Cognitive impairment and hippocampal neuronal damage in ß-thalassaemia mice.

ß-Thalassaemia is one of the most common genetic diseases worldwide. During the past few decades, life expectancy of patients has increased significantly owing to advance in medical treatments. Cognitive impairment, once has been neglected, has gradually become more documented. Cognitive impairment in ß-thalassaemia patients is associated with natural history of the disease and socioeconomic factors. Herein, to determined effect of ß-thalassaemia intrinsic factors, 22-month-old ß-thalassaemia mouse was used as a model to assess cognitive impairment and to investigate any aberrant brain pathology in ß-thalassaemia. Open field test showed that ß-thalassaemia mice had decreased motor function. However, no difference of neuronal degeneration in primary motor cortex, layer 2/3 area was found. Interestingly, impaired learning and memory function accessed by a Morris water maze test was observed and correlated with a reduced number of living pyramidal neurons in hippocampus at the CA3 region in ß-thalassaemia mice. Cognitive impairment in ß-thalassaemia mice was significantly correlated with several intrinsic ß-thalassaemic factors including iron overload, anaemia, damaged red blood cells (RBCs), phosphatidylserine (PS)-exposed RBC large extracellular vesicles (EVs) and PS-exposed medium EVs. This highlights the importance of blood transfusion and iron chelation in ß-thalassaemia patients. In addition, to improve patients' quality of life, assessment of cognitive functions should become part of routine follow-up.

[Analysis of rare mutations associated with Thalassemia and their hematological characteristics in Chenzhou region of Hunan Province].

OBJECTIVE: To explore the distribution and hematological characteristics of rare thalassemia-associated mutations in Chenzhou region of Hunan Province with an aim to provide a basis for genetic counseling and effective prevention. METHODS: A total of 37 370 individuals enrolled from January 2015 to December 2021 were screened by routine blood test and hemoglobin electrophoresis. The genotypes were determined with high-throughput sequencing. RESULTS: A total of 8 455 thalassemia mutations (including 185 rare ones) were detected, which had involved 27 mutational types. Rare type α-Thalassemia --THAI and CD31 (AGG>AAG) have the typical microcytic hypochromic hematological features, whilst SEA-HPFH, CD14 (CTG>-TG), CD37 (TGG>TAG), -90(C>T), Codon 15 (G>A), IVS-I-128 (T>G), CD86 (GCC>GC-) and Chinese Gγ+(Aγδß)0 had typical microcytic hypochromic and ß-thalassemia-associated hematological features of elevated HbA2 or HbF. In addition, the -50（G>A）heterozygotes of ß-thalassemia had normal or slightly decreased MCV and MCH without an increase in HbA2. CONCLUSION: Various forms of thalassemia-associated mutations have been identified in the Chenzhou region of Hunan Province. Above finding has facilitated development of preventive and control strategies for thalassemia as well as birth health programs.

ß-Thalassemia gene editing therapy: Advancements and difficulties.

ß-Thalassemia is the world's number 1 single-gene genetic disorder and is characterized by suppressed or impaired production of ß-pearl protein chains. This results in intramedullary destruction and premature lysis of red blood cells in peripheral blood. Among them, patients with transfusion-dependent ß-thalassemia face the problem of long-term transfusion and iron chelation therapy, which leads to clinical complications and great economic stress. As gene editing technology improves, we are seeing the dawn of a cure for the disease, with its reduction of ineffective erythropoiesis and effective prolongation of survival in critically ill patients. Here, we provide an overview of ß-thalassemia distribution and pathophysiology. In addition, we focus on gene therapy and gene editing advances. Nucleic acid endonuclease tools currently available for gene editing fall into 3 categories: zinc finger nucleases, transcription activator-like effector nucleases, and regularly interspaced short palindromic repeats (CRISPR-Cas9) nucleases. This paper reviews the exploratory applications and exploration of emerging therapeutic tools based on 3 classes of nucleic acid endonucleases in the treatment of ß-thalassemia diseases.

