A large cohort of Hb H disease in northeast Thailand: A molecular revisited, diverse genetic interactions and identification of a novel mutation.

BACKGROUND AND AIMS: To update the molecular characteristics of α-thalassemia in northeast Thailand, the molecular basis and genetic interactions of Hb H disease were examined in a large cohort of patients. MATERIALS AND METHODS: A study was done on 1,170 subjects with Hb H disease and various genetic interactions encountered during 2009-2023. Hb and DNA analyses were carried out. RESULTS: As many as 40 genotypes with several known, previously undescribed, and novel mutations were observed. These included 698 subjects (59.8 %) of Hb H disease, 357 (30.6 %) with EABart's disease, 63 (5.4 %) with EEBart's disease, 18 (1.7 %) with abnormal Hbs, 17 (1.5 %) with ß-thalassemia, and 4 (0.4 %) with EFBart's or EFABart's disease. The molecular basis of 13 subjects (1.1 %) remains unknown. The α0-thalassemia included --SEA (n = 1,139, 97.4 %) and --THAI (n = 21, 1.8 %). Two rare mutations were identified in 3 subjects (0.3 %) with --SA and --CR deletions. For α+-thalassemia, -α3.7 kb del (n = 626, 53.5 %), Hb Constant Spring (n = 415, 35.5 %), -α4.2 kb del (n = 44, 3.8 %), Hb Paksé (n = 36, 3.1 %), and Hb Q-Thailand (n = 19, 1.6 %), were detected. Ten rarer α+-thalassemia were identified, including a novel mutation, namely the Hb Chumphae (HBA2:c.32T>A). The Hb H-Lansing-Ramathibodi, Hb H-Jax, and Hb H-Chumphae are hitherto undescribed in this region. PCR-based diagnostic methods for these α-thalassemia defects were described. CONCLUSIONS: This study confirms the diverse heterogeneity and genetic interactions causing Hb H disease in northeast Thailand. The results should prove useful for laboratory diagnosis and genetic counseling of this genetic disorder in the region.

[Cases Analysis of Hemoglobin H Disease Caused by HBA2:c.2T>C and HBA2:c.2delT Mutations].

OBJECTIVE: To investigate two cases of rare pathogenic genes, initiation codon mutations in HBA2 gene, combined with Southeast Asian deletion and their family members to understand the relationship of HBA2:c.2T>C and HBA2:c.2delT mutations with clinical phenotype. METHODS: The peripheral blood of family members was obtained for blood cell analysis and capillary electrophoresis hemoglobin analysis. Gap-PCR and reverse dot blotting (RDB) were used to detect common types of mutations in ɑ-thalassaemia gene. Sanger sequencing was used to analyze HBA1 and HBA2 gene sequence. RESULTS: Two proband genotypes were identified as --SEA/αα with HBA2:c.2T>C and --SEA/αα with HBA2:c.2delT. HBA2:c.2T>C/WT and HBA2:c.2delT/WT was detected in family members. They all presented with microcytic hypochromic anemia. CONCLUSION: When HBA2:c.2T>C and HBA2:c.2delT are heterozygous that can lead to static α-thalassemia phenotype, and when combined with mild α-thalassemia, they can lead to the clinical manifestations of hemoglobin H disease. This study provides a basis for genetic counseling.

Hemoglobin H Disease and Growth: A Comparative Study of DHbH and NDHbH Patients.

