Alpha-globin Research Articles

GC-MS aided In silico molecular docking studies on the anti-sickling properties of Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler stem bark

Zanthoxylum zanthoxyloides is an important medicinal herb in African traditional medicine. It has been used to treat an array of pathological conditions, most notably is sickle cell anaemia, one of the most important genetic haematological conditions affecting individuals of African descent. This study identifies and evaluates Zanthoxylum zanthoxyloides bioactives with potential haemoglobin oxygen-affinity modulatory activity. Phytochemical constituents identified via gas chromatography-mass spectrometry of a 70% ethanolic stem bark extract of Zanthoxylum zanthoxyloides (Lam.) Zepern. and Timler were analyzed for their in silico anti-sickling activity. Gas chromatography-mass spectrometry aided in silico molecular docking studies identified the presence of two compounds, 7-(dimethylamino)-2,3-dihydro-1H cyclopenta[c]chromen-4-one and (8S,9S,10R,13S,14S,16R,17S)-17-acetyl-6,10,13,16-tetramethyl-8,9,11,12,14,15,16,17-octahydrocyclopenta[a]phenanthren-3-one, with a strong binding affinity for the active pocket located at the N-terminal region (valine 1) of the alpha globin subunit of haemoglobin S. Further analysis of the binding pose of the ligands demonstrated the formation of favourable interactions including hydrogen bonding, π-cation, van der Waals and hydrophobic contacts. Computational investigations on the pharmacokinetic profile of 7-(dimethylamino)-2,3-dihydro-1H cyclopenta[c]chromen-4-one and (8S,9S,10R,13S,14S,16R,17S)-17-acetyl-6,10,13,16-tetramethyl-8,9,11,12,14,15,16,17-octahydrocyclopenta[a]phenanthren-3-one reveals a favourable oral bioavailability and drug-likeness profile. The findings add to the evidence bolstering the role of Zanthoxylum zanthoxyloides in the management of sickle cell anemia.

Activin Receptor-Ligand Trap for the Treatment of β-thalassemia: A Serendipitous Discovery.

β-thalassemia is a hereditary disorder caused by defective production of β-globin chains of hemoglobin (Hb) that leads to an increased α/β globins ratio with subsequent free α-globins. Alpha globin excess causes oxidative stress, red blood cells membrane damage, premature death of late-stage erythroid precursors, resulting in ineffective erythropoiesis.The transforming growth factor β (TGF-β) superfamily signaling acts on biological processes, such as cell quiescence, apoptosis, proliferation, differentiation, and migration, and plays an essential role in regulating the hematopoiesis. This pathway can lose its physiologic regulation in pathologic conditions, leading to anemia and ineffective erythropoiesis. Activin receptor-ligand trap molecules such as Sotatercept and Luspatercept downregulate the TGF-β pathway, thus inhibiting the Smad2/3 cascade and alleviating anemia in patients with β-thalassemia and myelodysplastic syndromes.In this review, we describe in extenso the TGF-β pathway, as well as the molecular and biological basis of activin receptors ligand traps, focusing on their role in various β-thalassemia experimental models. The most recent results from clinical trials on sotatercept and luspatercept will also be reviewed.

δ-Globin Chain Variants Associated with Decreased Hb A2 Levels: A National Reference Laboratory Experience

High prevalence of hemoglobin (Hb) disorders mandates national programs for screening and genetic counseling in many countries. Increased Hb A2 levels are commonly associated with β-thalassemias, however, various disorders including alteration of δ chains may result in decreased production of Hb A2, thus hindering the diagnosis of β-thalassemias. The reported data reflect the experience of a large reference laboratory in the United States. In the current study, we have attempted to assess the prevalence and also tried to characterize the identified mutations in the HBD gene resulting in decreased Hb A2 levels. In our cohort, 1.6% of 6486 patients were found to have Hb A2 values of <1.9%. Bidirectional sequencing of the HBD gene demonstrated mutations in 20 cases (19.0% of the individuals with decreased Hb A2). In addition to the previously reported variants, one novel mutation (Hb A2-Utah or HBD: c.46T>C).

