Development of pre-implantation genetic testing protocol for monogenic disorders (PGT-M) of Hb H disease.

Hb H disease is the most severe form of α-thalassemia compatible with post-natal life. Compound heterozygous α0-thalassemia- SEA deletion/α+-thalassemia- 3.7kb deletion is the commonest cause of Hb H disease in Thailand. Preimplantation genetics testing for monogenic disorders (PGT-M) is an alternative for couples at risk of the disorder to begin a pregnancy with a healthy baby. This study aims to develop a novel PCR protocol for PGT-M of Hb H disease- SEA/-3.7kb using multiplex fluorescent PCR. A novel set of primers for α+-thalassemia- 3.7kb deletion was developed and tested. The PCR protocol for α0-thalassemia- SEA deletion was combined for Hb H disease- SEA/-3.7kb genotyping. The PCR protocols were applied to genomic DNA extracted from subjects with different thalassemia genotypes and on whole genome amplification (WGA) products from clinical PGT-M cycles of the families at risk of Hb Bart's. The results were compared and discussed. The results showed three PCR products from α+-thalassemia- 3.7kb primer set, and three from α0thalassemiaSEA primer set. The results were consistent with the known thalassemia genotypes. The novel -α3.7 primers protocol was also tested on 37 WGA products from clinical PGT-M cycles giving accurate genotyping results and a satisfying amplification efficiency with the ADO rates of 2.7%, 0%, and 0% for HBA2, HBA1, and internal control fragments, respectively. This novel PCR protocol can precisely distinguish Hb H disease- SEA/-3.7kb from other genotypes. Additionally, this is the first PCR protocol for Hb H disease- SEA/-3.7kb which is optimal for PGT-M.

De-novo ATR-16 syndrome associated with inherited hemoglobin Evanston causing HbH phenotype: a rare occurrence.

Abnormality of three α-globin genes, either deletion or point mutation results in symptomatic Hemoglobin H (HbH) phenotype. Most of such cases of α-globin defects are inherited from the parents, de-novo cases are exceedingly rare. Herein, a case of HbH is reported where the proband inherited one α-globin gene with a point mutation (αEvanston) from the mother. This was associated with large de-novo deletion of chromosome 16p13.3 resulting in α-thalassemia and mental retardation (ATR-16) syndrome. This deletion also encompassed two α-globin genes from chromosome 16, eventually leading to --/ααEvanston genotype, explaining the clinical presentation of the proband. The challenges in screening of such cases and confirming the molecular diagnosis along with the mode of inheritance has been discussed.

Support Vector Machine-Based Formula for Detecting Suspected α Thalassemia Carriers: A Path toward Universal Screening.

The blood counts of α thalassemia carriers (α-thal) are similar to those of ß thalassemia carriers, except for Hemoglobin A2 (Hb A2), which is not elevated. The objective of this study was to determine whether mathematical formulas are effective for detecting suspected α-thal. The data were obtained from the database of the prevention program for detecting couples at risk for having a child with hemoglobinopathy. Red Blood Cells (RBC) indices were analyzed using mathematical formulas, and the sensitivity and negative predictive value (NPV) were calculated. Among 1334 blood counts suspected of α-thal analyzed, only the Shine and Lal and the Support Vector Machine formulas revealed high sensitivity and NPV. Sensitivity was 85.54 and 99.33%, and NPV was 98.93 and 99.93%, respectively. Molecular defects were found in 291, and 81 had normal α genes. Molecular analysis was not performed in 962 of the samples. Based on these results, mathematical formulas incorporating one of these reliable formulas for detecting suspected α or ß thalassemia carriers in the program of the automatic analyzers can flag these results, increase the awareness of the primary physicians about the carrier risk, and send an alert with a recommendation for further testing.

A rare hemoglobinopathy duo: Hb Adana×Hb SEA in a 1-year-old patient - a case report and a brief literature review.