Relationship of TNFα-238 G/A (rs 361525) genotypes with TNFα gene expression in liver and pancreas disorders in sample of beta thalassemia major adult Iraqi patients.

BACKGROUND: Tumor necrosis factor-α (TNFα) is a crucial physiologic regulator of immune responses, and several disorders have been associated with its dysregulation. OBJECTIVE: This study aimed to understand TNFα gene expression in adult patients with liver and pancreas disorders and examine the impact of TNFα-238 genotypes on this population. METHODS: At the Ibn Al-Baladi Hospital in Baghdad, blood samples were collected from forty patients who were diagnosed with beta thalassemia together with pancreatic disease, forty patients who were diagnosed with thalassemia together with liver disorder, and forty patients who were diagnosed with thalassemia without pancreas or liver disorder. For the purpose of establishing a control group, forty samples were collected from persons who were of the same age and gender and seemed to be in good health. All of these individuals were deemed to be older than 18 years old. Through the utilization of real-time polymerase chain reaction (PCR), the level of TNF-α gene expression was investigated and assessed. The T-ARMS-PCR method was performed for detection and genotyping of TNFα-238 in thalassemia patients and healthy control samples. RESULTS: The result showed that TNF α gene expression assessment showed that group B (thalassemia patients with liver disorder) had higher folding than other groups while the lowest gene expression was in group D (as control group). Furthermore, the relationship between TNFα gene expressions folding with TNFα-238 genotypes in beta thalassemia major patients, discovered a considerable increase at GA genotype patients in TNFα gene expression level, followed by AA genotype compared to the GG genotype. Furthermore, the results of the current study showed an association between the presence of the mutant (A) allele whether heterozygous (GA) and homozygous (AA) with the TNF-α gene expression in thalassemia patients with liver and pancreatic disorders. CONCLUSION: Based on the results, it can be concluded that there is a relationship between the presence of the mutant (A) allele, whether heterozygous (GA) or homozygous (AA) of TNF-α 238, and TNF-α gene expression in liver and pancreatic diseases as well as in patients with thalassemia.

Molecular characterization of similar Hb Lepore Boston-Washington in four Chinese families using third generation sequencing.

Hemoglobin (Hb) Lepore is a rare deletional δß-thalassemia caused by the fusion between delta-beta genes, and cannot be identified by traditional thaltassemia gene testing technology. The aim of this study was to conduct molecular diagnosis and clinical analysis of Hb Lepore in four unrelated Chinese families using third generation sequencing. Decreased levels of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and an abnormal Hb band were observed in the probands of the four families. However, no common α and ß-thalassemia variants were detected in the enrolled families using polymerase chain reaction-reverse dot blot hybridization based traditional thalassemia gene testing. Further third-generation sequencing revealed similar Hb Lepore-Boston-Washington variants in all the patients, which were resulted from partial coverage of the HBB and HBD globin genes, leading to the formation of a delta-beta fusion gene. Specific gap-PCR and Sanger sequencing confirmed that all the patients carried a similar Hb Lepore-Boston-Washington heterozygote. In addition, decreased levels of MCH and Hb A2 were observed in the proband's wife of family 2, an extremely rare variant of Hb Nanchang (GGT > AGT) (HBA2:c.46G > A) was identified by third-generation sequencing and further confirmed by Sanger sequencing. This present study was the first to report the similar Hb Lepore-Boston-Washington in Chinese population. By combining the utilization of Hb capillary electrophoresis and third-generation sequencing, the screening and diagnosis of Hb Lepore can be effectively enhanced.

Newborn Screening for ß-Thalassemia Identifies a Complex Genotype Involving a Novel ß-Globin Gene Mutation (HBB:c.336dup).

Newborn screening identified a Chinese-Canadian infant who was positive for possible ß-thalassemia (ß-thal). Detailed family studies demonstrated that the proband was a compound heterozygote for the Chinese Gγ(Aγδß)0-thal deletion and a novel frameshift mutation within exon 3 (HBB:c.336dup), and heterozygous for the Southeast Asian α-thal deletion (--SEA/αα). This case illustrates the importance of follow-up molecular testing of positive newborn screening results to confirm the diagnosis and define risks for future pregnancies.

