Hemoglobin Shaare Zedek (Hb SZ) is a rare structural α-Hb variant. Characterizing its genotype-phenotype relationship and genetic origin enhances diagnostic and clinical management insights. We studied a proband and six family members using high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), PCR, and sequencing to analyze α- and β-globin genes and α-globin haplotypes. Pathogenicity predictions and a rapid diagnostic method were developed. The proband, his father, grandfather, and aunt had Hb migrating to the HbH-zone on CE and elevated fetal hemoglobin (HbF) on HPLC. Direct sequencing identified an A to G mutation at codon 56 of the α2-globin gene, characteristic of Hb SZ. Additionally, the proband carried a β-globin gene mutation [HBB.52A>T]. Mild thalassemia-like changes were observed in the proband, whereas individuals with only the Hb SZ variant did not exhibit these changes. Pathogenicity predictions indicated that Hb SZ is benign. The variant can be identified using restriction fragment length polymorphism (RFLP) and allele-specific PCR. The Thai variant of Hb SZ is associated with the haplotype [- - M - - - -]. Hb SZ is a non-pathological variant that minimally affects red blood cell parameters, even when it coexists with β0-thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis.

UNASSIGNED: This study aimed to evaluate the efficacy of α-thalassemia gene testing as a part of an antenatal intervention program over a 10-year period.
UNASSIGNED: All patients underwent α-thalassemia gene testing, which included the analysis of three types of deletions and mutations. Rare α-thalassemia gene testing was performed using Sanger sequencing, multiplex ligation-dependent probe amplification, and sequencing techniques. Prenatal diagnosis was performed in high-risk couples using chorionic villus sampling or amniocentesis.
UNASSIGNED: From 2010 to 2019, among the 91,852 patients examined, α-thalassemia mutations were identified in 41.78% of patients. The most frequent α0 gene mutation was--SEA, followed by--THAI. Two rare α0-thalassemia gene mutations at --32.8 and --230, were also observed. A total of 2,235 high-risk couples were identified, of which 562 were affected, including three with the--SEA/--THAI genotype and one with the--SEA/--230 genotype. Additionally, prenatal diagnosis revealed four cases of fetal anemia and/or mild edema, along with two cases of severe fetal edema. Chromosome and gene chip results were normal. Thalassemia gene testing showed an αCSα/αCSα genotype in four patients with anemia and/or mild edema, while two patients with severe fetal edema had one--SEA/αCSα genotype and one--SEA/--GX genotype. Using the cut-off points of 74.6 fL and 24.4 pg as criteria for identifying α0-thalassemia carriers and HbH disease, the detection rate of missed diagnoses in high-risk couples is consistent with national guidelines for standards, potentially saving 10,217,700 ¥.
UNASSIGNED: Routine molecular testing for α-thalassemia in high-risk prenatal populations effectively prevented severe α-thalassemia births. Despite the high cost, the cutoff points proposed by this study suggest that implementing screening using a new parameter has the potential to reduce current expenses.

UNASSIGNED: Thalassemia is a severe hereditary blood disorder that poses a significant threat to human health and leads to mortality and disability. It is one of the most prevalent monogenic diseases worldwide. The aim of this study was to analyze the molecular epidemiological data of individuals of childbearing age from the Han ethnic group with thalassemia in Southwest China and to explore the application of next-generation sequencing (NGS) technology in screening thalassemia carriers.
UNASSIGNED: The participants were Han males and females of childbearing age who sought medical advice at the West China Second University Hospital, Sichuan University from June 2022 to June 2023. We detected α- and β-thalassemia mutations using full-length capture of the thalassemia genes and NGS technology.
UNASSIGNED: In a cohort of 1,093 participants, 130 thalassemia carriers were identified, with an overall detection rate of 11.89% (130/1,093). Among these, 0.91% (10/1,093) had mutations that could not be detected using traditional PCR techniques. The proportions of carriers with α-, β-, and α-complexed β-thalassemia gene mutations were 7.68% (84/1,093), 3.93% (43/1,093), and 0.27% (3/1,093), respectively. We identified a novel HBA2 c.166del variant that has not been previously reported.
UNASSIGNED: Using NGS technology, we found that the mutation-carrying rate of thalassemia genes was 11.89% in the Han population of childbearing age in Southwest China. Compared with the results of traditional PCR techniques, NGS detected an additional 0.91% (10/1,093) rare genetic variants. NGS technology should be utilized as the primary screening method for thalassemia carriers among Han nationality people of childbearing age in Southwest China.