Introduction and importance: Alpha thalassemia, resulting from nondeletional mutations, typically presents a more severe clinical manifestation compared to deletional mutations. Severe outcomes, such as hydrops fetalis, are associated with two specific nondeletional mutations. Therefore, DNA-based investigation is crucial for suspected carriers exhibiting subtle hematological abnormalities to facilitate proper diagnosis and effective family counseling. Case presentation: In this report, the authors describe a phenotypically normal 1-year-old girl with a rare and unique alpha-thalassemia genotype due to the presence of Hb Adana, a nondeletional alpha-chain mutation compounded with Hb SEA, an alpha-globin gene deletion. Clinical discussion: Mutations determine the clinical manifestations of alpha-thalassemia. DNA testing is recommended for suspected carriers with relatively small hematological abnormalities, for precise diagnosis and family counseling. To provide clinicians with a reference for diagnostic assessment, the authors established a genotype-phenotype correlations based on reported cases of Hb Adana following an exhaustive literature review. Being interested in determining which ethnicities and genotypes are associated with a higher risk of complications, including hydrops fetalis and transfusion dependence, the authors formalized a diagnostic evaluation guide and a guide for early screening to improve outcomes. Conclusion: Precise genetic evaluation is important for the diagnosis of alpha thalassemia. Hematologists play a critical role in managing these disorders, understanding genotype-phenotype correlations, and highlighting the significance of genetic counseling for high-risk patients. Extensive studies on these various genophenotypes are required to improve the diagnosis and prognosis of such medical conditions and advocate preventative strategies.

Ancestry of the major long-range regulatory site of the α-globin genes in the Portuguese population with the common 3.7 kb α-thalassemia deletion.

BACKGROUND: The α-Major Regulatory Element (α-MRE), also known as HS-40, is located upstream of the α-globin gene cluster and has a crucial role in the long-range regulation of the α-globin gene expression. This enhancer is polymorphic and several haplotypes were identified in different populations, with haplotype D almost exclusively found in African populations. The purpose of this research was to identify the HS-40 haplotype associated with the 3.7 kb α-thalassemia deletion (-α3.7del) in the Portuguese population, and determine its ancestry and influence on patients' hematological phenotype. METHODS AND RESULTS: We selected 111 Portuguese individuals previously analyzed by Gap-PCR to detect the presence of the -α3.7del: 50 without the -α3.7del, 34 heterozygous and 27 homozygous for the -α3.7del. The HS-40 region was amplified by PCR followed by Sanger sequencing. Four HS-40 haplotypes were found (A to D). The distribution of HS-40 haplotypes and genotypes are significantly different between individuals with and without the -α3.7del, being haplotype D and genotype AD the most prevalent in patients with this deletion in homozygosity. Furthermore, multiple correspondence analysis revealed that individuals without the -α3.7del are grouped with other European populations, while samples with the -α3.7del are separated from these and found more closely related to the African population. CONCLUSION: This study revealed for the first time an association of the HS-40 haplotype D with the -α3.7del in the Portuguese population, and its likely African ancestry. These results may have clinical importance as in vitro analysis of haplotype D showed a decrease in its enhancer activity on α-globin gene.

Association of ZBTB38 gene polymorphism (rs724016) with height and fetal hemoglobin in individuals with sickle cell anemia.

Objectives: Our study evaluated the association of the polymorphism rs724016 in the ZBTB38 gene, previously associated with height in other populations, with predictors of height, clinical outcomes, and laboratory parameters in sickle cell anemia (SCA). Methods: Cross-sectional study with individuals with SCA and aged between 3 and 20 years. Clinical, laboratory, molecular, and bone age (BA) data were evaluated. Levels of IGF-1 and IGFBP-3 were adjusted for BA, target height (TH) was calculated as the mean parental height standard deviation score (SDS), and predicted adult height (PAH) SDS was calculated using BA. Results: We evaluated 80 individuals with SCA. The homozygous genotype of the G allele of rs724016 was associated with a lower height SDS (p < 0.001) and, in a additive genetic model, was negatively associated with HbF levels (p = 0.016). Lower adjusted IGF-1 levels were associated with co-inheritance of alpha-thalassemia and with the absence of HU therapy. Elevated HbF levels were associated with a lower deficit in adjusted growth potential (TH minus PAH). Conclusion: Our analysis shows that SNP rs724016 in the ZBTB38 is associated with shorter height and lower HbF levels, an important modifier of SCA.

Third-generation sequencing identified a novel complex variant in a patient with rare alpha-thalassemia.