Premarital Screening is Pivotal in Reducing the Births of Babies Affected with Thalassemia Major in Iraq.

Thalassemia major is one of the health problems in Iraq, especially in Kurdistan. Pre-marriage mandatory preventive screening program was established in Kurdistan in 2008, which allowed us to study the prevalence of different hemoglobinopathies among newly married young adults in this region. A total of 1154 subjects (577 couples) attending the Koya district, premarital Health center, were screened using red cell indices. Those who had mean corpuscular volume (MCV)<80 fl and mean corpuscular hemoglobin (MCH)<27 pg had high-performance liquid chromatography and iron studies. Out of 1154 individuals that were evaluated, 183 (11.9%) had low MCV and MCH. Of the former 183 subjects, 69 (5.97%) had ß-thalassemia trait, 10 (0.86%) had δß-thalassemia trait, and no other hemoglobinopathies were recorded in our study. There was second-degree consanguinity in 4.7% of all 577 couples. In two couples, both partners had ß-thalassemia trait and both were consanguineous. Both couples decided to separate after counseling. Based on the current study, the role of the premarital screening program in decreasing the number of new thalassemia major cases among the Kurdish population is laudable. Therefore, mandatory premarital screening is advised in all parts of Iraq.

[Advance of research on the role of BCL11A in the occurrence and treatment of ß-Thalassemia].

ß-Thalassemia is a single-gene disease caused by mutations in ß-globin and has a distinct geographical characteristics. Current treatment for patients with moderate to severe thalassemia has mainly relied on long-term blood transfusion and/or hematopoietic stem cell transplantation. B cell lymphoma/leukemia 11A (BCL11A) as a transcriptional repressor plays a vital role in monitoring γ/ß hemoglobin switching, maintaining the normal function of hematopoietic stem cells, and regulating erythrocyte differentiation and lymphocyte development. With the rapid progress in gene editing technology, the BCL11A as a therapeutic target for ß-thalassemia has shown promising results. This article has systematically summarized the regulatory mechanism and therapeutic potential of the BCL11A, with an aim to provide new ideas for the treatment of ß-thalassemia.

[Study of the types of mutations of Thalassemia in Shanghai area].

OBJECTIVE: To analyze the mutations of globin genes among patients suspected for thalassemia from the Shanghai area. METHODS: A total of 4 644 patients diagnosed at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine between June 2016 and December 2019 were selected as the study subjects. The patients were tested for common mutations associated with thalassemia gene by Gap-PCR and reverse dot blotting (RDB). Patients were suspected to harbor rare mutations based on the inconsistency between hematological phenotypes and results of common mutation detection, and were further analyzed by Gap-PCR and Sanger sequencing. RESULTS: Among the 4 644 patients, 2 194 (47.24%) were found to carry common thalassemia mutations, among which 701 (15.09%) were α-thalassemia, 1 448 (31.18%) were ß-thalassemia, and 45 (0.97%) were both α- and ß-thalassemia. Forty six samples were found to harbor rare mutations, which included 17 α-globin gene and 29 ß-globin gene mutations. CD77(CCC>ACC) (HBA2: c.232C>A) of the α-globin gene, NG_000007.3: g.70567_71015del449, codon 102(-A) (HBB: c.308_308delA) and IVS-Ⅱ-636 (A>G) (HBB: c.316-215A>G) of the ß-globin gene were previously unreported new types of globin gene mutations. CONCLUSION: Among the 4 644 patients, the detection rate for common thalassemia mutations was 47.24%, whilst 46 samples were detected with rare gene mutations. The type of gene mutation types were diverse in the Shanghai area. The study has provided more accurate results for genetic diagnosis and counseling.

Impact of genotype on multi-organ iron and complications in patients with non-transfusion-dependent ß-thalassemia intermedia.