Background: Hemoglobin H disease (HbH), a hemoglobinopathy resulting from abnormal alpha globin genes, is classified into two categories: deletional HbH (DHbH) and non-deletional HbH (NDHbH). The alpha-mutation genotypes exhibit a range of clinical anemias, which differentially impact patient growth. Objectives: This retrospective study assessed the growth of HbH patients at Siriraj Hospital, Mahidol University. Methods: Patients diagnosed with HbH between January 2005 and April 2021 were analyzed using growth standard scores of the Thai Society for Pediatric Endocrinology (2022 version) and BMI-for-age Z scores of the World Health Organization. Growth failure was defined as a patient's height for age exceeding two standard deviations below the mean. Results: Of the 145 HbH patients, 75 (51.7%) had NDHbH, with --SEA/αCSα being the most common genotype (70 patients; 93.3%). The mean baseline hemoglobin level was significantly lower in NDHbH patients than in DHbH patients (8.16 ± 0.93 g/dL vs. 9.51 ± 0.68 g/dL; P < 0.001). Splenomegaly and growth failure prevalences were higher in NDHbH patients (37.3% vs. 0%, with P < 0.001, and 22.7% vs. 8.6%, with P = 0.020, respectively). Multivariable analysis revealed splenomegaly > 3 cm was associated with growth failure (OR = 4.28; 95% CI, 1.19-15.39; P = 0.026). Conclusions: NDHbH patients exhibited lower hemoglobin levels and more pronounced splenomegaly than DHbH patients. Growth failure can occur in both HbH types but appears more prevalent in NDHbH. Close monitoring of growth velocity is essential, and early treatment interventions may be required to prevent growth failure.

Detecting rare thalassemia in children with anemia using third-generation sequencing.

BACKGROUND: In China, conventional genetic testing methods can only detect common thalassemia variants. Accurate detection of rare thalassemia is crucial for clinical diagnosis, especially for children that need long-term blood transfusion. This study aims to explore the application value of third-generation sequencing (TGS) in the diagnosis of rare thalassemia in children with anemia. METHODS: We enrolled 20 children with anemia, excluding from iron deficiency anemia (IDA). TGS was employed to identify both known and novel thalassemia genotypes, while sanger sequencing was used to confirm the novel mutation detected. RESULTS: Among the 20 samples, we identified 5 cases of rare thalassemia. These included ß-4.9 (hg38,Chr11:5226187-5231089) at HBB gene, α-91(HBA2:c.*91delT), αCD30(HBA2:c.91-93delGAG), Chinese Gγ+(Aγδß)0(NG_000007.3: g .48795-127698 del 78904) and delta - 77(T > C)(HBD:c.-127T>C). Notably, the -SEA/α-91α genotype associated with severe non-deletional hemoglobin H disease (HbH disease) has not been previously reported. Patients with genotypes ß654/ß-4.9 and -SEA/α-91α necessitate long-term blood transfusions, and those with the -SEA/αCD30α, Chinese Gγ+(Aγδß)0 and delta thalassemia demonstrate mild anemia. CONCLUSIONS: TGS demonstrates promising potential as a diagnostic tool for suspected cases of rare thalassemia in children, especially those suspected to have transfusion-dependent thalassemia (TDT).

Analysis of Hematological Indices and Splenectomy Rates in 2,130 Patients with Hemoglobin H Diseases or ß-Thalassemia.

INTRODUCTION: Splenomegaly and hypersplenism are common complications of thalassemia patients due to the excessive clearance of defective red blood cells from the spleen. To date, splenectomy has been considered one of the most effective treatments for splenomegaly, reducing clinical severity among thalassemia patients. Thus, we aim to investigate the differences in splenectomy rates and hematological indices among thalassemia patients with different genotypes. METHOD: In this study, we analyzed the clinical data of thalassemia in 2,130 patients admitted to the 923rd Hospital of the People's Liberation Army from January 2006 to December 2020, and the statistical software SPSS 26.0 was applied to analyze the data. RESULT: Of the 2,130 patients with thalassemia, 265 patients underwent splenectomy. It was determined that significantly more patients with hemoglobin H (HbH) disease, a form of α-thalassemia, have undergone splenectomy than ß-thalassemia patients (20% vs. 7%). Further, HbH disease patients were diagnosed at a significantly older age than ß-thalassemia patients. CONCLUSION: The greater probability of HbH disease patients undergoing splenectomy is likely influenced by multiple factors, including their lower dependency on transfusion, leading to high spleen compensatory stress on the spleen, and the destruction of defective erythrocytes. In contrast, ß-thalassemia is clinically more severe and less tolerant of hemoglobin fluctuations. Based on these findings, clinicians are suggested to pay more attention to HbH disease patients as many of them are still under-transfused, which could lead to chronic hemolysis and more severe hepatosplenomegaly. These results might offer insight for improving the clinical management of patients with different types of thalassemia.