Heterozygosity for the Novel HBA2: c.*91_*92delTA Polyadenylation Site Variant on the α2-Globin Gene Expanding the Genetic Spectrum of α-Thalassemia in Iran

There are four copy numbers of α-globin genes (16p13.3) in the human genome and the number of defective α-globin genes dictates the severity of α-thalassemia (α-thal). Mutations that occur in the 3′ untranslated region (3′UTR), and especially at the polyadenylation (polyA) sites, affect the translation, stability and export of mRNA. A patient with hypochromic microcytic anemia was referred to the Kariminejad-Najmabadi Pathology & Genetics Center, Tehran, Iran by the health network. Molecular analysis of genomic DNA for the evaluation of mutations on the α- and β-globin genes was performed. Direct sequencing of the hemoglobin (Hb) subunit α2 (HBA2) gene revealed a two nucleotide deletion between +816 and +817 in the 3′UTR, located at the polyA site, which seems to be a novel pathogenic variant. This novel variant expands the genetic spectrum of α-thal in the 3′UTR of the HBA2 gene.

Molecular Genetic Analysis of α-Thalassemia in Hamadan Province, West Iran

Identifying couples who are carriers of thalassemia-causing mutations, followed by prenatal diagnosis (PND), is undoubtedly an effective way to prevent the birth of children with the disease. Our aim in this study was to report for the first time the spectrum of α-globin gene mutations in the population living in Hamadan Province, West Iran. Multiplex gap-polymerase chain reaction (gap-PCR), amplification refractory mutation system (ARMS)-PCR, and direct DNA sequencing of HBA1 and HBA2 genes were used to identify the α-thalassemia (α-thal)-causing mutations in a cohort of 389 individuals including 328 α-thal carriers and 61 normal subjects. A total of 17 different mutations and 25 different genotypes were detected. The –α3.7 (rightward) deletion (NG_000006.1: g.34164_37967del3804) was the most frequent mutation, accounting for more than half of all mutations (61.04%). This study revealed that there is a variety of α-thal mutations and α-thal genotypes in Hamadan Province, West Iran. This observation is probably due to the complexity of the Hamadan Province population that is composed of Persians (Fars), Turks, Kurds, and Lurs/Laks. In conclusion, our results demonstrated the spectrum of mutations in α-globin genes in Iran and increased our understanding of their distribution in this country.

Molecular Characterization of β- and α-Globin Gene Mutations in Individuals with Borderline Hb A2 Levels

Elevated Hb A2 level (≥4.0%) is considered to be reliable parameter to identify β-thalassemia (β-thal) carriers. However, some β-thal carriers have been misdiagnosed as their Hb A2 levels are below 4.0%. In addition, coinheritance of α-thalassemia (α-thal) and β-thal might affect Hb A2 levels. Therefore, the aim of this study was to investigate the mutations of β- and α-globin genes in individuals with borderline Hb A2 levels in Thailand. Three hundred samples from individuals with Hb A2 levels of 3.5–3.9% were collected for molecular diagnosis of β-globin gene mutations. In addition, the α0-thal, α+-thal, Hb Constant Spring (Hb CS, HBA2: c.427T>C), and Hb Paksé (HBA2: c.429A>T) diagnostics were also performed. Sixteen samples (5.33%) had β-globin gene mutations, and codon 41/42 (–TTCT) (HBB: c.126_129delCTTT) was the most prevalent mutation. Ninety-eight samples (32.67%) had α-globin gene mutations including four Hb H (β4)-Hb CS disease, two Hb H disease, 13 heterozygous α0-thal, 11 homozygous α+-thal, two α+-thal/Hb CS, one α+-thal/Hb Paksé, 61 heterozygous α+-thal, and four Hb CS. Furthermore, seven cases of β-thal carriers coinheriting α-thal were observed, and five of them carried Hb H disease. High prevalence of both α- and β-thal in subjects with borderline Hb A2 levels suggested that molecular diagnosis of α- and β-thal should be performed, especially in a high prevalence area of thalasssemia carriers, for accurate diagnosis and genetic counseling to prevent and control new severe thalassemia cases. Moreover, β-thal carriers who coinherited Hb H disease might have reduced Hb A2 levels, leading to a misdiagnosis of β-thal in analysis programs.