We evaluated the impact of the genotype on clinical and hematochemical features, hepatic and cardiac iron levels, and endocrine, hepatic, and cardiovascular complications in non-transfusion-dependent (NTD) ß-thalassemia intermedia (TI) patients. Sixty patients (39.09 ± 11.11 years, 29 females) consecutively enrolled in the Myocardial Iron Overload in Thalassemia project underwent Magnetic Resonance Imaging to quantify iron overload, biventricular function parameters, and atrial areas and to detect replacement myocardial fibrosis. Three groups of patients were identified: homozygous ß+ (N = 18), heterozygous ß0ß+ (N = 22), and homozygous ß0 (N = 20). The groups were homogeneous for sex, age, splenectomy, hematochemical parameters, chelation therapy, and iron levels. The homozygous ß° genotype was associated with significantly higher biventricular end-diastolic and end-systolic volume indexes and bi-atrial area indexes. No difference was detected in biventricular ejection fractions or myocardial fibrosis. Extramedullary hematopoiesis and leg ulcers were significantly more frequent in the homozygous ß° group compared to the homozygous ß+ group. No association was detected between genotype and liver cirrhosis, hypogonadism, hypothyroidism, osteoporosis, heart failure, arrhythmias, and pulmonary hypertension. Heart remodelling related to a high cardiac output state cardiomyopathy, extramedullary hematopoiesis, and leg ulcers were more pronounced in patients with the homozygous ß° genotype compared to the other genotypes analyzed. The knowledge of the genotype can assist in the clinical management of NTD ß-TI patients.

Pharmacogenomics of Drugs Used in ß-Thalassemia and Sickle-Cell Disease: From Basic Research to Clinical Applications.

In this short review we have presented and discussed studies on pharmacogenomics (also termed pharmacogenetics) of the drugs employed in the treatment of ß-thalassemia or Sickle-cell disease (SCD). This field of investigation is relevant, since it is expected to help clinicians select the appropriate drug and the correct dosage for each patient. We first discussed the search for DNA polymorphisms associated with a high expression of γ-globin genes and identified this using GWAS studies and CRISPR-based gene editing approaches. We then presented validated DNA polymorphisms associated with a high HbF production (including, but not limited to the HBG2 XmnI polymorphism and those related to the BCL11A, MYB, KLF-1, and LYAR genes). The expression of microRNAs involved in the regulation of γ-globin genes was also presented in the context of pharmacomiRNomics. Then, the pharmacogenomics of validated fetal hemoglobin inducers (hydroxyurea, butyrate and butyrate analogues, thalidomide, and sirolimus), of iron chelators, and of analgesics in the pain management of SCD patients were considered. Finally, we discuss current clinical trials, as well as international research networks focusing on clinical issues related to pharmacogenomics in hematological diseases.

Exagamglogene Autotemcel for Transfusion-Dependent ß-Thalassemia.

BACKGROUND: Exagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis through ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of the erythroid-specific enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs). METHODS: We conducted an open-label, single-group, phase 3 study of exa-cel in patients 12 to 35 years of age with transfusion-dependent ß-thalassemia and a ß0/ß0, ß0/ß0-like, or non-ß0/ß0-like genotype. CD34+ HSPCs were edited by means of CRISPR-Cas9 with a guide mRNA. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was transfusion independence, defined as a weighted average hemoglobin level of 9 g per deciliter or higher without red-cell transfusion for at least 12 consecutive months. Total and fetal hemoglobin concentrations and safety were also assessed. RESULTS: A total of 52 patients with transfusion-dependent ß-thalassemia received exa-cel and were included in this prespecified interim analysis; the median follow-up was 20.4 months (range, 2.1 to 48.1). Neutrophils and platelets engrafted in each patient. Among the 35 patients with sufficient follow-up data for evaluation, transfusion independence occurred in 32 (91%; 95% confidence interval, 77 to 98; P<0.001 against the null hypothesis of a 50% response). During transfusion independence, the mean total hemoglobin level was 13.1 g per deciliter and the mean fetal hemoglobin level was 11.9 g per deciliter, and fetal hemoglobin had a pancellular distribution (≥94% of red cells). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No deaths or cancers occurred. CONCLUSIONS: Treatment with exa-cel, preceded by myeloablation, resulted in transfusion independence in 91% of patients with transfusion-dependent ß-thalassemia. (Supported by Vertex Pharmaceuticals and CRISPR Therapeutics; CLIMB THAL-111 ClinicalTrials.gov number, NCT03655678.).