Severity scoring system to guide transfusion management in pediatric non-deletional HbH.

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, ß-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.

Bone Mineral Density and Dickkopf-1 in Adolescents with Non-Deletional Hemoglobin H Disease.

BACKGROUND: Low bone mineral density (BMD) is prevalent in individuals with ß-thalassemia and is associated with increased circulating dickkopf-1 concentration. These data are limited in α-thalassemia. Therefore, we aimed to determine the prevalence of low BMD and the association between BMD and serum dickkopf-1 in adolescents with non-deletional hemoglobin H disease, a form of α-thalassemia whose severity is comparable to ß-thalassemia intermedia. METHODOLOGY: The lumbar spine and total body BMD were measured and converted into height-adjusted z-scores. Low BMD was defined as BMD z-score ≤ -2. Participant blood was drawn for measurement of dickkopf-1 and bone turnover marker concentrations. RESULTS: Thirty-seven participants with non-deletional hemoglobin H disease (59% female, mean age 14.6 ± 3.2 years, 86% Tanner stage ≥2, 95% regularly transfused, 16% taking prednisolone) were included. Over one year prior to the study, mean average pretransfusion hemoglobin, ferritin and 25-hydroxyvitamin D concentrations were 8.8 ± 1.0 g/dL, and 958 ± 513 and 26 ± 6 ng/mL, respectively. When participants taking prednisolone were excluded, the prevalence of low BMD at the lumbar spine and total body was 42% and 17%, respectively. BMD at both sites was correlated positively with body mass index z-score, and negatively with dickkopf-1 (all p-values <0.05). There were no correlations among dickkopf-1, 25-hydroxyvitamin D, osteocalcin and C-telopeptide of type-I collagen. Multiple regression analysis showed dickkopf-1 inversely associated with total body BMD z-score adjusting for sex, bone age, body mass index, pre-transfusion hemoglobin, 25-hydroxyvitamin D, history of delayed puberty, type of iron chelator and prednisolone use (p-value = 0.009). CONCLUSIONS: We demonstrated a high prevalence of low BMD in adolescents with non-deletional hemoglobin H disease. Moreover, dickkopf-1 inversely associated with total body BMD suggesting it may serve as a bone biomarker in this patient population.

The Clinical Phenotypes of Alpha Thalassemia.

Clinical manifestations of α-thalassemia range from no symptoms to severe transfusion-dependent anemia. Alpha thalassemia trait is deletion of 1 to 2 α-globin genes, whereas α-thalassemia major (ATM; Barts hydrops fetalis) is the deletion all 4 α genes. All other genotypes of intermediate severity are categorized as HbH disease, a vastly heterogenous group. Clinical spectrum is classified as mild, moderate, and severe by symptoms and need for intervention. Anemia in prenatal period may be fatal without intrauterine transfusions. New therapies to modify HbH disease or provide cure for ATM are under development.

Hematological phenotype analysis of fetuses and patients with hemoglobin H disease / 中华地方病学杂志