Association of Hb Shenyang [α26(B7)Ala→Glu, GCG>GAG, HBA2: c.80C>A (or HBA1)] with Several Types of α-Thalassemia in Thailand

Hb Shenyang [α26(B7)Ala→Glu, HBA2: c.80C>A (or HBA1)] is a rare α chain variant. Its genotype-phenotype relationship and origin have not been described in Thailand before. Three Thai subjects (P1–P3) carrying this variant were studied. Hemoglobin (Hb) analysis was performed by capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) as well as molecular characterization using appropriate polymerase chain reaction (PCR) techniques and DNA sequencing. Hemoglobin analysis by HPLC revealed fast-moving abnormal peaks at a retention time (RT) of 1.59–1.62 min., while CE revealed a fast-moving abnormal Hb at zone 12 and ahead of Hb A2 in three subjects. DNA analysis revealed a C>A transition at codon 26 of the α2-globin gene glutamic acid to replace alanine, corresponding to Hb Shenyang. The Southeast Asian [– –SEA α-thalassemia-1 (α-thal-1)] deletion was also identified in P1 and his mother, while Hb Constant Spring (Hb CS, HBA2: c.427T > C) was identified in P2. The Hb Shenyang concentration measured by CE revealed 5.1–17.2% heterozygosity with normal red blood cell (RBC) parameters. The α haplotype [+ – S + – + –] [S signifies the inter ζ hypervariable region (HVR)] was associated with the Thai Hb Shenyang. The genotype-phenotype relationship indicates Hb Shenyang is likely a non pathological Hb variant that has neither dramatic clinical symptoms nor hematological anomalies. A simple multiplex allele-specific PCR for rapid diagnosis of Hb Shenyang has been developed.

Pedigree Analysis of Nonhomologous Sequence Recombination of HBA1 and HBA2 Genes

The aim of this study was to investigate a family with nonhomologous sequence recombination of HBA1 and HBA2 genes and provide a favorable basis for genetic counseling and eugenics. Peripheral blood of family members was collected. Hematological parameters were determined by an automated cell counter and hemoglobin (Hb) analysis was performed using high performance liquid chromatography (HPLC). Villus samples were taken for prenatal diagnosis (PND). Gap-polymerase chain reaction (gap-PCR) and reverse dot-blot were used for thalassemia genotyping. DNA sequencing was used to analyze the gene sequence of HBA1 (α1-globin) and HBA2 (α2-globin). The nonhomologous sequence recombination allelic variant of HBA1 and HBA2 genes were identified, namely, a gene conversion on the HBA2 gene called α12 (HBA12). The α12 allele consists primarily of the HBA2 gene sequence except for a segment of the IVS-II in which HBA2-specific sequences have been replaced by HBA1-specific sequences. The following genotypes were observed: – –SEA/αα12 (Southeast Asian deletion), αα/αα12 and αQSα/αα12 (Hb Quong Sze or Hb QS; HBA2: c.377T>C), and all manifested as small cell hypochromic anemia. To find the α12 allele in the Chinese population and clarify the influence of the α12 allele and its common inheritance with abnormal Hb and α-thalassemia (α-thal) on α-globin gene expression can help guide clinical diagnosis and genetic counseling.

Thalassemia and erythroid transcription factor KLF1 mutations associated with borderline hemoglobin A2 in the Thai population.