Impact of α-Globin Gene Expression and α-Globin Modifiers on the Phenotype of ß-Thalassemia and Other Hemoglobinopathies: Implications for Patient Management.

In this short review, we presented and discussed studies on the expression of globin genes in ß-thalassemia, focusing on the impact of α-globin gene expression and α-globin modifiers on the phenotype and clinical severity of ß-thalassemia. We first discussed the impact of the excess of free α-globin on the phenotype of ß-thalassemia. We then reviewed studies focusing on the expression of α-globin-stabilizing protein (AHSP), as a potential strategy of counteracting the effects of the excess of free α-globin on erythroid cells. Alternative processes controlling α-globin excess were also considered, including the activation of autophagy by ß-thalassemia erythroid cells. Altogether, the studies reviewed herein are expected to have a potential impact on the management of patients with ß-thalassemia and other hemoglobinopathies for which reduction in α-globin excess is clinically beneficial.

Molecular Characterization of α- and ß-Thalassemia Among Children Less Than 18 Years Old in Guizhou, China.

BACKGROUND: Thalassemia is an inherited hemolytic disease, the complications and sequelae of which have posed a huge impact on both patients and society. But limited studies have investigated the molecular characterization of α- and ß-thalassemia in children from Guizhou, China. METHODS: Between January 2019 and December 2022, a total of 3301 children, aged 6 months to 18 years, suspected of having thalassemia underwent molecular analysis. RESULTS: Out of the total sample, 824 (25%) children were found to carry thalassemia mutations. The carrier rates of α-thalassemia, ß-thalassemia, and α + ß-thalassemia were determined as 8.1%, 15.6%, and 1.3%, respectively. Approximately 96.5% of the α-thalassemia gene mutations were --SEA (51%), ααCS (20.9%), -α3.7 (19.6%), and -α4.2 (5.0%). The most prevalent mutations of ß-thalassemia were ßCD17(A>T) (41.5%), ßCD41-42(-TTCT) (37.7%), and ßIVS-II-654(C>T) (11.3%). Additionally, we identified rare cases, including one case with ααHb Nunobiki/αα, two cases with triplicated α-thalassemia (one case with ααα/ααα and ßCD41-42/ßN and the other with ααα-3.7/αα and ßE CD26/ßN), and also one case with α Q-Thailandα/-α4.2 and ßCD41-42/ßN. CONCLUSIONS: Our study findings provide important insights into the heterogeneity of thalassemia carrier rates and molecular profiles among children in the Guizhou region. The findings support the development of prevention strategies to reduce the incidence of severe thalassemia in the future.

Abnormal hemoglobin anti-Lepore Hong Kong compound with ß0-thalassemia ameliorate thalassemia severity when co-inherited with α-thalassemia.

Abnormal hemoglobin anti-Lepore Hong Kong is a rare ßδ fusion variants resulting from non-homologous crossover during meiosis. Anti-Lepore Hong Kong is known to consistently exhibit significantly increased level of HbA2. In this study, we used multiplex ligation-dependent probe amplification (MLPA) and single molecular real-time (SMRT) sequencing, as well as Sanger sequencing, to identify variants in five unrelated families with abnormal elevated HbA2 level. All probands in these five families were found to be heterozygous for anti-Lepore Hong Kong. Among them, two families showed co-occurrence of ß0-thalassemia and α-thalassemia (-SEA/ or αCSα/). Heterozygotes for anti-Lepore Hong Kong displayed an average HbA2 level of 17.7% and behaved normal. However, when combined with ß0-thalassemia and α-thalassemia, the probands exhibited higher HbA2 level (30.2-40.8%) and behaved with ß-thalassemia trait. Furthermore, determination of the α/ß-mRNA ratio revealed a slight downregulation of ß-globin, similar to that of ß-thalassemia minor. Our study is the first to identify compound heterozygotes for anti-Lepore Hong Kong, ß0-thalassemia and α-thalassemia, provide valuable information for prenatal counseling.