Objective:To analyze the relationship between hematological and genotype characteristics of fetuses and patients with hemoglobin (Hb) H disease and their natural disease progression.Methods:From 2010 to 2022, a total of 1 252 blood samples from fetuses and patients with Hb H disease who visited the Guangxi Zhuang Autonomous Regional Maternal and Child Health Hospital were collected (including 174 umbilical cord blood samples, 1 062 peripheral blood samples from patients over 2 years old, and 16 peripheral blood samples from patients with rare cases of genotype Hb H). Additionally, 278 peripheral blood samples were collected from patients aged 0 - 2 years old with Hb H 3.7, Hb H 4.2, Hb H CS, and Hb H WS disease for the study of trends in red blood cell development. Multiple probe hybridization and microarray comparative genomic hybridization technology combined with first-generation Sanger sequencing were used for rare mutation detection.Results:Among the 1 062 Hb H disease patients over 2 years old, 62.34% (662/1 062) had gene deletion (--/-α), of which Hb H 3.7 (-- SEA/-α 3.7) and Hb H 4.2 (-- SEA/-α 4.2) were the most common, accounting for 42.28% (449/1 062) and 19.11% (203/1 062) of the total, respectively. Among the non-deletion genotypes (--/αα T or α Tα/αα T), Hb H CS (-- SEA/α CS), Hb H WS (-- SEA/α WS) and α CSα/α CSα accounted for 16.85% (179/1 062), 16.48% (175/1 062) and 1.98% (21/1 062), respectively. The 81.12% (537/662) of patients with deletional Hb H disease showed mild to moderate anemia, with Hb H detection rates ranging from 75% to 80%. Among non-deletional Hb H disease, Hb H WS disease showed the mild (blood Hb concentration > 95 g/L in 90% of patients) phenotype while Hb H CS and Hb H QS (-- SEA/αα QS) patients had moderate to severe anemia, with Hb H detected in peripheral blood at higher levels than in other types of Hb H disease patients. Except for Hb H CS and Hb H QS, which did not show a significant increase in Hb A2 levels when complicated with β-thalassemia, Hb A2 levels were increased (> 3.5%) in all other types of Hb H disease patients. When Hb H disease was complicated with β-thalassemia, Hb H peaks were not detected in either type of Hb H disease. The results of red blood cell development trend detection showed that erythrocyte counts were elevated in patients with Hb H disease compared to their normal counterparts; whereas, blood Hb, mean erythrocyte volume (MCV) and mean erythrocyte hemoglobin content (MCH) were lower than in their normal counterparts ( P < 0.05) and decreased to the minimum at 6 months to 1 year of age. Patients with Hb H CS disease, as the most severe form of anemia, had the highest MCV values ( P < 0.001). The results of fetal cord blood with Hb H disease showed that α CSα/α CSα caused severe intrauterine anemia, followed by Hb H QS and Hb H CS. The content of Hb Bart's in umbilical cord blood was negatively correlated with the severity of anemia ( rs = - 0.58, P < 0.001). When Hb H disease was complicated with β-thalassemia, there was no significant improvement in fetal anemia, and the Hb Bart's content did not change significantly ( P > 0.05). In addition, Hb H 21.9 (-α 21.9kb/-- SEA) and Hb H 2.4 (-α 2.4/-- SEA) were common in patients with deletion rare Hb H. In patients with non-deletion rare Hb H, αα Amsterdam-A1/-- SEA and αα Hb G-Georgia/-- SEA were both first reported. Conclusions:There is heterogeneity in clinical manifestations of patients with different types of Hb H disease or same type of Hb H disease at different developmental stages. When patients with Hb H are complicated with β-thalassemia, the phenotype of patients with the deletion type is improved, while that of patients with the non-deletion type is not. Compared to normal individuals, patients with Hb H disease have lower blood Hb concentration, MCV and MCH, and more rapid physiological changes in red blood cells.

Hydrops Fetalis Associated with Fetal Hemoglobin H-Pakse Disease.

INTRODUCTION: Hemoglobin H-Pakse (Hb H-PS) disease is a variant of non-deletional Hb H disease associated with various degrees of anemia. The disorder is rare but commonly seen in Southeast Asia. However, the prenatal course of Hb H-PS disease has never been published. The objective of this report was to describe prenatal diagnosis and management of Hb H-PS disease, which is theoretically much more critical in fetal life than adult life. CASE PRESENTATION: The prenatal courses of two fetuses affected by Hb H-PS were comprehensively explored. Both of them showed sonographic signs of fetal anemia at 19-20 weeks of gestation (increased cardiac size and increase middle cerebral artery peak systolic velocity [MCA-PSV]). On follow-up scans, both revealed frank hydropic signs at 22-24 weeks. One fetus died at 24 weeks, shortly before the scheduled intrauterine blood transfusion (IUT). The other one underwent IUT at 22 weeks, leading to completely reversed hydropic signs, which resulted in successful outcomes that ended with the delivery of a healthy baby at term. The fetus needed only one IUT, and the course of anemic status improved in late pregnancy. IUT in this case was possibly beneficial to adult life. CONCLUSION: Fetuses with Hb H-PS may be associated with hydrops fetalis, usually occurring at mid-pregnancy. The hydrops tends to improve in late gestation. If they can pass through this most critical period in utero without anemic insults in developing organs, good long-term prognosis can be expected. This successful prenatal diagnosis and intrauterine treatment may encourage care providers to pay more attention to fetal Hb H-PS disease, to prevent anemic hypoxia in developing organs and adult diseases of fetal origin.

