommended before radical treatment against vivax malaria. Phenotypic assays have been widely used for screening G6PD deficiency, but in heterozygous females, the random lyonization causes difficulty in interpreting the results. Over 200 G6PD variants have been identified, which form genotypes associated with differences in the degree of G6PD deficiency and vulnerability to haemolysis. This study aimed to assess the frequency of G6PD mutations using a newly developed molecular genotyping test., Methods: A multiplexed high-resolution melting (HRM) assay was developed to detect eight G6PD mutations, in which four mutations can be tested simultaneously. Validation of the method was performed using 70 G6PD-deficient samples. The test was then applied to screen 725 blood samples from people living along the Thai-Myanmar border. The enzyme activity of these samples was also determined using water-soluble tetrazolium salts (WST-8) assay. Then, the correlation between genotype and enzyme activity was analysed., Results: The sensitivity of the multiplexed HRM assay for detecting G6PD mutations was 100 % [95 % confidence interval (CI): 94.87-100 %] with specificity of 100 % (95 % CI: 87.66-100 %). The overall prevalence of G6PD deficiency in the studied population as revealed by phenotypic WST-8 assay was 20.55 % (149/725). In contrast, by the multiplexed HRM assay, 27.17 % (197/725) of subjects were shown to have G6PD mutations. The mutations detected in this study included four single variants, G6PD Mahidol (187/197), G6PD Canton (4/197), G6PD Viangchan (3/197) and G6PD Chinese-5 (1/197), and two double mutations, G6PD Mahidol + Canton (1/197) and G6PD Chinese-4 + Viangchan (1/197). A broad range of G6PD enzyme activities were observed in individuals carrying G6PD Mahidol, especially in females., Conclusions: The multiplexed HRM-based assay is sensitive and reliable for detecting G6PD mutations. This genotyping assay can facilitate the detection of heterozygotes, which could be useful as a supplementary approach for high-throughput screening of G6PD deficiency in malaria endemic areas before the administration of primaquine and tafenoquine.

Increased expression of fetal hemoglobin (HbF) improves the clinical severity of β-thalassemia patients. EHMT1/2 histone methyltransferases are epigenetic modifying enzymes that are responsible for catalyzing addition of the repressive histone mark H3K9me2 at silenced genes, including the γ-globin genes. UNC0638, a chemical inhibitor of EHMT1/2, has been shown to induce HbF expression in human erythroid progenitor cell cultures. Here, we report the HbF-inducing activity of UNC0638 in erythroid progenitor cells from β-thalassemia/HbE patients. UNC0638 treatment led to significant increases in γ-globin mRNA, HbF expression, and HbF-containing cells in the absence of significant cytotoxicity. Moreover, UNC0638 showed additive effects on HbF induction in combination with the immunomodulatory drug pomalidomide and the DNMT1 inhibitor decitabine. These studies provide a scientific proof of concept that a small molecule targeting EHMT1/2 epigenetic enzymes, used alone or in combination with pomalidomide or decitabine, is a potential therapeutic approach for HbF induction. Further development of structural analogs of UNC0638 with similar biological effects but improved pharmacokinetic properties may lead to promising therapies and possible clinical application for the treatment of β-thalassemia.

Patients with severe thalassemia commonly have a survival that is significantly shorter than that of the general population. Allogeneic hematopoietic stem cell transplantation (allo-SCT) is the only established treatment that is potentially curative, but it is limited by the availability of donors and the medical condition of the patient. To expand the donor pool to include haploidentical related donors, we introduced a program consisting of a pharmacologic pretransplant immune suppression phase (PTIS) and 2 courses of dexamethasone and fludarabine, followed by pretransplant conditioning with fludarabine-i.v. busulfan and post-transplant graft-versus-host disease (GVHD) prophylaxis with cyclophosphamide, tacrolimus, and mycophenolate mofetil. We transplanted 83 consecutive transfusion-dependent patients with thalassemia (median age, 12 years; range, 1 to 28 years) with a minimum follow-up of 6 months (median, 15 months; range, 7 to 53 months); the 3-year projected overall and event-free survival is over 96%, and there have been no secondary graft failures. Of the first 31 patients, we had 2 graft failures, both of them occurring in patients with extremely high titers of anti-donor-specific HLA antibodies (anti-DSAs), but after adjusting the PTIS to include bortezomib and rituximab for patients with high titers of anti-DSAs and using pharmacologic dose guidance for busulfan, we had no graft failures in the last 52 patients. Six (7%) of 83 patients developed severe GVHD. We conclude that this is a safe and efficacious approach to allogeneic SCT in thalassemia, yielding results comparable to those available for patients with fully matched donors., (Copyright © 2020. Published by Elsevier Inc.)