IntroductionElevated hemoglobin (Hb) A2 is an important diagnostic marker for β-thalassemia carriers. However, diagnosis of cases with borderline Hb A2 may be problematic. We described the molecular characteristics found in a large cohort of Thai subjects with borderline Hb A2.Material and methodsExamination was done on 21,657 Thai subjects investigated for thalassemia at Khon Kaen University, Thailand. A total of 202 subjects with borderline Hb A2 (3.5–4.0%) were selectively recruited and hematological parameters were recorded. DNA variants in α-, β-, δ-globin, and Krüppel-like factor 1 (KLF1) genes were examined using PCR.ResultsAmong 202 subjects, DNA analysis identified carriers of α+-thalassemia (n = 48; 23.8%), β-thalassemia (n = 22; 10.9%) and KLF1 mutations (n = 48; 23.8%). No molecular defect was observed in the remaining 84 (41.5%) subjects. Interaction of KLF1 and α-thalassemia was observed in 10 cases. Of the 22 β-thalassemia carriers, five β+-thalassemia mutations were identified with lower MCV and higher Hb A2. Seven KLF1 mutations were detected in 10 genotypes in subjects with higher MCV and Hb F. No β0-thalassemia, α-globin gene triplication or δ-globin gene mutation was detected.ConclusionsA large proportion of subjects with borderline Hb A2 are not β-thalassemia carriers and for those with β-thalassemia, only mild β+-thalassemia mutations were detected. Evaluation of the patients using Hb A2, Hb F and MCV values will help in selecting cases for further molecular analysis. The results should explain the unusual phenotype of the cases and facilitate a thalassemia screening program in the region.

First Report of Hemoglobin Le Lamentin [Alpha 20 (B1) His → Gln] in the Alpha1 Globin Gene in an Indian Patient and a Brief Update

Alpha globin gene structural variants are caused mainly due to point mutations in the alpha globin gene. They are generally asymptomatic but in rare cases cause problems in association with other structural variants of thalassemia. We report here for the first time in Indian population a rare alpha globin gene structural variant named Hb Le Lamentin.

Characterization of a Novel 71.8 kb α0-Thalassemia Deletion and Subsequent Summary of a Practical Procedure for Thalassemia Molecular Diagnosis

Thalassemia is the most common monogenic disorder around the world. Based on the principle of genotype–phenotype correlation, identification of thalassemia mutations is the essential prerequisite for clinical diagnosis and management. Because only common mutations are routinely detected, the identification of rare or undetermined mutations is a challenge for clinical laboratories. Herein, a proband presenting with inconsistent phenotype–genotype correlation after routine molecular screening was investigated by multiplex ligation-dependent probe amplification (MLPA), targeted-next generation sequencing (targeted-NGS), gap-polymerase chain reaction (gap-PCR) and Sanger sequencing. Eventually, a novel 71.8 kb deletion (– −71.8) was identified and characterized, which included HBZ (ζ), HBA2 (α2), and HBA1 (α1) genes and was causing α0-thalassemia (α0-thal). Furthermore, we summarized a practical procedure based on accumulated experience in studies and clinical practice, which can be a guide for molecular screening and clinical diagnosis of thalassemia, especially for identification of undetermined or novel mutations.

In-Silico structural analysis of wild-type human hemoglobin and its mutation resulting in sickle cell anemia

Background: Hemoglobin (HbA) is a metalloprotein having a heme prosthetic group, two α-globin chains, and two β-globin chains. A point mutation in the 6th position of wild-type HBB gene (GAG&gt;GTG) substitutes Glutamic acid (E) to Valine (V) in the β-chains of hemoglobin molecule (HbS) resulting in Sickle Cell Anemia. Structural deformities in the β-globin gene leads to disruptive conformation of red blood cells, disturbs the oxygen transportation and causes functional abnormalities.&#x0D; Aims and Objective: The purpose of the study was to understand the structural, genetic and metabolic effect of the point mutation on HBB gene that causes sickle cell disease.&#x0D; Materials and Methods: The 3D structures of HbA and HbS proteins were retrieved from PDB and the changes in their physical properties were analyzed using Swiss PDB viewer and Molegro molecule viewer. Gene networks were constructed using GeneMANIA server to study genetic and metabolic interactions of the HBB gene.&#x0D; Results: In-silico studies showed that the normal bond length between Glu6-Glu7 in HbA molecule is 1.32Å and that of HbS is 1.33Å. After comparing the two proteins, it was observed that sickle cell hemoglobin suffers a change in bond angle from 122.4º in HbA to 119.35º in HbS. Comparative energy minimization of Glu6 and Val6 in wild-type and mutant hemoglobin respectively yielded 8.78 and -9.083 KJ/mol net energy values, suggesting a more reactive HbA and less reactive HbS. Gene networks were determined on the basis of physical, genetic and co-expressive interactions of HBB gene which revealed a strong connection between HBB and HBA1 genes within the association constituted of various metabolic functions.&#x0D; Conclusion: Sickle cell disease results from a sequence of events which start with a single nucleotide substitution that ultimately leads to severe anemia and other cardiovascular problems. Incorporation of computational exercises correlates to a better probability of discovering precision medicine through target site-specific drug designing.