Caracterización clínica y epidemiológica de población pediátrica costarricense con alfa-talasemia

Objetivo: Describir las características clínicas y epidemiológicas de pacientes con alfatalasemia atendidos en un hospital nacional pediátrico. Métodos: Estudio observacional descriptivo de corte transversal. Se estudia a 60 pacientes del Servicio de Hematología del Hospital Nacional de Niños "Dr. Carlos Sáenz Herrera", Caja Costarricense de Seguro Social, del 1° de enero de 2018 al 31 de enero de 2019, cuyas edades están comprendidas entre los 0 meses y 12 años 11 meses de edad, con índices hematimétricos sugestivos de alfa-talasemia y con electroforesis de hemoglobina patrón AA con HbA2 normal o disminuida. Análisis molecular: identificación de 21 mutaciones y deleciones más frecuentes para el gen de alfa-globina: 3.7, 4.2, 20.5, MED, FIL, SEA, THAI, anti-3.7 triplicación, HbConstant Spring, HbQuonSze, Hb Adana, HbKoya Dora, HbIcara, HbPakse, a2 poli A-1/2, a2-cd142, a1-cd14, a2-init-cd, a2-cd19, a2-IVS1, a2-cd59. Se utiliza el método de amplificación por reacción en cadena de la polimerasa e hibridación inversa del ácido desoxirribonucleico genómico en leucocitos de sangre periférica de los pacientes. Resultados: Se confirma la enfermedad en 44/60 casos (73%). La edad media al diagnóstico para estos casos es de 4.9 años (desviación estándar 3.0), predominó el sexo femenino en 52.3% de los casos. La provincia de Guanacaste reportó la mayor prevalencia de la enfermedad. El defecto genético delecional -3.7 Kb es el genotipo más frecuente. El fenotipo en el 77.2% de los casos indicó portador silente de alfa-talasemia. El 84.1% de los sujetos positivos para alfa-talasemia correlacionó con hipocromía, microcitosis y eritrocitosis en el hemograma inicial. El 9% de los casos evidenció la coexistencia de alfa-talasemia y anemia por deficiencia de hierro. Conclusiones: Este estudio demuestra que los hallazgos de índices eritrocitarios que indiquen hipocromía y microcitosis con aumento del cómputo de eritrocitos, índices férricos normales y una electroforesis de hemoglobina con patrón normal (AA) sugieren ser estudiados molecularmente por alfa-talasemia. La electroforesis de hemoglobina reportada como normal no excluye la condición de alfa-talasemia y debe realizarse el estudio molecular.