Compound heterozygosity for Southeast Asian hereditary persistence of fetal hemoglobin and ß0-thalassemia results in thalassemia intermedia: Pedigree analysis and genetic research in a family from South China. A case report.

RATIONALE: Compound heterozygotes for deletional ß-thalassemia can be difficult to diagnose due to its diverse clinical presentations and no routine screenings. This can lead to disease progression and delay in treatment. PATIENT CONCERNS: We reported pedigree analysis and genetic research in a family with rare ß-thalassemia. DIAGNOSIS: Pedigree analysis and genetic research demonstrated that the patient was a compound heterozygote for ß-thalassemia CD17/Southeast Asian hereditary persistence of fetal hemoglobin deletion, inherited from the parents. Magnetic resonance imaging T2* examination revealed severe iron deposition in the liver. Echocardiography revealed endocardial cushion defect. INTERVENTIONS: The patient was treated with Deferasirox after receiving the final molecular genetic diagnosis. The initial once-daily dose of Deferasirox was 20 mg/kg/d. OUTCOMES: The patient discontinued the medication three months after the first visit. Two years later, the patient visited the Department of Hepatobiliary and Pancreatic Diseases. He was recommended to undergo splenectomy after surgical repair of the congenital heart disease. However, the patient refused surgical treatment because of the economic burden. LESSONS: We report that fetal hemoglobin is a sensitive indicator for screening large deletions of the ß-globin gene, which can be effectively confirmed by the multiplex ligation-dependent probe amplification assay. In non-transfusion-dependent thalassemia patients, iron status assessment should be regularly performed, and iron chelation treatment should be initiated early. This case will provide insights for the diagnosis of rare genotypes of ß-thalassemia and has important implications for genetic counseling.

ß0-Thalassemia Caused by a Novel Nonsense Mutation [HBB:c.199A > T].

We report two hemoglobinopathy cases involving a novel ß-thalassemia (ß-thal) nonsense mutation, HBB:c.199A > T. One patient had Hb S/ß-thal, and a second unrelated patient had Hb D-Punjab/ß-thal. The HBB:c.199A > T mutation introduces a premature termination codon at amino acid codon 66 (AAA→TAA) in exon 2, resulting in typical high Hb A2 ß0-thal.

RNA therapeutics for ß-thalassemia.

ß-thalassemia is an autosomal recessive disease, caused by one or more mutations in the ß-globin gene that reduces or abolishes ß-globin chain synthesis causing an imbalance in the ratio of α- and ß-globin chain. Therefore, the ability to target mutations will provide a good result in the treatment of ß-thalassemia. RNA therapeutics represents a promising class of drugs inclusive antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA) and APTAMER have investigated in clinical trials for treatment of human diseases as ß-thalassemia; Especially, ASO therapeutics can completely treat ß-thalassemia patients by the way of making ASO infiltrating through erythrocyte progenitor cells, migrating to the nucleus and hybridizing with abnormal splicing sites to suppress an abnormal splicing pattern of ß-globin pre-mRNA. As a result, the exactly splicing process is restored to increase the expression of ß-globin which increases the amount of mature hemoglobin of red blood cells of ß-thalassemia patients. Furthermore, current study demonstrates that RNA-based therapeutics get lots of good results for ß-thalassemia patients. Then, this chapter focuses on current advances of RNA-based therapeutics and addresses current challenges with their development and application for treatment of ß-thalassemia patients.