Thalassemias are a group of inherited blood disorders that affects 5-7% of the world population. Comprehensive screening strategies are essential for the management and prevention of this disorder. Today, many clinical and research laboratories have widely utilized next-generation sequencing (NGS) technologies to identify diseases, from germline and somatic disorders to infectious diseases. Yet, NGS application in thalassemia is limited and has just recently surfaced due to current demands in seeking alternative DNA screening tools that are more efficient, versatile, and cost-effective. This review aims to understand the several aspects of NGS technology, including its most current and expanding uses, advantages, and limitations, along with the issues and solutions related to its integration into routine screening and diagnosis of thalassemias. Hitherto, NGS has been a groundbreaking technology that offers tremendous improvements as a diagnostic tool for thalassemia in terms of its higher throughput, accuracy, and adaptability. The superiority of NGS in detecting rare variants, solving complex hematological problems, and providing non-invasive alternatives to neonatal diagnosis cannot be overlooked. However, several pitfalls still preclude its use as a stand-alone technique over conventional methods.

Moyamoya syndrome (MMS) refers to the moyamoya vascular disease associated with various systemic diseases and conditions, including sickle cell anemia, Fanconi anemia and iron deficiency anemia. However, the association between MMS and other hemoglobinopathies is less frequently observed. MMS, like moyamoya disease, is a cerebrovascular condition that is characterized by chronic progressive stenosis or occlusion at the ends of the bilateral internal carotid arteries, anterior cerebral arteries and the beginning of the middle cerebral arteries, and is secondary to the formation of an abnormal vascular network at the base of the skull. Patients with MMS are prone to thrombosis, aneurysm and bleeding. The present study reports the case of a 43-year-old man with α-thalassemia who presented with moyamoya vessels with a ruptured aneurysm bleeding into the ventricle. α-thalassemia is considered as an extremely rare but potential cause of MMS. Since MMS is a progressive disease, early diagnosis and treatment is vital to prevent the disease from worsening.

Carriers of α-thalassemia exhibit hypochromic microcytosis with mean corpuscular volume (MCV) < 80 fL, mean corpuscular hemoglobin (MCH) < 27 pg, and reduced hemoglobin A2 (HbA2). We studied the distribution and diagnostic efficiencies of these indicators and their combinations in patients with and without alpha-thalassemia. Based on genetic diagnosis, 10,883 participants were divided into alpha-thalassemia group (n = 1655) and negative-for-alpha-thalassemia group (n = 9228). Erythrocyte parameters and hemoglobin analysis of the groups were analyzed. Moreover, we compared the four screening schemes (MCV/MCH, MCV/MCH/HbA2, MCV + MCH, MCV + MCH + HbA2) to find the best for α-thalassemia screening. The genotypes of --SEA/αα, and -α3.7/αα are the most prevalent with 54.9% and 27.6% in Fujian Province, China. There were significant differences in the distribution of MCV, MCH, and HbA2 in the two groups. Among the three, MCH exhibited the highest sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy. Although the four screening schemes have their advantages, there are significant differences in their sensitivity and specificity. MCV + MCH had the best diagnostic performance (72.6% sensitivity, 89.0% specificity) as well as the highest Youden index (61.59%). Our results showed that MCH could be used to screen α-thalassemia instead of MCV and HbA2. However, it is recommended that MCV/MCH/HbA2 screening be used in areas with high α-thalassemia incidence to increased sensitivity.

OBJECTIVE: A co-inheritance of α0-thalassemia can ameliorate the clinical severity of the hemoglobin (Hb) E-β-thalassemia disease. This information should be provided at prenatal diagnosis. Identification of α0-thalassemia in an affected fetus is therefore valuable. We have explored this genetic interaction in a large cohort of affected fetuses with hemoglobin (Hb) E-β-thalassemia in northeast Thailand.
METHODS: A study was done retrospectively on 1,592 couples at risk of having fetuses with Hb E-β0-thalassemia, encountered from January 2011 to December 2019. A total of 415 left-over DNA specimens of the affected fetuses with Hb E-β0-thalassemia disease were further investigated. Examination of α0-thalassemia was done using gap-PCR or a multiplex PCR assay for simultaneous detection of Hb E and α0-thalassemia mutations.
RESULTS: Of the 415 affected fetuses, the two most common β0-thalassemia genes found were the codons 41/42 (-TTCT) (199/415; 48.0%) and codon 17 (A-T) (115/415; 27.7%). α0-thalassemia was found unexpectedly in 21 (5.1%) fetuses. Hematologic phenotypes of the parents indicated that it was impossible to differentiate a pure β0-thalassemia carrier from a double β0-thalassemia/α0-thalassemia heterozygote unless DNA analysis is performed. In contrast, a reduced level of Hb E in the Hb E carrier (<25%) is a valuable marker for predicting double heterozygosity for Hb E/α0-thalassemia. This could be further confirmed using a multiplex PCR assay.
CONCLUSIONS: There is a high prevalence of co-inheritance of α0-thalassemia in fetuses with Hb E-β0-thalassemia disease. In a high-risk population such as Thailand, we recommend screening for α0-thalassemia in all affected fetuses with Hb E-β0-thalassemia disease and providing complete genetic information to the parents to make appropriate decisions at prenatal diagnosis and genetic counseling.