Objective: Describe the clinical and epidemiological characteristics of patients with alpha thalassemia in the Hematology Service of a national pediatric hospital. Methods: Cross-sectional descriptive observational study. 60 patients from the Hematology Service of the National Children's Hospital "Dr. Carlos Sáenz Herrera", Costa Rican Social Security Fund, from January 1, 2018 to January 31, 2019, with hematometric indices suggestive of alphaThalassemia, with AA standard hemoglobin electrophoresis with normal or decreased HbA2 with ages between 0 months and 12 years 11 months old. Molecular analysis: Identification of 21 mutations and deletions that includes the detection of the most frequent deletions/mutations for the alpha globin gene: 3.7, 4.2, 20.5, MED, FIL, SEA, THAI, anti-3.7 tripling, HbConstant Spring, HbQuonSze, Hb Adana, HbKoya Dora, HbIcara, HbPakse, a2 poli A-1/2, a2-cd142, a1-cd14, a2-init-cd, a2-cd19, a2-IVS1, a2-cd59. The reverse hybridization PCR amplification method of genomic DNA in peripheral blood leukocytes of patients isused. Results: Of the 60 cases studied, in 44/60 (73%) cases the disease is confirmed. The average age at diagnosis for these cases is 4.9 years (SD 3.0), the female sex predominated in 52.3% of the cases. Guanacaste reported the highest prevalence of the disease. The deletional genetic defect -3.7 Kb was the most frequent genotype and the phenotype in 77.2% of the cases was he silent carrier of alpha thalassemia. In 84.1% of subjects positive for alpha thalassemia, it correlated with hypochromia, microcytosis, and erythrocytosis in the initial blood count. 9% of the cases showed the coexistence of alpha thalassemia and iron deficiency anemia. Conclusions: This study demonstrates that the findings of erythrocyte indices that indicate hypochromia and microcytosis with increased erythrocyte count; normal iron indices and a normal hemoglobin (AA) electrophoresis pattern suggest that they should be studied molecularly for alpha thalassemia. Hemoglobin electrophoresis reported as normal does not exclude the condition of alpha thalassemia and the molecular study must be carried out.

DeepThal: A Deep Learning-Based Framework for the Large-Scale Prediction of the α+-Thalassemia Trait Using Red Blood Cell Parameters.

Objectives: To develop a machine learning (ML)-based framework using red blood cell (RBC) parameters for the prediction of the α+-thalassemia trait (α+-thal trait) and to compare the diagnostic performance with a conventional method using a single RBC parameter or a combination of RBC parameters. Methods: A retrospective study was conducted on possible couples at risk for fetus with hemoglobin H (Hb H disease). Subjects with molecularly confirmed normal status (not thalassemia), α+-thal trait, and two-allele α-thalassemia mutation were included. Clinical parameters (age and gender) and RBC parameters (Hb, Hct, MCV, MCH, MCHC, RDW, and RBC count) obtained from their antenatal thalassemia screen were retrieved and analyzed using a machine learning (ML)-based framework and a conventional method. The performance of α+-thal trait prediction was evaluated. Results: In total, 594 cases (female/male: 330/264, mean age: 29.7 ± 6.6 years) were included in the analysis. There were 229 normal controls, 160 cases with the α+-thalassemia trait, and 205 cases in the two-allele α-thalassemia mutation category, respectively. The ML-derived model improved the diagnostic performance, giving a sensitivity of 80% and specificity of 81%. The experimental results indicated that DeepThal achieved a better performance compared with other ML-based methods in terms of the independent test dataset, with an accuracy of 80.77%, sensitivity of 70.59%, and the Matthews correlation coefficient (MCC) of 0.608. Of all the red blood cell parameters, MCH < 28.95 pg as a single parameter had the highest performance in predicting the α+-thal trait with the AUC of 0.857 and 95% CI of 0.816−0.899. The combination model derived from the binary logistic regression analysis exhibited improved performance with the AUC of 0.868 and 95% CI of 0.830−0.906, giving a sensitivity of 80.1% and specificity of 75.1%. Conclusions: The performance of DeepThal in terms of the independent test dataset is sufficient to demonstrate that DeepThal is capable of accurately predicting the α+-thal trait. It is anticipated that DeepThal will be a useful tool for the scientific community in the large-scale prediction of the α+-thal trait.

Clinical and genetic characteristics of hemoglobin H disease in Iran.