Dizygotic twins with prolonged jaundice and microcytic, hypochromic, hemolytic anemia with pyropoikilocytosis

Dizygotic twin males, born at 34 weeks gestation, had prolonged jaundice, microcytic, hypochromic anemia, FABarts hemoglobin, elevated end-tidal CO, and blood films consistent with hereditary pyropoikilocytosis. DNA sequencing revealed both had a heterozygous alpha spectrin (SPTA1) mutation (c.460_462dup) inherited from their asymptomatic mother, plus a 3-base pair duplication in alpha globin (HBA2) (c.364_366dupGTG) inherited from their asymptomatic father.

Hemoglobinopatias

formados pela combinação de duas cadeias polipeptídicas do “tipo α” com duas cadeias do “tipo β”, sendo Hb A1, Hb A2 e Hb Fetal os três principais tipos de hemoglobinas no adulto normal. As hemoglobinopatias são doenças causadas por mutações que afetam os genes de globinas, que resultam em alterações estruturais e/ou funcionais das moléculas de hemoglobina. Dentre as hemoglobinas variantes, as mais frequentes na população brasileira são a hemoglobina S e C. O objetivo é detectar hemoglobinas anormais em uma população acadêmica de Alfenas-MG. Para a realização do estudo, foram coletadas 336 amostras de sangue periférico, hemolisadas com saponina e clorofórmio. Em seguida, foi realizada a eletroforese de hemoglobina em pH alcalino para qualificação de hemoglobinas normais e grande parte das anormais. A confirmação da hemoglobina S foi feita pela prova de falcização. Do total de amostras analisadas, 97,62% (n=328) apresentam perfil eletroforético compatível com hemoglobinas AA e 2,38% (n=8) compatível com hemoglobinas AS (traço falciforme). No Brasil, a prevalência média de Hb AS é próxima de 2% na população total. Portanto, os resultados encontrados reafirmam a média encontrada na população brasileira de pessoas com o traço falciforme.

Sodium, Magnesium, Calcium, Manganese, Iron, Copper, and Zinc in Serums of Beta Thalassemia Major Patients

Thalassemia major is the most severe form of thalassemia and occurs with the impaired synthesis of β-globin which causes the accumulation of unpaired alpha globin chain. Patients with beta thalassemia major can only survive with periodically safe blood transfusions leading to the accumulation of iron in the bloods of patients, and this causes several endocrinopathies. Although iron overload in thalassemic patients has been extensively studied, there is little information about the levels of other trace elements. The aim of this study was to investigate the differences of serum concentrations of sodium, magnesium, calcium, manganese, iron, copper, and zinc for patients with major β-thalassemia. Concentration of elements was determined by inductively coupled plasma-mass spectrometry system. The statistical difference between the elemental concentrations of the patient and control groups was found by the Mann-Whitney U test. In addition, the relationship between concentrations of the measured elements for each group was determined by the Spearman correlation test. The results revealed that iron, zinc, magnesium, and manganese serum levels of thalassemic patients were significantly higher than the control group while calcium concentration was statistically lower than the control group. There was no significant difference observed for copper and sodium levels of patients when compared to the healthy control group.

Update in Laboratory Diagnosis of Thalassemia.