BACKGROUND: Thalassemias represent some of the most common monogenic diseases worldwide and are caused by variations in human hemoglobin genes which disrupt the balance of synthesis between the alpha and beta globin chains. Thalassemia gene detection technology is the gold standard to achieve accurate detection of thalassemia, but in clinical practice, most of the tests are only for common genotypes, which can easily lead to missing or misdiagnosis of rare thalassemia genotypes. CASE PRESENTATION: We present the case of an 18-year-old Chinese female with abnormal values of routine hematological indices who was admitted for genetic screening for thalassemia. Genomic DNA was extracted and used for the genetic assays. Gap polymerase chain reaction and agarose gel electrophoresis were performed to detect HBA gene deletions, while PCR-reverse dot blot hybridization was used to detect point mutations in the HBA and HBB genes. Next-generation sequencing and third-generation sequencing (TGS) were used to identify known and potentially novel genotypes of thalassemia. We identified a novel complex variant αHb WestmeadαHb Westmeadαanti3.7/-α3.7 in a patient with rare alpha-thalassemia. CONCLUSIONS: Our study identified a novel complex variant that expands the thalassemia gene variants spectrum. Meanwhile, the study suggests that TGS could effectively improve the specificity of thalassemia gene detection, and has promising potential for the discovery of novel thalassemia genotypes, which could also improve the accuracy of genetic counseling. Couples who are thalassemia carriers have the opportunity to reduce their risk of having a child with thalassemia.

Detection of 13 Novel Variants and Investigation of Mutation Distribution by Next Generation Sequencing in Hemoglobinopathies: A Single Center Experience.

Hemoglobinopathies are the most common monogenic disorders in the world. Traditional diagnostic algorithms generated by conventional methods for thalassemia can be labor-intensive and time-consuming due to the complexities of the genes involved and the variability in disease-causing mutations. With the advantages of next-generation sequencing (NGS) technology, molecular analysis of highly complex diseases such as hemoglobinopathies has become easier. Next-generation sequencing is a highly sensitive and effective method due to its capacity to sequence many gene regions simultaneously while allowing good read depths. In this study, single nucleotide changes, small deletions and copy number variations in HBA1, HBA2 and HBB in 914 patients with suspected hemoglobinopathy were analysed with NGS. At least one HBA1, HBA2, HBB or HBD variant was detected in 483 (52.8%) patients. Ten novel variants were detected in HBA1 and HBA2, three in HBB, and one in HBD. From these variants, c.*76T > A, c.301-24 G > A, c.301-24G > C c.-41C > G, c.-37-40C > G, c.-9G > C, c. 95 + 9C > T, c.95 + 26C > A, c.95 + 38C > T and c.*18C > G variants were located in α-globin genes, c.-25T > C, c.*103T > C and c92 + 39A > G variants were located in ß-globin genes, and c.-43C > A was located in HBD. This is the first comprehensive study using NGS for the molecular diagnosis of hemoglobinopathies in Turkey. Accurate molecular diagnosis is of critical importance in hemoglobinopathies which are a public health problem due to their increased prevalence, high burden to society, and lack of curative treatment. Currently, NGS appears to be an advanced option over conventional methods to detect all variants occurring by molecular mechanisms and simultaneously analyse many genomic sequences.

Severe Transfusion-Dependent Thalassemia in Compound Heterozygote Palestinian Siblings with Two α-Globin Gene Defects, Hb Taybe D HBA1: C.119_121delCCA Mutation and HBA2: C.*94A > G Mutation.

Alpha and Beta Thalassemia are autosomal recessive anemias that cause significant morbidity and mortality worldwide, especially in the Middle East and North Africa (MENA) region where carrier rates reach up to 50%. We report the case of two siblings of Palestinian origin born who presented to our tertiary healthcare center for the management of severe transfusion dependent hemolytic anemia. Before presentation to our center, the siblings were screened for a-thalassemia using the Alpha-globin StripAssay. They were found to carry the α2 polyA-1 [AATAAA > AATAAG] mutation in the heterozygous form, which was insufficient to make a diagnosis. No pathogenic variants were detected on Sanger sequencing of the HBB gene. Full sequencing of the a-gene revealed compound heterozygous variants (HBA1:c.119_121delCCA and the previously detected HBA2:c.*+94A > G Poly A [A->G]) with trans inheritance. This report highlights the impact of non-deletional mutations on α-globin chain stability. The compound heterozygosity of a rare α-globin chain pathogenic variant with a polyadenylation mutation in the probands leads to clinically severe a-thalassemia. Due to the high carrier status, the identification of rare mutations through routine screening techniques in our populations may be insufficient. Ongoing collaboration among hematologists, medical geneticists, and counselors is crucial for phenotypic-genotypic correlation and assessment of adequate genetic testing schemes.

Idiopathic Thrombocytosis in Alpha Thalassemia Trait Patient.