Hemoglobin H (Hb H) disease is a subtype of α-thalassemia caused by deletional and/or non-deletional mutations in three alpha-globin genes in which the various genotypes determine the disease severity. This study was aimed to investigate the frequency of alpha gene mutations and genotypes and their correlation with hematological and clinical characteristics in Iran. Among 202 patients diagnosed with Hb H disease through a national study in Iran according to standard methods, we had access to the hematologic and clinical findings and genetic data of 101 patients in whom genetic study was performed. Genomic DNA from peripheral blood was extracted and analyzed for identification of α-globin gene mutations using Multiplex Gap Polymerase Chain Reaction, Reverse Hybridization Assay, and finally Direct DNA Sequencing method. Twenty-one different mutations and thirty genotypes were detected in 101 patients with Hb H disease. In total, 39 patients (38.6%) were deletional and 62 patients (61.4%) were non-deletional type of the disease. The --MED mutation was highly prevalent in almost half of the patients (56.4%). Among various genotypes, -MED/-a3.7 (29.7%) and -α20.5/-α5NT (6.9%) were the most prevalent genotypes found in the studied group. Patients with non-deletional type presented with more severe hematological and clinical findings. Hb H percentage and serum ferritin levels were significantly higher in non-deletional patients in comparison to the deletional group (p < 0.05). 12 (11.9%) and 40 (39.6%) out of 101 patients were on regular and occasional transfusions, respectively. 83% of those with regular transfusion belonged to the non-deletional group. Among transfusion-dependent patients, -MED/αCSα and α20.5/-α5NT were the most common genotypes. In this study, two patients with -α20.5/αCSα and -MED/α-5NT genotypes experienced thrombotic events. This study indicated that although non-deletional genotypes of Hb H disease were responsible for more clinical severity of the disease, due to the presence of severe phenotypes even in deletional types, no definite correlation was found between genotype and phenotype.

Image Analysis Using Machine Learning for Automated Detection of Hemoglobin H Inclusions in Blood Smears - A Method for Morphologic Detection of Rare Cells.

BACKGROUND: Morphologic rare cell detection is a laborious, operator-dependent process which has the potential to be improved by the use of image analysis using artificial intelligence. Detection of rare hemoglobin H (HbH) inclusions in red cells in the peripheral blood is a common screening method for alpha-thalassemia. This study aims to develop a convolutional neural network-based algorithm for the detection of HbH inclusions. METHODS: Digital images of HbH-positive and HbH-negative blood smears were used to train and test the software. The software performance was tested on images obtained at various magnifications and on different scanning platforms. Another model was developed for total red cell counting and was used to confirm HbH cell frequency in alpha-thalassemia trait. The threshold minimum red cells to image for analysis was determined by Poisson modeling and validated on image sets. RESULTS: The sensitivity and specificity of the software for HbH+ cells on images obtained at ×100, ×60, and ×40 objectives were close to 91% and 99%, respectively. When an AI-aided diagnostic model was tested on a pilot of 40 whole slide images (WSIs), good inter-rater reliability and high sensitivity and specificity of slide-level classification were obtained. Using the lowest frequency of HbH+ cells (1 in 100,000) observed in our study, we estimated that a minimum of 2.4 × 106 red cells would need to be analyzed to reduce misclassification at the slide level. The minimum required smear size was validated on 78 image sets which confirmed its validity. CONCLUSIONS: WSI image analysis can be utilized effectively for morphologic rare cell detection. The software can be further developed on WISs and evaluated in future clinical validation studies comparing AI-aided diagnosis with the routine diagnostic method.

Telomere shortening correlates with disease severity in hemoglobin H disease patients.

Hemoglobin H (Hb H) disease is the most significant health problem of the α-thalassemia syndromes. The Hb disease patients are categorized based on their genotype to deletional and nondeletional, with the latter genotype presents the more severe clinical symptoms. Since telomere length is an indicator of biological aging and health, we hypothesized that telomere length could reflect Hb H disease's severity. In this study, we recruited 48 deletional and 47 nondeletional Hb H disease patients, along with 109 normal controls, for telomere length assessment. The leukocyte telomere length was assessed by monochromatic multiplex real-time PCR and reported as the telomere to single-copy gene (T/S) ratio. When telomere length was adjusted for age, the analysis of covariance between the control and the two Hb H disease groups revealed no significant difference. However, the telomere shortening rate was more rapid in the nondeletional Hb H disease group than those of the control and deletional Hb H disease groups. Gender analysis found that male patients have a significantly lower T/S ratio than females in the nondeletional group but not in the control and deletional groups. In the two disease groups, the T/S ratio was not influenced by ferritin level or transfusion burden but was positively correlated with the absolute reticulocyte count.