OBJECTIVE: To analyze genetic mutations in a Chinese pedigree affected with Alpha-thalassemia X-linked intellectual disability syndrome, providing a precise diagnosis and genetic counseling.
METHODS: Clinical data was collected. A novel alternative splicing variant detected by whole-exome sequencing was validated by Sanger sequencing. The functional effect of the mutation was predicted with Mutation Tasting. The analysis of 5\' splice site score was estimated with MaxEntScan. Changes in amino acid sequencing were predicted with Mutalyzer. The tertiary structures of the wild type and mutation-carrying protein were predicted by I-TASSER. RNA was extracted from peripheral blood lymphocytes from the proband, his mother and a healthy control. Quantitative Real-Time PCR was used to detect mRNA expression.
RESULTS: The proband presented with severe intellectual disability, developmental delay, characteristic facies, seizures and cryptorchidism. A novel hemizygous duplication mutation in the ATRX gene in a splice site between exons 3 and 4, NM_000489: c.189+1dupG, was identified with WES in the proband. Sanger sequencing confirmed that the mutation was inherited from his mother, who carried a heterozygous mutation, while his father was not affected. Bioinformatics analysis indicated that the splicing region where the mutation was located is highly conserved and the variant was damaging, producing a truncated protein due to the premature translation of a stop codon. Sanger sequencing with the Quantitative Real-Time PCR product containing a G base inserted between bases 189 and 190. The level of mRNA expression showed that ATRX gene transcription decreased due to the mutation (P < 0.05).
CONCLUSIONS: A novel mutation in ATRX was found in this pedigree and was confirmed to be pathogenic through functional studies. Our research expanded the spectrum of ATRX gene mutations, providing a precise diagnosis and a basis for genetic counseling.

Thalassemia is one of the major inherited haematological disorders in the Southeast Asia region. This study explored the potential utility of red blood cell (RBC) parameters and reticulocyte cell population data (CPD) parameters in the differential diagnosis of α and β-thalassaemia traits as a rapid and cost-effective tool for screening of thalassemia traits. In this study, a total of 1597 subjects (1394 apparently healthy subjects, 155 subjects with α-thalassaemia trait, and 48 subjects with β-thalassaemia trait) were accrued. The parameters studied were the RBC parameters and reticulocyte CPD parameters derived from Unicel DxH800. A novel algorithm named αβ-algorithm was developed: (MN-LMALS-RET × RDW) - MCH) to discriminate α from β-thalassaemia trait with a cut-off value of 1742.5 [AUC = 0.966, sensitivity = 92%, specificity = 90%, 95% CI = 0.94-0.99]. Two prospective studies were carried: an in-house cohort to assess the specificity of this algorithm in 310 samples comprising various RBC disorders and in an interlaboratory cohort of 65 α-thalassemia trait, and 30 β-thalassaemia trait subjects to assess the reproducibility of the findings. We propose the αβ-algorithm to serve as a rapid, inexpensive surrogate evaluation tool of α and β-thalassaemia in the population screening of thalassemia traits in geographic regions with a high burden of these inherited blood disorders.

An insertion or deletion of a nucleotide (nt) in the penultimate or the last exon can result in a frameshift and premature termination codon (PTC), giving rise to an unstable protein variant, showing a dominant phenotype. We described two α-globin mutants created by the deletion of a nucleotide in the penultimate or the last exon of the α1-globin gene: the Hb Campania or α1 cod95 (-C), causing a frameshift resulting in a PTC at codon 102, and the Hb Sciacca or α1 cod109 (-C), causing a frameshift and formation of a PTC at codon 133. The carriers showed α-thalassemia alterations (mild microcytosis with normal Hb A2) and lacked hemoglobin variants. The 3D model indicated the α-chain variants\' instability, due to the severe structural alterations with impairment of the chaperone alpha-hemoglobin stabilizing protein (AHSP) interaction. The qualitative and semiquantitative analyses of the α1mRNA from the reticulocytes of carriers highlighted a reduction in the variant cDNAs that constituted 34% (Hb Campania) and 15% (Hb Sciacca) of the total α1-globin cDNA, respectively. We developed a workflow for the in silico analysis of mechanisms triggering no-go decay, and its results suggested that the reduction in the variant mRNA was likely due to no-go decay caused by the presence of a rare triplet, and, in the case of Hb Sciacca, also by the mRNA\'s secondary structure variation. It would be interesting to correlate the phenotype with the quantity of other frameshift mRNA variants, but very few data concerning α- and β-globin variants are available.