Platelet count increases are typically a reactionary response to one of a variety of pathophysiological events. We present here a case of microcytic hypochromic red blood cells and thrombocytosis in an adolescent female that we have monitored for three years. The patient was positive for alpha thalassemia trait; negative for mutation in Janus kinase 2, calreticulin, or myeloproliferative leukemia virus oncogene; and negative for reactive causes of thrombocytosis. Noticeably, a variant in atypical chemokine receptor 1 (ACKR1) (c.-67T>C, rs2814778) was found to be homozygous. Accordingly, the case was diagnosed as idiopathic thrombocytosis, and treatment was given to restore platelet levels to normal. Our findings highlight the possibility of an unknown association between alpha thalassemia trait and idiopathic thrombocytosis in the presence of ACKR1 mutation, which could be implicated in disease pathogenesis.

Algorithmic approach utilizing histology and immunohistochemistry for the current classification of diffuse glioma.

INTRODUCTION: Diffuse glioma constitutes 28% of primary brain tumors. Until recently morphologic appearance was the only criterion for classifying these tumors. However, WHO 2016 incorporates molecular information in the primary diagnosis of gliomas such as Isocitrate dehydrogenase 1 (IDH1), Alpha thalassemia/mental retardation syndrome X inked (ATRX) as well as 1p/19q codeletion on FISH. In a resource-limited setup where FISH is not available, Alpha internexin (INA) has been suggested as a surrogate IHC marker. MATERIAL AND METHODS: Cross-sectional study conducted in the Department of Pathology for two years. Tissue blocks and clinical as well as radiological details were obtained from departmental archives. After assessing the morphologic details, routine IHC markers such as GFAP, Ki67 and P53 along with molecular markers like IDH-1, ATRX, and lNA were applied. RESULTS: Out of 55 cases of diffuse glioma, 23 cases of astrocytoma and 32 cases of oligodendroglioma with an overall mean age of presentation of 41.49 ± 12.47 years. IDH-1 expression among diffuse glioma was 89.1% in our study. Alteration in the ATRX gene expression was observed in 95.7% of astrocytomas. 75% of oligodendrogliomas expressed INA with no significant difference in expression between the two grades. Based on the algorithmic approach using molecular surrogate markers, diffuse gliomas were categorized into six distinct groups. IDH-mutant, ATRX loss of expression astrocytoma and IDH-mutant, INA positive oligodendroglioma are two categories that do not require further molecular testing. This comprises 72.7% of the cases and these do not warrant further workup. CONCLUSION: Implementation of combined phenotypic-genotypic diagnosis with the use of histomorphology and immunohistochemical surrogates for molecular genetic alterations will yield more homogeneous and narrowly defined diagnostic entities which will provide better prognostication and definitive treatment. It also is cost-effective in a resource-limited setup.

Clinical, laboratory, and molecular characteristics of a cohort of children with hemoglobinopathy S/beta-thalassemia.

INTRODUCTION: Hemoglobinopathy Sß-thalassemia (HbSß-thal) has a wide range of clinical and laboratory severity. There is limited information on the natural history of HbSß-thal and its modulating factors. We described the molecular, hematological, and clinical characteristics of a cohort of children with HbSß-thal and estimated its incidence in Minas Gerais, Brazil. METHODS: Laboratory and clinical data were retrieved from medical records. Molecular analysis was performed by HBB gene sequencing, PCR-RFLP, gap-PCR, and MLPA. RESULTS: Eighty-nine children were included in the study. Fourteen alleles of ß-thal mutations were identified. The incidence of HbSß-thal in the state was 1 per 22,250 newborns. The most common ßS-haplotypes were CAR and Benin. The most frequent ßthal-haplotypes were V, II, and I. Coexistence of 3.7 kb HBA1/HBA2 deletion was present in 21.3 % of children. ß-thalassemia mutations were associated with several clinical and laboratory features. In general, the incidence of clinical events per 100 patient-years was similar for children with HbSß0-thal, IVS-I-5 G>A, and IVS-I-110 G>A. Children with HbSß+-intermediate phenotypes had a more severe laboratory and clinical profile when compared with those with HbSß+-mild ones. ßS-haplotypes and α-thalassemia did not meaningfully influence the phenotype of children with HbSß-thal. CONCLUSION: The early identification of ß-thalassemia alleles may help the clinical management of these children.

Αlpha-thalassemia: A practical overview.