Association of hemoglobin H (HbH) disease with hemoglobin A1c and glycated albumin in diabetic and non-diabetic patients.

OBJECTIVES: Hemoglobin A1c (HbA1c) and glycated albumin (GA) are glycemic control status indicators in patients with diabetes mellitus. Hemoglobin H (HbH) disease is a moderately severe form of α-thalassemia. Here we examine the usefulness of HbA1c and GA in monitoring glycemic control in patients with HbH disease. METHODS: HbA1c, GA, and an oral glucose tolerance test were performed in 85 patients with HbH disease and 130 healthy adults. HbA1c was measured using five methods, including two systems based on cation-exchange high-performance liquid chromatography (Variant II Turbo 2.0 and Bio-Rad D100), a capillary zone electrophoresis method (Capillarys 3 TERA), a boronate affinity HPLC method (Premier Hb9210), and an immunoassay (Cobas c501). RESULTS: Significant lower levels of HbA1c were observed in patients with HbH disease than in healthy adults. In contrast, GA showed no statistically significant differences between participants with and without HbH disease. A considerable number of diabetic patients with HbH disease would be missed if using HbA1c as a diagnostic criterion for diabetes mellitus. CONCLUSIONS: GA but not HbA1c is suitable for monitoring glycemic control in patients with HbH disease that can modify the discriminative ability of HbA1c for diagnosing diabetes.

[Regulation effect of siRNA on ß-globin in erythrocytes of hemoglobin H disease].

Objective: To investigate the regulatory effect of targeted siRNA on ß-globin in erythroid cells cultured by targeted differentiation in vitro and provide new theoretical support for gene therapy for hemoglobin H (HbH) disease. Methods: Based on the ß-globin gene expression results, the optimal siRNA sequence and its effective action dose were screened in erythroid cells, and the effect of the effective dose of the optimal siRNA on the regulation of ß-globin expression and apoptosis in erythroid cells was examined. The effective dose of the optimal siRNA was applied to erythroid cells with HbH disease. The effects of transfected siRNAs on red line cells with HbH disease were comprehensively evaluated by measuring the expression of ß-globin, reactive oxygen species (ROS) , and apoptosis rates. Results: Within 96 hours after transfection, siRNA2 significantly downregulated ß-globin expression in in vitro cultured erythroid cells, but not α-globin. siRNA silencing effect and duration of effect were dose-dependent. siRNA2 downregulated ß-globin expression, reduced intracellular ROS production, and decreased apoptosis rate in erythroid cells with HbH disease. Conclusion: Targeted siRNAs can downregulate ß-globin expression, reduce intracellular ROS production, and downregulate apoptosis rate in erythroid cells with HbH disease.

Abnormal hemoglobin H band in myelodysplastic syndromes (MDS): A case report.

Myelodysplastic syndrome (MDS) is a group of heterogeneous diseases derived from hematopoietic stem cells characterized by hemolytic anemia and high risk of conversion to acute leukemia. MDS is an age-related disease in which approximately 80% of patients are over 60years of age, male and female. Anemia is the most common clinical condition, and many patients are also associated with infection and bleeding. When the amount of α globin synthesis is insufficient, the remaining ß chain forms tetramer ß4, i.e. HbH. The latter forms a precipitate in red blood cells, leading to hemolytic anemia, called HbH disease, the majority of which is congenital, a small number of patients with myelodysplastic syndrome and acute myeloid leukemia may appear HbH (called acquired HbH disease). We reported a 71years old male patient diagnosed as myelodysplastic syndromes (MDS) in our hospital. The patient has a negative α-thalassemia gene test. The H band is detected by hemoglobin electrophoresis. This article analyzed and discussed this case with MDS, as well reviewed MDS.