Alpha- and β-thalassemias and abnormal hemoglobin (Hb) are common in tropical countries. These abnormal globin genes in different combinations lead to many thalassemic diseases including three severe thalassemia diseases, i.e., homozygous β-thalassemia, β-thalassemia/Hb E, and Hb Bart’s hydrops fetalis. Laboratory diagnosis of thalassemia requires a number of tests including red blood cell indices and Hb and DNA analyses. Thalassemic red blood cell analysis with an automated hematology analyzer is a primary screening for thalassemia since microcytosis and decreased Hb content of red blood cells are hallmarks of all thalassemic red blood cells. However, these two red blood cell indices cannot discriminate between thalassemia trait and iron deficiency or between α- and β-thalassemic conditions. Today, Hb analysis may be carried out by either automatic high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) system. These two systems give both qualitative and quantitative analysis of Hb components and help to do thalassemia prenatal and postnatal diagnoses within a short period. Both systems have a good correlation, but the interpretation under the CE system should be done with caution because Hb A2 is clearly separated from Hb E. In case of α-thalassemia gene interaction, it can affect the amount of Hb A2/E. Thalassemia genotypes can be characterized by the intensities between alpha-/beta-globin chains or alpha-/beta-mRNA ratios. However, those are presumptive diagnoses. Only DNA analysis can be made for specific thalassemia mutation diagnosis. Various molecular techniques have been used for point mutation detection in β-thalassemia and large-deletion detection in α-thalassemia. All of these techniques have some advantages and disadvantages. Recently, screening for both α- and β-thalassemia genes by next-generation sequencing (NGS) has been introduced. This technique gives an accurate diagnosis of thalassemia that may be misdiagnosed by other conventional techniques. The major limitation for using NGS in the screening of thalassemia is its cost which is still expensive. All service labs highly recommend to select the technique(s) they are most familiar and most economic one for their routine use.

Rare β-Globin Gene Mutations including a de novo Mutation of Hemoglobin Hammersmith in Southern Thailand

Objective: The aim of this study was to characterize unknown β-globin gene mutations in individuals who attended Songklanagarind Hospital for thalassemia screening and genetic counseling.Material and Methods: β-thalassemia mutations in individuals with hemoglobin (Hb) A2 levels &gt;3.5% originating from various provinces in southern Thailand were characterized by reverse dot blot hybridization (RDB) and multiplex gappolymerase chain reaction using a panel of 30 allele-specific probes for point mutations and 6 sets of specific primers for large deletions. Mutations which could not be identified by these two methods were further analyzed by direct deoxyribonucleic acid (DNA) sequencing.Results: Nineteen subjects found to have uncharacterized β-globin gene mutations were analyzed by direct DNA sequencing. Nine different rare mutations were identified, four of which have not been to date described in Thailand: -30 (T&gt;C), codon 5 (-CT), Hb Monroe (codon 30, G&gt;C) and Hb Hammersmith (codon 42, T&gt;C). An Hb Hammersmith mutation detected in one subject appeared to be a spontaneous mutation, unrelated to family history. The other five mutations have been reported previously within Thailand, but here they were identified in the southern part of Thailand for the first time: -31 (A&gt;G), codon 15 (-T), codon 35 (C&gt;A), codon 95 (+A) and Hb Dhonburi (codon 126, T&gt;G). The presence of the mutations was confirmed by RDB.Conclusion: In addition to the already reported β-globin gene mutations, 9 other different types of mutations were identified. This information should be useful for planning genetic counseling and prenatal diagnosis programs for prevention and control of thalassemia diseases.

Association of Hb A2 Variants with Several Forms of α- and β-Thalassemia in Thailand