α-Thalassemia is an inherited blood disorder characterized by decreased synthesis of α-globin chains that results in an imbalance of α and ß globin and thus varying degrees of ineffective erythropoiesis, decreased red blood cell (RBC) survival, chronic hemolytic anemia, and subsequent comorbidities. Clinical presentation varies depending on the genotype, ranging from a silent or mild carrier state to severe, transfusion-dependent or lethal disease. Management of patients with α-thalassemia is primarily supportive, addressing either symptoms (eg, RBC transfusions for anemia), complications of the disease, or its transfusion-dependence (eg, chelation therapy for iron overload). Several novel therapies are also in development, including curative gene manipulation techniques and disease modifying agents that target ineffective erythropoiesis and chronic hemolytic anemia. This review of α-thalassemia and its various manifestations provides practical information for clinicians who practice beyond those regions where it is found with high frequency.

CETP gene polymorphisms and haplotypes are explanatory variables for HDL cholesterol level in sickle cell disease

Variations in lipid profile have been observed in sickle cell disease (SCD) and understanding their relationship with disease severity is crucial. This study aimed to investigate the association of polymorphisms of the CETP gene and laboratory markers of disease severity with lipid profile in a pediatric population with SCD. Biochemical and anthropometric analyses and CETP and alpha-thalassemia genotyping were performed. The study included 133 children and adolescents with sickle cell anemia (SCA) or hemoglobin SC disease (SCC), in steady-state. The SCA and no hydroxyurea (no HU) groups had higher values of ApoB, total cholesterol, low-density lipoprotein cholesterol (LDL-C), and non-high-density lipoprotein cholesterol (non-HDL-C) compared to the SCC and HU groups. However, there were no significant differences in ApoA1 and HDL-C levels between the groups based on genotype. Furthermore, the groups with altered levels of ApoA1, HDL-C, and the triglyceride/HDL ratio exhibited lower hemoglobin (Hb) levels and higher white blood cell counts. Hb level was associated to HDL-C levels. Analysis of CETP gene variants showed that the minor alleles of rs3764261 (C>A), rs247616 (C>T), and rs183130 (C>T), as well as the TTA haplotype, are explanatory variables for HDL-C levels. These findings suggested that dyslipidemia in SCD, specifically related to HDL-C levels, may be influenced by individual genetic background. Additionally, further investigation is needed to determine if clinical manifestations are impacted by CETP gene variants.

Random forest classifiers trained on simulated data enable accurate short read-based genotyping of structural variants in the alpha globin region at Chr16p13.3.

In regions where reads don't align well to a reference, it is generally difficult to characterize structural variation using short read sequencing. Here, we utilize machine learning classifiers and short sequence reads to genotype structural variants in the alpha globin locus on chromosome 16, a medically-relevant region that is challenging to genotype in individuals. Using models trained only with simulated data, we accurately genotype two hard-to-distinguish deletions in two separate human cohorts. Furthermore, population allele frequencies produced by our methods across a wide set of ancestries agree more closely with previously-determined frequencies than those obtained using currently available genotyping software.

Molecular patterns of alpha-thalassemia in the kingdom of Saudi Arabia: identification of prevalent genotypes and regions with high incidence.

BACKGROUND: Alpha-thalassemia (α-thalassemia) is one of the most common monogenic diseases in Saudi Arabia and is associated with significant morbidity. Premarital testing programs in Saudi Arabia reduce the burden of hemoglobinopathy disorders, and ongoing monitoring is required. We aimed to explore the molecular nature of α-globin genes and identify the most common genotypes and regions with a high risk of α-thalassemia in Saudi Arabia. METHODS: This retrospective study was conducted between January 2021 and December 2022. Six hundred twenty-five samples from patients with microcytic hypochromic anemia in Saudi Arabia were analyzed using reverse dot blot hybridization (RDBH)-based multiplex-PCR, which screens for the known 21 mutations of α-globin genes. RESULTS: Seven mutations in the α-globin gene were identified in 88.96% (556) patients. The most frequent abnormality of a-globin genes was -α3.7 (62.3%), followed by α2IVS1(-5nt) (20.7%) and α2 polyA-1 (α2T.Saudi) (14.1%). Interestingly, α2 polyA-2 (α2T.Turkish) was identified in Saudi and presented with -MED, causing Haemoglobin H disease. The incidence of α-thalassemia in Saudi Arabia's cities showed significant differences (P = 0.004). Jeddah City had the highest percentage of cases (25%), followed by Makkah (23%), Taif (13.3%), and Al-Ahassa (12.4%). CONCLUSION: The study provides current knowledge about the molecular nature of α- thalassemia, highlights the common genotypes that could contribute to disease occurrence in the Saudi population, and sheds light on Saudi regions with a high incidence. It also recommends further studies in a larger population and with differently composed molecular assays to verify these findings.