Background: Induction chemotherapy with carboplatin followed by radiotherapy has been used for many years for treating intracranial germ-cell tumors (IC-GCTs) in Thailand. The objective of this study was to assess treatment outcomes, focusing on survival and ototoxicity., Methods: The outcomes of all patients with IC-GCT treated at Ramathibodi Hospital and the Prasat Neurological Institute between 2000 and 2017 were reviewed and analyzed, including all patient characteristics and treatment modalities. Five-year overall survival (OS) and event-free survival (EFS) were analyzed using the Kaplan-Meier method, and factors affecting survival were compared using the log-rank test., Results: Fifty-three patients age 1-14 years (median, 11 years) were included in this study. The median follow-up time was 63 months. The 5-year EFS and OS rates were 94.3% and 96.2% for all patients, respectively. No statistical difference in OS or EFS was observed between the data of recipients in the carboplatin-based and historical cisplatin-based therapies in our institutes. Concerning radiotherapy, omission of radiotherapy or focal irradiation results in worse long-term survival outcomes, but reduction in dose of radiotherapy to less than 40 Gy did not cause any negative impact on survival rates. Furthermore, carboplatin was associated with lower rates of hearing loss than cisplatin (5.7% vs 87.5%)., Conclusions: Induction chemotherapy with carboplatin-based regimens was associated with excellent survival rates and low ototoxicity in patients with IC-GCT. Radiotherapy should be given to all patients with a minimal volume equivalent to whole-ventricular radiotherapy, during which doses of lower than 40 Gy can be effectively used., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology and the European Association of Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Protein C (PC) deficiency, caused by mutations of the PROC gene, is a common inherited risk factor of thromboembolism (TE) among Thai people. This study aimed to investigate the association of 3 single nucleotide polymorphisms (SNPs; -1654 C/T, -1641 A/G, -1461A/T) at the PROC promoter region with PC activity and the risk of developing TE. A total of 216 patient s with TE, diagnosed at aged 0 to 20 years, and 102 healthy adults were enrolled. The SNPs were identified by Sanger sequencing. Protein C activity was measured using an automated functional clotting assay. Linear and logistic regression analyses were used to determine the association of SNPs with PC activity and the risk of TE. Patients and controls with homozygous TAA (119.6% ± 26.1%) and CGT haplotypes (102.7% ± 22.6%) had significantly lower PC activity than those with a homozygous CAA haplotype (140.4% ± 44.9%); P = .027 and .016, respectively. However, none of these haplotypes increased the risk of TE. This study suggested that the 3 PROC promoter SNPs were shown to be associated with lower PC activity but did not increase the risk of TE.

Background: We studied long-term outcomes and prognostic features of Thai children with acute lymphoblastic leukemia treated with modified St Jude Children's Research Hospital (SJCRH) protocols., Patients and Methods: Pediatric patients newly diagnosed with acute lymphoblastic leukemia were included. From 1997 to 2003, the first group received modified Total Therapy XIIIB (previous protocol). From 2004 to 2014, the latter had modified Total Therapy XV (current protocol)., Results: In 250 patients, the event-free survival rates (± standard error) of the previous protocol (n = 95) were 82.8 ± 3.9%, 81.7 ± 4.0%, and 81.7 ± 4.0% at 5, 10, and 15 years, respectively; current protocol event-free survival rates (n = 155) were 84 ± 3.0%, 80.8 ± 3.4%, and 80.8 ± 3.4%, respectively. Previous protocol overall survival rates for the same years were 89.2 ± 3.2%, 84.8 ± 3.8%, and 84.8 ± 3.8%, and for the current protocol were 90 ± 2.5%, 86.9 ± 3.2%, and 83.7 ± 4.4%. Previous protocol relapses were 10.5% (10 patients), with 7 having isolated hematologic and 3 isolated/combined central nervous system relapses. Current protocol relapses were 9.7% (15 patients), with 7 having isolated hematologic, 6 isolated/combined central nervous system, and 2 extramedullary relapses. Patients with leukocyte counts over 100 × 10 9 /L and who had disease classified as high risk had worse event-free survival using the previous protocol. However, only initial leukocyte counts of ≥ 100 × 10 9 /L predicted adverse outcomes under the current protocol. Minimal residual disease positivity was a prognostic factor of worse overall survival only for previous protocol patients., Conclusion: Favorable outcomes of childhood acute lymphoblastic leukemia occurred using adapted SJCRH protocols, perhaps because of multidisciplinary care teams and improved parent advocacy. Inferior outcomes might be prevented by addressing predictive factors to ameliorate monitoring and care., (Copyright © 2019 Elsevier Inc. All rights reserved.)