In this study, Hb A2 variants and their association with α- and β-thalassemia (α- and β-thal) were analyzed. We performed molecular analyses to identify α-thal [– –SEA (Southeast Asian), – –THAI (Thai), –α3.7 (rightward) and –α4.2 (leftward)] deletions, and Hb Constant Spring (Hb CS; HBA2: c.427T>C), Hb A2-Melbourne (HBD: c.130G>A), Hb A2′ (HBD: c.49G>C), Hb A2-Lampang (HBD: c.142G>A). β0-Thalassemia mutations included codon 17 (A>T) (HBB: c.52A>T), codons 41/42 (–TCTT) (HBB: c.126_129delCTTT), codons 71/72 (+A) (HBB: c.216_217insA) and IVS-I-1 (G>T) (HBB: c.92+1G>T) in 23 samples which had a Hb A2 variant peak in zone 1 of the capillary electrophoresis (CE) electropherogram. Results showed that 20 patients (87.0%) carried Hb A2-Melbourne with seven different genotypes for α- and β-thal, two (8.7%) carried Hb A2′ and one (4.3%) carried Hb A2-Lampang. All three samples doubly heterozygous for Hb A2-Melbourne/β0-thal had Hb A2 levels lower than 4.0%, while summation of Hb A2 and Hb A2-Melbourne ranged from 4.9–5.3%, reaching the accepted range (4.0–10.0%) for β-thal trait. Hb A2-Melbourne is the most common δ-globin variant in the Thai population. Hb A2 variant and Hb A2 levels must be combined in order to diagnose carriers of β-thal. β-Globin haplotype analysis showed an association with a single β-globin haplotype [+ – – – – + +] of Hb A2-Melbourne, Hb A2′ and Hb A2-Lampang, indicating that they were of the same origin. We developed a multiplex allele-specific polymerase chain reaction (ASPCR) for simultaneous detection of these three Hb A2 variants.

A Wide Spectrum Study of α-Globin Chain Variants: Cases from the UK

Over many years, cases of suspected α-globin chain variants were collected from different parts of the UK. The suspicion was based on the clinical picture, high performance liquid chromatography (HPLC) variant percentage, retention time (RT) and isoelectric focusing (IEF). DNA sequencing and the restriction enzyme EaeI were used for definitive diagnosis. One hundred and forty-eight variants were confirmed on one or both of the two α-globin genes (HBA2, HBA1). These cases were identified as 46 different α-globin chain variants. The most common variants were Hb J-Meerut [HBA2: c.362C>A (or HBA1)] (10.1%) and Hb Q-India (HBA1: c.193G>C) (8.1%), followed by Hb J-Paris-I [HBA2: c.38C>A (or HBA1)] and Hb Manitoba II (HBA1: c.309C>A) (7.4% for each). Other α variants were detected at lower frequencies. Two novel alleles were also detected: Hb Walsgrave [α116(GH4)Glu→Val (HBA2: c.350A>T)] and Hb Coombe Park [α127(H10)Lys→Glu (HBA2: c.382A>G)]. The majority of the ethnic origin was Indian. The positive predictive value for α variant identification by HPLC-RT analysis was 65.9%, 41.9% by IEF, and using both RT and IEF, the value was 72.1%. The number of variants was higher in HBA1 than in HBA2 genes and in exons 1 and 2 than in exon 3. There was no clustering of mutations in consecutive codons. This study, the characterization of a wide spectrum of α-globin chain variants, can facilitate the presumptive diagnosis of these variants prior to screening by a panel of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), and a definitive diagnosis by DNA sequencing.

Analysis of beta-globin gene variants in Liuzhou area of Guangxi

To determine the composition and distribution of beta-thalassemia-associated genotypes in Liuzhou area of Guangxi, China. From January to December 2017, 13 847 individuals who came for premarital examination, maternity examination or health check were recruited with informed consent. The subjects were analyzed by reverse dot blotting (RDB) for 17 common beta-thalassemia-associated variants among the Chinese population. Individuals with inconsistent results by blood test, electrophoresis, and RDB were subjected to Sanger sequencing to detect rare variants of the beta globin gene. In total 2098 individuals were found to harbor beta-thalassemia-associated variants, which included 2075 heterozygotes (98.90%), 12 compound heterozygotes (0.57%) and 11 homozygotes (0.52%). CD41-42 (48.43%) and CD17 (31.45%) were the most common variants. Three hundred and thirty eight-individuals were found to also carry heterozygous variants of the alpha globin gene, with the most common types being --SEA/aa, -a3.7/aa, aCSa/aa, -a4.2/aa. Through Sanger sequencing, rare genotypes such as beta-32/betaN, betaCD41-42/betaIVS-II-5 and betaCD30/betaN were detected. Liuzhou area has a high incidence of beta-thalassemia, but with a complex variant spectrum and clinical phenotypes different from other regions. Genetic counseling and prenatal diagnosis for the carrier population is crucial for the reduction of the related birth defects. Our result may provide valuable information for the prevention and control of beta-thalassemia in this area.