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.

Development of a Quantitative Multiplex PCR to Detect Three Common Alpha Thalassemia Deletions.

Alpha thalassemia is an autosomal recessive genetic disorder with a high prevalence in the Middle East. The severe form of alpha-thalassemia is incompatible with life and can cause significant obstetric complications in the mother. Therefore, it is important to determine the genotype in parents who have a chance of having a fetus with one of the severe forms of this disease. A total of 112 samples that were previously analyzed for common alpha thalassemia mutations in Iran were used in this study. A new multiplex PCR including quantitative polymerase chain reaction to amplify the homologous regions of the alpha-globin gene cluster and fluorescent gap PCR was designed to identify -α3.7, -α4.2, --MED deletions. The ROC curve was used to determine the optimum cutoff points. Statistical analysis showed that there is a significant difference between the peak height ratios for different genotypes. The peak corresponding to the 297 bp fragment resulting from the amplification of the allele with MED-I deletion was detected in all the samples with this deletion. Different cutoffs for a range of sensitivities and specificities were determined by the ROC curve. The suggested method can identify three common large deletions in the alpha-globin gene cluster. A study with a larger sample size can provide more accurate information about the sensitivity and specificity of this test.

Alpha globin gene copy number and incident ischemic stroke risk among Black Americans.

Introduction: People with African ancestry have greater stroke risk and greater heritability of stroke risk than people of other ancestries. Given the importance of nitric oxide (NO) in stroke, and recent evidence that alpha globin restricts nitric oxide release from vascular endothelial cells, we hypothesized that alpha globin gene (HBA) deletion would be associated with reduced risk of incident ischemic stroke. Methods: We evaluated 8,947 participants self-reporting African ancestry in the national, prospective Reasons for Geographic And Racial Differences in Stroke (REGARDS) cohort. Incident ischemic stroke was defined as non-hemorrhagic stroke with focal neurological deficit lasting ≥ 24 hours confirmed by the medical record or focal or non-focal neurological deficit with positive imaging confirmed with medical records. Genomic DNA was analyzed using droplet digital PCR to determine HBA copy number. Multivariable Cox proportional hazards regression was used to estimate the hazard ratio (HR) of HBA copy number on time to first ischemic stroke. Results: Four-hundred seventy-nine (5.3%) participants had an incident ischemic stroke over a median (IQR) of 11.0 (5.7, 14.0) years' follow-up. HBA copy number ranged from 2 to 6: 368 (4%) -α/-α, 2,480 (28%) -α/αα, 6,014 (67%) αα/αα, 83 (1%) ααα/αα and 2 (<1%) ααα/ααα. The adjusted HR of ischemic stroke with HBA copy number was 1.04; 95%CI 0.89, 1.21; p = 0.66. Conclusions: Although a reduction in HBA copy number is expected to increase endothelial nitric oxide signaling in the human vascular endothelium, HBA copy number was not associated with incident ischemic stroke in this large cohort of Black Americans.

Concomitant Presence of Hb Agrinio and - -Med Deletion in a Greek Male Patient with Hemoglobinopathy H: More Severe Phenotype and Literature Review.

Hemoglobin (Hb) Agrinio is a rare non-deletional a-globin mutation observed almost exclusively in Greek, Spanish or other Mediterranean families. The clinical manifestations of a carrier of a single Hb Agrinio mutation (single heterozygosity) depend on the concomitant presence or absence of other mutations or variants in the beta, alpha or other modifying genes. We present a Greek patient harboring a Hb Agrinio variant plus the - -Med alpha deletional allele, having an infrequent severe form of alpha thalassemia, in contrast to the typical alpha thalassemic patient and requiring regular red blood cell (RBC) transfusions and chelation treatment. We also provide a concise literature review regarding alpha thalassemic hemoglobin variants and their molecular and clinical combinations. A phase 2, double-blind, randomized, placebo-controlled, multicenter clinical trial to determine the efficacy and safety of luspatercept (BMS-986346/ACE-536) for the treatment of anemia in adults with alpha thalassemia with the participation of our center is currently recruiting patients (NCT05664737).